# data[].attributes.headline
another article on using chloride dioxide to control LD

# data[].attributes.subject
another article on using chloride dioxide to control LD

# data[].attributes.attachment_link.readable_text
Summary

This paper describes the results of a five-year monitoring programme applied to the water distribution system of the University Hospital of Pisa (Italy). The purpose of the programme was to evaluate the efficacy of an integrated water safety plan in controlling spp. colonisation of the potable water system. The impact of the safety plan on the ecology of legionella in the water network was evaluated by studying the genetic variability and the chlorine susceptibility of the strains isolated prior to, and throughout, the application of continuous , the risk management plan adopted appeared to discourage further cases of nosocomial chlorine dioxide treatment. After 45 months of water hyperchlorination, Legionella spp. were still present but the positive supply points were reduced by 79.4%. The samples exceeding 10Legionella pneumophila serogroup 1 (overall positivity rate: 161/423; 38%). Molecular typing was performed on 61 isolates (37.9% of the positive samples) selected on spatial and temporal criteria. This revealed the circulation and the persistence in the hospital environment of three prevalent types of ³ cfu/L were reduced by 83.8% and the mean counts showed a decrease of 94.6%. The majority of the isolates belonged to L. pneumophila Wadsworth, demonstrating allelic and electrophoretic characteristic profiles and different chlorine susceptibility. Two of these, one predominant and pre-dating the sanitation regimen, and one other isolated after three years of water treatment, were chlorine tolerant. Despite the ineffectiveness of chlorine dioxide in eradicating legionellosis. L. pneumophila

Introduction

Since the early 1980s, it has been known that healthcare-acquired Legionnaires' disease occurs due to exposure to Legionella spp. colonising hospital water distribution systems.1, 2, 3 The incidence of Legionnaires' disease reported in Italy over the last few years amounts to ∼9.7 cases per million. Of all these cases, 20-32% were nosocomial with a fatality rate exceeding 30%. ³

For prevention of nosocomial Legionella spp. infections, a structured quality management and adequate security system has to be established. Elements of the risk management plan are active surveillance for infections caused by waterborne pathogens, standard operating procedures for sanitisation, maintenance and operation of water systems, and scheduled controls on water samples collected from significant environmental sources. ⁴ Understanding pathogen distribution and relatedness is essential for determining the epidemiology of nosocomial infections and for aiding in the choice of effective control methods. Molecular typing assists accurate assessment of strain inter-relationship, and helps to elucidate the evolutionary response of pathogens to the environment when disinfection procedures are undertaken. Many genotypic methods have been applied to the epidemiological typing of L. pneumophila, even though their efficiency varies, and most of them are not standardised.5, 6, 7 Pulsed-field gel electrophoresis (PFGE) is probably the most widely used method, owing to its relative ease and high discriminatory power, although the inter-gel reproducibility of this technique is rather poor. ⁸ Sequence-based typing (SBT) is a powerful epidemiological method, rapid and easy to perform and provides unambiguous results. This method is particularly useful for classification within the 39 Legionella species when applied to the identification of the mip gene nucleotide sequences. ⁹ An SBT protocol developed by the European Working Group on Legionella Infection (EWGLI) is currently proposed as the new 'gold standard' for L. pneumophila typing. ⁵

The current study describes the results of a five-year monitoring programme applied to the water distribution system of the University Hospital of Pisa (Italy) to evaluate the efficacy of an integrated disinfection-filtration strategy in controlling Legionella spp. colonisation of the hospital water system. The impact of the water network safety plan adopted by the hospital trust was evaluated by the study of the genetic variability and the chlorine susceptibility of Legionella spp. strains isolated prior to and throughout the application of continuous chlorine dioxide treatment systems.

Section snippets

Setting

The Azienda Ospedaliero - Universitaria Pisana (AOUP), a 1605-bed teaching hospital, is composed of several buildings on two different sites (Site A and Site B) of the town of Pisa. As a result of two nosocomial cases of Legionnaires' disease in Site A (3.2 per 100 000 ordinary admissions) a control and risk management plan was begun in March 2002. The plan included: (i) active clinical surveillance for Legionella spp. infections; (ii) standard operating procedures for maintenance and operation

Results

The environmental monitoring revealed the presence of Legionella spp. in all the seven buildings of Site A of the University Hospital. Before the activation of the chlorine dioxide treatment system (March 2002 to March 2003) Legionella spp. were isolated in 54 of the 81 (66.7%) water samples analysed with a mean count of 31 868 cfu/L (range: 200-560 000 cfu/L), 41 (78.8%) of these samples exceeding 10 ³ cfu/L. Following 45 months of the application of the safety plan, including planned maintainance

Discussion

The five-year monitoring programme of the water-distribution system of the AOUP showed that L. pneumophila was ubiquitous and that the colonisation was persisting despite the disinfection measures taken. The molecular typing of the isolates showed the circulation and the persistence in the hospital environment of three prevalent types of L. pneumophila Wadsworth.

The two molecular methods adopted showed a 100% concordance, although a low degree of genetic heterogeneity characterised the analysed

Acknowledgements

We are grateful to Prof. Christopher Tyack for language revision of the final manuscript.

Role of environmental surveillance in determining the risk of hospital-acquired legionellosis: a national surveillance study with clinical correlations

La legionellosi in Italia nel 2005

World Health Organization
Guidelines for drinking-water quality

2020, International Journal of Infectious Diseases

The water samples collected from the involved wards just after the cases occurred were all negative for legionellae, also due to the disinfection treatments adopted in the hospital and described previously (Marchesi et al., 2013). The molecular typing of Lp1 isolated in the hospital water during the period 2009-2019 showed a high grade of similarity and stability over time, regardless of disinfection, in line with other authors (Casini et al., 2008). The molecular stability of these environmental isolates and the lack of correspondence with the clinical strains did not support the hypothesis of a healthcare-associated cluster.

Three cases of pneumonia caused by Legionella pneumophila serogroup 1 (Lp1) in immunosuppressed patients with repeated hospitalization were suspected as a healthcare-associated cluster. The environmental investigation did not reveal the presence of legionellae in the hospital patient rooms. Water samples collected from the homes of two patients were also negative for Legionella spp. In the absence of environmental strains potentially involved in the infections, we proceeded to genotype environmental Lp1 strains isolated in the hospital during routine water sampling during the decade 2009-2019 and recovered after long-term storage at −20 °C. These 'historical' strains exhibited a high grade of similarity and stability over time, regardless of the disinfection systems. The different molecular profiles shown among the clinical and environmental strains excluded a nosocomial outbreak. The study suggests that the application of molecular typing may be a useful tool to discriminate hospital vs community-acquired cases, mostly for severely immunosuppressed patients in whom the symptomatology could be insidious and the incubation period could be prolonged. Moreover, the genotyping allowed us to exclude any link between the cases.

2019, Decontamination in Hospitals and Healthcare

Healthcare-acquired Legionnaires' disease is caused by exposure to Legionella in hospital drinking water. Disinfecting the drinking water system is an effective preventive measure. The efficacy of any disinfection measures should be validated in a step-wise fashion from laboratory assessment to a controlled multihospital evaluation over a prolonged period of time. In this chapter, we describe long-term systemic disinfection methods (copper‑silver ionization, chlorine dioxide, monochloramine, ultraviolet light, hyperchlorination), a localized disinfection method (point-of-use filtration), and short-term disinfection methods in outbreak situations (superheat-and-flush plus or minus hyperchlorination). Controlled evaluations by other hospitals with experience with the system under consideration are the strongest evidentiary demonstration of efficacy. We also discuss the importance of risk assessment and infection preventionists in the selection of hospital disinfection strategies. The infection preventionist should be consulted in the selection of a commercial disinfection system. Their input will assure that healthcare facilities personnel make an evidence-based selection. Regulations and guidelines pertaining to Legionella control and Legionnaires' disease prevention can affect the need for and choice of disinfectant. These disinfectant technologies have also demonstrated efficacy against other problematic waterborne pathogens. Routine surveillance cultures of water for Legionella and monitoring of disinfectant concentrations are necessary for long-term efficacy.

2016, American Journal of Infection Control

The modern built environment (BE) design creates unique ecological niches ideal for the survival and mutual interaction of microbial communities. This investigation focused on the synergistic relations between Legionella and the fungal species commonly found in BEs and the impact of these synergistic relationships on the survival and transmission of Legionella.

A field study was conducted to identify the types and concentrations of fungi in BEs. The fungal isolates purified from BEs were cocultured with Legionella to study their synergistic association. Cocultured Legionella cells were aerosolized in an air-tight chamber to evaluate the efficacy of ultraviolet (UV) to inactivate these cells.

Aspergillus, Alternaria, and Cladosporium were the most common fungi detected in samples that tested positive for Legionella. After coculturing, Legionella cells were detected inside fungal hyphae. The microscopic observations of Legionella internalization in fungal hyphae were confirmed by molecular analyses. UV disinfection of the aerosolized Legionella cells that were cocultured with fungi indicated that fungal spores and propagules act as a shield against UV radiation. The shield effect of fungal spores on Legionella cells was quantified at >2.5 log ₁₀.

This study provides the first evidence, to our knowledge, of Legionella cell presence inside fungi detected in an indoor environment. This symbiotic relationship with fungi results in longer survival of Legionella under ambient conditions and provides protection against UV rays.

2016, Environmental Research

Additionally, molecular typing aids in the accurate assessment of strains' interrelationships, as well as the appearance of new STs (Fontana et al., 2014). Molecular typing also enables understanding the evolutionary response of pathogens to the environment when disinfection procedures are undertaken (Casini et al., 2008). To date, there are no specific indications with regard to the protocol to be used in air sampling.

Legionella is an intracellular microorganism living in natural and artificial aquatic environments. Although its transmission to humans is linked to the inhalation of contaminated aerosols, there is no validated air sampling method for the control and prevention of the disease. The aim of the present study was to provide more information on the distribution of Legionella spp. in indoor environments and to determine whether the same Legionella strains are isolated from air and water samples.

Ten healthcare facilities located in seven regions of Italy were enrolled. The serological typing of Legionella spp. from water samples and the surrounding air by active and passive sampling was assessed using polyvalent and monovalent antisera. Subsequently, the strains identified as Legionella pneumophila ( Lpn) underwent molecular typing by sequence-based typing (SBT) using seven genes ( flaA, pilE, asd, mip, mompS, proA, and neuA). The allelic profile number was assigned using the European Working Group for Legionella Infections-SBT database.

Lpn serogroup 6 was the most prevalent serogroup; it was found simultaneously in the air and water samples of three different healthcare facilities. In the remaining seven hospitals, Lpn serogroups 1, 6, 7, 9, and 12 were isolated exclusively from water samples. The molecular investigation showed that Lpn strains in the water and air samples of each positive healthcare facility had the same allelic profile. Strains, identified as sequence types (STs) 728 and ST 1638+ST 1324, were isolated in two respective healthcare facilities, and a new strain, identified as ST 1989, was obtained in one healthcare facility.

The application of the SBT method allowed to verify the homology among Legionella strains from water samples and the surrounding air. The results showed that the same Lpn strains were present in the air and water samples, and a new Legionella strain was identified.

2015, Molecular and Cellular Probes

Currently, more than 50 Legionella species are described and 70 serogroups [35] are identified, but Legionella pneumophila serogroup 1 is responsible for 39-70% of all Legionella nosocomial infections in Europe and for more than 84% of all Legionnaire's disease cases that occur worldwide, [9,32]. This is, primarily, due to the fact that L. pneumophila serogroup 1 is the most frequent bacterium of the species circulating in water systems [10]. Various parameters support the presence of L. pneumophila in water, such as hardness, temperature (between 25 and 45 °C), corrosion, scale, flow regimes [9,19], ability to produce monospecies biofilms [27] and resistance to disinfectant [18] particularly when chlorine is adopted in Legionella risk control.

The aim of the present work was to validate the performances of a new molecular method comprehensive of water sample filtration, DNA extraction and Real-Time PCR for the quantification of Legionella spp. in clear water samples, in accordance with the recent ISO Technical Specification 12869:2012. All criteria and requirements were verified considering inclusivity and exclusivity, check of the calibration function, limit of detection and limit of quantification, recovery calculation, robustness and uncertainty of the entire method. The performances were validated as all parameters resulted to be in compliance with values detailed by the above mentioned standard. The described method proved to be specific, sensitive, accurate and it has been fully validated according to ISO/TS 12869:2012. The possibility of using a validated molecular method will improve the reliability of the results making it a promising tool that should be used in addition to cultural analysis. Moreover, these findings make it particularly suitable for a relatively inexpensive screening of water samples, reducing the turnaround time and the workload.

2014, International Journal of Hygiene and Environmental Health

Legionella occurrence in premise drinking water (DW) systems contributes to legionellosis outbreaks, especially in the presence of suitable protozoan hosts. This study examined L. pneumophila behavior within DW biofilms grown on copper (Cu) and unplasticized polyvinylchloride (uPVC) surfaces in the presence of Acanthamoeba polyphaga. One year-old DW biofilms were established within six CDC biofilm reactors: three each containing Cu or uPVC coupons. Biofilms were then inoculated with L. pneumophila (uPVC-Lp and Cu-Lp), or L. pneumophila and A. polyphaga (uPVC-Lp/Ap and Cu-Lp/Ap) and compared to sterile water inoculated controls (uPVC- and Cu-Control) over a 4 month period. L. pneumophila appeared more persistent by qPCR within Cu biofilms in the presence of A. polyphaga compared to uPVC biofilms with or without A. polyphaga, but maintained their cultivability in uPVC biofilms compared to Cu biofilms. Also, persistent shedding of L. pneumophila cells (assayed by qPCR) in the effluent water implied colonization of L. pneumophila within Cu-coupon reactors compared to no detection from uPVC-coupon reactor effluent 14 days after inoculation. Hence, L. pneumophila appeared to colonize Cu surfaces more effectively and may be shed from the biofilms at a greater frequency and duration compared to L. pneumophila colonized uPVC surfaces with host amoebae playing a role in L. pneumophila persistence within Cu biofilms.

View all citing articles on Scopus
View full text

# data[].attributes.attachment_link.title
Molecular epidemiology of Legionella pneumophila serogroup 1 isolates following long-term chlorine dioxide treatment in a university hospital water system

# data[].attributes.attachment_link.description
This paper describes the results of a five-year monitoring programme applied to the water distribution system of the University Hospital of Pisa (Ital...

# data[].attributes.attachment_link.description
About ScienceDirect Shopping cart Contact and supportTerms and conditionsPrivacy policy

# data[].attributes.headline
using plasma to deactivate LD in wastewater 

# data[].attributes.subject
using plasma to deactivate LD in wastewater 

# data[].attributes.attachment_link.title
Deactivation of Legionella Pneumophila in municipal wastewater by ozone generated in arrays of microchannel plasmas - IOPscience

# data[].attributes.headline
preventing Healthcare acquired cases of LD 

# data[].attributes.subject
preventing Healthcare acquired cases of LD 

# data[].attributes.attachment_link.readable_text
2016, Infectious Disease Clinics of North America

The CDC position is that there is no known safe level of Legionella in building water systems and recommends attempts to reduce Legionella to undetectable levels if routine testing is done.19,60 Other guidance is to use Legionella concentration21,61 or a percentage of positive outlets62 to initiate corrective actions. The US Veterans Health Administration HCA LD Prevention Directive23 is an example of a policy that attempts to address all Legionella-positive outlets, following the recommendations of the CDC and other external stakeholders.

2013, Decontamination in Hospitals and Healthcare

This chapter provides an overview of safe water provision, treatment technologies and on-site water risk control to ensure patient safety in healthcare facilities. It reviews water and public health patient safety in prevention of avoidable waterborne-infections addressing water hygiene, and detailing water treatment methods, technologies, and sustainbility in future development. Water use, applications and key relevant guidance are included, addressing requirements for water quality, integrating augmented care units clinical practice management and tools addressing the risk of infection to patients, using a whole team approach to microbial water safety.

View all citing articles on Scopus

# data[].attributes.attachment_link.title
A proactive approach to prevention of health care-acquired Legionnaires' disease: The Allegheny County (Pittsburgh) experience

# data[].attributes.attachment_link.description
The Allegheny County Health Department (ACHD) in Pennsylvania distributed the first guidelines for prevention and control of health care-acquired Legi...

# data[].attributes.headline
flushing stagnant water reduces the presence of LD in water premises

# data[].attributes.subject
flushing stagnant water reduces the presence of LD in water premises

# data[].attributes.headline
another mention on monochloramine and residual disenfection

# data[].attributes.subject
another mention on monochloramine and residual disenfection

# data[].attributes.headline
the effect of LD in environments involving water 

# data[].attributes.subject
the effect of LD in environments involving water 

# data[].attributes.attachment_link.readable_text
Although Legionella spp. are often isolated from natural aquatic habitats, outbreaks of legionellosis are rarely traced to these sources. To determine the fate of Legionella pneumophila in the environment, filtered and unfiltered river water and seawater microcosms, incubated at 4 degrees C and 26 degrees C, were inoculated with [3H]thymidine-labeled L. pneumophila cells. Survival in these microcosms was monitored using [3H]thymidine labeling and culture on buffered-charcoal yeast extract agar amended with alpha-ketoglutarate (BCYE alpha). Immunofluorescent microscopy, direct fluorescent antibody staining, and acridine orange direct counts were also employed. To assess effects of grazing on Legionella, a duplicate set of samples was filtered through 2.0-microns Nuclepore filters to trap large protozoa. Over the test period, in the microcosms incubated at 4 degrees C, the culturable counts decreased ca. 1 log on BCYE alpha agar, with no substantial decline in thymidine count. Autoclaved seawater and river water controls held at 15 degrees C also showed no change in thymidine count. At 26 degrees C, a 3-log decline was observed in culturable counts, with ca. 1-log decline in thymidine counts. These results indicate that, although culturability declined by one to three orders of magnitude, when L. pneumophila microcosms were incubated at 4 degrees C and 26 degrees C, the cells remained metabolically active for extended periods, especially at 4 degrees C.

# data[].attributes.attachment_link.title
Effect of temperature on survival of Legionella pneumophila in the aquatic environment.

# data[].attributes.attachment_link.description
Effect of temperature on survival of Legionella pneumophila in the aquatic environment.

# data[].attributes.headline
this article talks about the effects of Citrox BCL (a combination of natural antimicrobials) and LDs biofilm 

# data[].attributes.subject
this article talks about the effects of Citrox BCL (a combination of natural antimicrobials) and LDs biofilm 

# data[].attributes.attachment_link.title
The Effect Citrox BCL on Legionella pneumophila Mechanisms of Biofilm Formation, Oxidative Stress and Virulence

# data[].attributes.attachment_link.description
Legionella pneumophila is responsible for causing Legionnaires’ disease and Pontiac fever, also known as legionellosis. The aim of this study was to investigate the mechanistic effect of a mixture of natural antimicrobials (Citrox BCL) in preventing L. pneumophila biofilm formation and reducing its…

# data[].attributes.headline
in the abstract section it mentions a fall in the incidences of LD and a decline in specific months

# data[].attributes.subject
in the abstract section it mentions a fall in the incidences of LD and a decline in specific months

# data[].attributes.attachment_link.title
LEGIONELLACEAE IN THE HOSPITAL WATER-SUPPLY: Epidemiological Link with Disease and Evaluation of a Method for Control of Nosocomial Legionnaires' Disease and Pittsburgh Pneumonia

# data[].attributes.attachment_link.description
An epidemiological link was found between contamination of a hospital water-supply by Legionella pneumophila and by Pittsburgh pneumonia agent (PPA) a...

# data[].attributes.headline
in the treatment section LD has lower mortality rates

# data[].attributes.subject
in the treatment section LD has lower mortality rates

# data[].attributes.attachment_link.readable_text
Summary

Infection with spp. is an important cause of community- and hospital-acquired pneumonia, occurring both sporadically and in outbreaks. Infection with spp. ranks among the three most common causes of severe pneumonia in the community setting, and is isolated in 1-40% of cases of hospital-acquired pneumonia. There are no clinical features unique to Legionnaires' disease. and in Legionella Macrolides fluoroquinolones are the most widely used drugs treatment. The availability of a good diagnostic repertoire, suitable for accurately diagnosing LD, constitutes the basis for the early recognition and treatment of the individual patient as well as for effective measures for prevention and control. This review summarizes the available information regarding the microbiology, clinical presentation, diagnosis and treatment of LD, with an emphasis on the laboratory diagnosis of infection with Legionella spp.

Introduction

An explosive, common-source outbreak of pneumonia caused by a previously unrecognized bacterium affected primarily persons attending an American Legion convention in Philadelphia in July, 1976. Thirty-four of 221 cases were fatal. Despite intensive laboratory investigation, the cause of the outbreak was undetected for months. An epidemiologic investigation determined that the disease most likely was airborne and focused primarily at one convention hotel, which subsequently had to be closed because of adverse publicity. ¹ About 6 months later, two investigators at the United States Centers for Disease Control and Prevention, Joseph McDade and Charles Shepard, announced that they had discovered the etiologic agent, a fastidious Gram-negative bacillus. ² Because of the historical association with the American Legion convention, this disease was called Legionnaires' disease (LD) and the etiologic agent Legionella pneumophila. L. pneumophila belongs to the family Legionellaceae. Use of an antibody test for the disease soon learned that several prior unsolved outbreaks of pneumonia had been LD, including outbreaks in the 1950s and 1960s. In addition, members of the genus had been isolated some 25 years earlier from sporadic cases of pneumonia. ³ An unsolved outbreak of a non-pneumonic febrile illness was also found to have resulted from exposure to Legionella bacteria; this illness was called Pontiac fever, after Pontiac, Michigan, where this had occurred. ⁴ As with LD, prior epidemics of Pontiac fever had occurred as early as 1949 without solved etiology.

There are three lessons from the Philadelphia outbreak. First and foremost was the dismissal of the idea that infectious diseases and their causes are a thing of the past or even that all infectious diseases are known. Second, LD occurs as a consequence of altering the environment for human benefit. Cases of LD have been traced to a wide variety of man-made water sources, including cooling towers, whirlpools and spas, fountains, ice machines and vegetable misters. A third lesson is how difficult it can be to break out of traditional patterns of thought when confronted with the unknown. In Philadelphia, initially, viral and toxic etiologies were sought, because of the clinical resemblance of the pneumonia to severe influenza. When bacteria that had the morphology of Gram-negative bacilli were subsequently demonstrated with electron microscopy in lung tissues of fatal LD cases, the significance of the observation was obscured by the consideration that they were merely secondary invaders. ³

This review summarizes the available information regarding the microbiology, clinical presentation, diagnosis and treatment of LD, with an emphasis on the laboratory diagnosis of infection with Legionella spp.

Section snippets

Microbiology

The family Legionellaceae consists of the single genus Legionella. Legionella are Gram-negative coccobacilli that measure 0.3-0.9 μm in width and 2-20 μm in length. In tissue and clinical specimens, the organisms are coccobacillary, measuring 1-2 μm. Elongated filamentous forms may be seen after growth on some culture media. Soluble iron and l-cysteine are required for optimal growth and for the initial isolation of the bacterium from both clinical and environmental sources. Iron, l-cysteine,

Clinical spectrum

Legionellosis classically presents as two distinct clinical entities, LD, a pneumonia with severe multisystem disease, ² and Pontiac fever, a self-limited flu-like illness. ⁴ In addition, many persons who are infected with legionellae, as proven by seroconversion, will remain asymptomatic. ²⁰ LD is transmitted from the environment to man by inhalation of an infectious aerosol. ²¹ In an unknown fraction of cases, microaspiration of contaminated water into the lungs could be the mode of nosocomial

Diagnosis

Although diagnostic methods have improved since L. pneumophila was first described in 1976, no currently available test is able to diagnose all Legionella spp. in a timely fashion with a high degree of sensitivity and specificity. Most of the data are applicable to L. pneumophila, since sensitivity and specificity estimates for non- pneumophila species are not known.

Treatment

LD is regarded by many as a plague with a high mortality. It must be remembered that the original cases were patients with severe disease who did not receive appropriate antibiotics. In this millennium, mortality has decreased with the increased index of suspicion by physicians, early empirical treatment with antibiotics that cover Legionella spp. and the advent of rapid laboratory tests. Delay in starting appropriate therapy has been associated with increased mortality. ⁹⁷ Benin et al. reported

Conclusions

When systematically sought, Legionella species are consistently recognized as one of the more common causes of pneumonia. The failure to diagnose LD in routine clinical practice largely depends on 3 factors: the inability to clinically and radiographically distinguish LD from other causes of pneumonia, the omission to order specific diagnostic tests for Legionella infection, and the shortcomings of available diagnostic tests. During an epidemic or in a setting with an unusual high prevalence, a

Acknowledgements

I thank Prof. Dr. Jan Kluytmans (Laboratory for Microbiology and Infection Control, Amphia Hospital, Breda, The Netherlands and Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands), Dr. Marcel Peeters (Laboratory for Medical Microbiology and Immunology, St. Elisabeth Hospital, Tilburg, The Netherlands) and Prof. Dr. Christina Vandenbroucke-Grauls (Department of Medical Microbiology and Infection Control, VU University Medical

T.J. Rowbotham
Legionella-like amoebal pathogens

T.J. Rowbotham
Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae

Typing of Legionella pneumophila and its role in elucidating the epidemiology of Legionnaires' Disease

P.H. Edelstein
Clinical features of Legionnaires' Disease: a selected review

View full text

# data[].attributes.attachment_link.title
Legionella spp. and Legionnaires' disease

# data[].attributes.attachment_link.description
Infection with Legionella spp. is an important cause of community- and hospital-acquired pneumonia, occurring both sporadically and in outbreaks. Infe...

# data[].attributes.headline
urinary antigen tests has lead to a reduction in mortality due to early diagnosis of legionnaires thanks to the tests

# data[].attributes.subject
urinary antigen tests has lead to a reduction in mortality due to early diagnosis of legionnaires thanks to the tests

# data[].attributes.attachment_link.readable_text
Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

References

1. Brenner DJ, Steigerwalt AG, McDade JE (1979) Classification of the Legionnaires' disease bacterium: Legionella pneumophila, genus novum, species nova, of the family Legionellacea e, familia nova. Ann Intern Med 90:656-658

2. Marston BJ, Plouffe JF, File TM, Hackman BA, Salstrom SJ, Lipman HB, et al (1997) Incidence of community-acquired pneumonia requiring hospitalization. Results of a population-based active surveillance study in Ohio. The Community-Based Pneumonia Incidence Study Group. Arch Intern Med 157:1709-1718

3. Fields BS, Benson RF, Besser RE (2002) Legionella and Legionnaires' disease: 25 years of investigation. Clin Microbiol Rev 3:506-526

Article Google Scholar

4. Hoge CW, Breiman RF (1991) Advances in the epidemiology and control of Legionella infections. Am J Epidemiol 13(Suppl L):329-340

5. Pasculle AW, Feeley JC, Gibson RJ, Cordes LG, Myerowitz RL, Patton CM, Gorman GW, Carmack CL, Ezzell JW, Dowling JN (1980) Pittsburgh pneumonia agent: direct isolation from human lung tissue. J Infect Dis 6:727-732

Google Scholar

6. Taylor TH, Albrecht MA (1995) Legionella bozemanii cavitary pneumonia poorly responsive to erythromycin: case report and review. Clin Infect Dis 2:329-334

Google Scholar

7. Harrison TG, Taylor AG (1988) The diagnosis of Legionnaires' disease by estimation of antibody levels. In: Harrison TG, Taylor AG (eds) A laboratory manual for Legionella. Wiley, Chichester, pp 130-136

Google Scholar

8. Nagington J, Wreghitt TG, Tobin JO, Macrae AD (1979) The antibody response in Legionnaires' disease. J Hyg (Lond) 2:377-381

Google Scholar

9. Bornstein N, Janin N, Bourguignon G, Surgot M, Fleurette J (1987) Prevalence of anti- Legionella antibodies in a healthy population and in patients with tuberculosis or pneumonia. Pathol Biol (Paris) 35:353-356

10. Di Capua A, Temperanza AM, Ciarrochi S, Castellani Pastoris M (1985) Upper limit of normal titer for Legionella pneumophila group 1 by indirect immunofluorescence and microagglutionation tests in healthy population in Italy. Boll Ist Sierotoer Milan 64:475-478

Google Scholar

11. Aguero-Rosenfeld ME, Edelstein PH (1998) Retrospective evaluation of the Du Pont radioimmunoassay kit for detection of Legionella pneumophila serogroup 1 antigenuria in humans. J Clin Microbiol 26:1775-1778

Google Scholar

12. Helbig JH, Uldum SA, Bernander S, Luck PC, Wewalka G, Abraham B, Gaia V, Harrison TG (2003) Clinical utility of urinary antigen detection for diagnosis of community-acquired, travel-associated, and nosocomial Legionnaires' disease. J Clin Microbiol 2:838-840

Article Google Scholar

13. Ramirez JA, Ahkee S, Tolentino A, Miller RD, Summersgill JT (1996) Diagnosis of Legionella pneumophila, Mycoplasma pneumoniae, or Chlamydia pneumoniae lower respiratory infection using the polymerase chain reaction on a single throat swab specimen. Diagn Microbiol Infect Dis 24:7-14

14. Murdoch DR, Chambers ST (2000) Detection of Legionella DNA in peripheral leukocytes, serum, and urine from a patient with pneumonia caused by Legionella dumoffii. Clin Infect Dis 30:382-383

15. England AC, Fraser DW, Plikaytis BD, Tsai TF, Storch G, Broome CV (1994) Sporadic legionellosis in the United States: the first thousand cases. Ann Intern Med 94:164-170

Google Scholar

16. Harrison TG, Doshi N (2001) Evaluation of the Bartels Legionella Urinary Antigen enzyme immunoassay. Eur J Clin Microbiol Infect Dis 10:738-740

Google Scholar

17. Pasculle AW, Veto GE, Krystofiak S, McKelvey K, Vrsalovic K (1989) Laboratory and clinical evaluation of a commercial DNA probe for the detection of Legionella spp. J Clin Microbiol 10:2350-2358

Google Scholar

18. Reischl U, Linde HJ, Lehn N, Landt O, Barratt K, Wellinghausen N (2002) Direct detection and differentiation of Legionella spp. and Legionella pneumophila in clinical specimens by dual-color real-time PCR and melting curve analysis. J Clin Microbiol 40:3814-3817

Download references

# data[].attributes.attachment_link.title
Diagnosis of Legionella infection in Legionnaires' disease - European Journal of Clinical Microbiology & Infectious Diseases

# data[].attributes.attachment_link.description
Since 1977, the diagnostic tools for Legionnaires' disease have been culture and serological investigation. Both methods require considerable time to produce results and have low to reasonable sensitivity. Since the introduction of urinary antigen tests in the mid 1990s, underdiagnosis has diminished and mortality has declined, thanks to early diagnosis.

# data[].attributes.headline
the prevention of healthcare-Acquired legionnaires  

# data[].attributes.subject
the prevention of healthcare-Acquired legionnaires  

# data[].attributes.attachment_link.readable_text
2016, Infectious Disease Clinics of North America

The CDC position is that there is no known safe level of Legionella in building water systems and recommends attempts to reduce Legionella to undetectable levels if routine testing is done.19,60 Other guidance is to use Legionella concentration21,61 or a percentage of positive outlets62 to initiate corrective actions. The US Veterans Health Administration HCA LD Prevention Directive23 is an example of a policy that attempts to address all Legionella-positive outlets, following the recommendations of the CDC and other external stakeholders.

2013, Decontamination in Hospitals and Healthcare

This chapter provides an overview of safe water provision, treatment technologies and on-site water risk control to ensure patient safety in healthcare facilities. It reviews water and public health patient safety in prevention of avoidable waterborne-infections addressing water hygiene, and detailing water treatment methods, technologies, and sustainbility in future development. Water use, applications and key relevant guidance are included, addressing requirements for water quality, integrating augmented care units clinical practice management and tools addressing the risk of infection to patients, using a whole team approach to microbial water safety.

View all citing articles on Scopus

# data[].attributes.attachment_link.title
A proactive approach to prevention of health care-acquired Legionnaires' disease: The Allegheny County (Pittsburgh) experience

# data[].attributes.attachment_link.description
The Allegheny County Health Department (ACHD) in Pennsylvania distributed the first guidelines for prevention and control of health care-acquired Legi...

# data[].attributes.headline
the Rise but fall of legionnaires in Nottingham England 

# data[].attributes.subject
the Rise but fall of legionnaires in Nottingham England 

# data[].attributes.attachment_link.readable_text
Summary

The annual number of confirmed cases of Legionnaires' disease in both Nottingham, and England and Wales, reached a peak in 1980 and has since declined. Legionella infection is a rare cause of community-acquired pneumonia managed at home (accounting for less than 1 % of cases), more common in those admitted to hospital (5-15%) and more common still in patients with severe pneumonia as seen on an intensive care unit (up to 30% of cases). Antibiotic therapy for any patient with moderate or severe pneumonia of uncertain aetiology should cover legionella infection.

Lower respiratory tract infection and upper respiratory tract infection (URTI) are very common, but the etiology is not diagnosed in routine practice. The objective of this study was to determine and compare the frequency distribution of the various infectious etiologies for these diseases. One hundred seventy five adults in the community with febrile LRTI and 75 with febrile URTI were included in a purely serologically based prospective study. Paired sera were obtained for each of the patients and were tested by EIA or immunofluorescence methods to identify 14 different pathogens. Only a significant change in antibody titers between the paired sera was considered diagnostic. At least one infectious etiology was identified in 167 patients (67%). In the LRTI group, infection with at least one of 7 respiratory viruses was found in 88 patients (50%). One of the atypical pathogens was found in 40 patients (23%), of these Legionella spp. in 19 (11%) and Mycoplasma pneumoniae in 18 (10%). A bacterial etiology was found in 19 patients (11%), of these Streptococcus pneumoniae in 8 (5%) and β-hemolytic streptococci group A in 5 (3%). The frequency distribution of etiologies in the URTI group was not significantly different from the LRTI group, except for M. pneumoniae that was identified in only one patient with URTI ( p = 0.015). More than one etiologic agent was found in 42 (17%) of the patients. LRTI is caused by a broad spectrum of etiologies, with respiratory viruses predominating and a moderate, but significant, prevalence of atypical pathogens. The frequency distribution of etiologies for URTI is similar to LRTI. In a significant proportion of patients with URTI and LRTI there is serologic evidence of infection with more than one pathogen. The justification and benefit of distinguishing between URTI and LRTI in routine clinical work is doubtful. When a decision is reached to treat RTI patients with an antibiotic, it is logical to use a macrolide or tetracycline.

To undertake a multileft study to evaluate the Biotest legionella urinary antigen enzyme immunoassay (EIA) performance against those EIAs already in use in 14 European laboratories.

Each laboratory examined urine specimens from appropriate patients using both their current assay and the Biotest EIA. Each examined: a standard panel of 12 coded urine samples (distributed by Biotest); a panel of 10 coded urine samples provided as part of a European external quality assurance (EQA) scheme; urine samples from patients with proven legionnaires' disease (LD); urine samples from patients with pneumonia of microbiologically proven cause other than LD; and urine samples submitted for routine examination. Thus, the performance of the Biotest assay (in comparison with current EIAs), its specificity and utility, and the inter-laboratory agreement were assessed.

Inter-laboratory agreement was excellent, with all participants obtaining the expected results for 20 of 22 coded urine specimens. Specificity, determined using 123 specimens from patients with infections of known etiology, was 100%. The Biotest EIA gave positive results in 86% of specimens which had been positive in the laboratories' current EIAs, and in 94.6% of those specimens which were positive for Legionella pneumophila serogroup 1.

The Biotest EIA is simple to use and specific and the results obtained in different laboratories show excellent agreement. The assay compares well with existing EIAs, at least for L. pneumophila serogroup 1.

A prospective one-year study ofcommunity pneumonia was conducted in Nottingham. 236 of 251 episodes of pneumonia (defined as an acute lower respiratory tract infection, for which antibiotics were prescribed, associated with new focal signs on examination of the chest) were investigated. Acute radiographic changes were present in 93 (39%). A pathogen was identified in 129 (55%) episodes, with Streptococcus pneumoniae, Haemophilus influenzae, and influenza viruses those most frequently identified. Mycoplasma pneumoniaewas uncommon and infection with Legionella pneumophilawas found in only 1 episode. Hospital admission was required in 52 (22%) episodes. 7 patients died (3%), all but one of the deaths occurring in patients who had been admitted to hospital. Pneumonia in the community is common but few people die of it. Initial antibiotic therapy should always cover S pneumoniae and H influenzae.

View all citing articles on Scopus

Recommended articles (6)

Inhalation exposes the upper and lower respiratory tracts of humans to a variety of airborne particles and vapors. Airborne transmission of pathogenic microorganisms to humans from the environment, animals, or other humans can result in disease. Inhalation is an important route of exposure as the lung is more susceptible to infection than the gastrointestinal tract. Ingested microorganisms must pass through the acidic environment of the stomach before they can colonize tissue while inhaled microorganisms are deposited directly on the moist surfaces of the respiratory tract. Inhalation of microbial aerosols can elicit adverse human health effects including infection, allergic reaction, inflammation, and respiratory disease. Following inhalation, infectious viruses, bacteria, and fungi can establish in host cells of the respiratory tract. Some are translocated and infect the gastrointestinal tract and other tissues. This article discusses human viral, bacterial, and fungal diseases transmitted via aerosols. Viral diseases presented are influenza, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), enteric viruses related infections, hantavirus disease, measles, and varicella. Bacterial diseases are Legionnaires' disease, tuberculosis, and nontubercule mycobacterial disease. Exposure to some Gram-negative and Gram-positive bacteria, endotoxin, and actinomycetes when dispersed through the air can result in disease following inhalation. Fungal diseases included are histoplasmosis, coccidiomycosis, blastomycosis, cryptococcosis, and aspergillosis. The threat of bioterrorism with airborne infectious agents is also briefly presented.

Immunology Letters, Volume 186, 2017, pp. 33-40

Legionella pneumophila is the main causative agent of Legionnaires' disease, which is a severe multi-system disease with pneumonia as the primary manifestation. We designed a recombinant Mip-PilE-FlaA dominant epitopes vaccine against Legionella pneumophila to prevent the disease and evaluated its immunogenicity and protective immunity. The protein structures of Mip, PilE and FlaA were analyzed using a computer, and the gene sequences of the dominant epitopes of the three proteins were selected to construct and optimize the vaccine. The optimized mip, pilE, flaA and recombinant mip-pilE-flaA gene sequences were cloned, expressed and purified. The purified proteins were used as dominant epitopes vaccines to immunize BALB/c mice and determine the protective immunity and immunogenicity of these purified proteins. The identification confirmed that the recombinant mip-pilE-flaA was successfully cloned and expressed. ELISA revealed that the Mip-PilE-FlaA group produced the highest IgG response, and this protein may considerably improve the production of some cytokines in BALB/c mice. Histopathology analyses of lungs from mice immunized with Mip-PilE-FlaA revealed a certain protective effect. Our work demonstrated that the recombinant dominant epitopes of Mip-PilE-FlaA exhibited strong immunogenicity and immune protection, and this protein may be an efficient epitopes vaccine candidate against Legionella pneumophila.

Journal of Microbiological Methods, Volume 139, 2017, pp. 1-7

Legionella spp. are important waterborne pathogens. Molecular typing has become an important method for outbreaks investigations and source tracking of Legionnaires. In a survey program conducted by the Guangzhou Center for Disease Control and Prevention, multiple serotypes Legionella pneumophila ( L. pneumophila) were isolated from waters in air-conditioning cooling towers in urban Guangzhou region, China between 2008 and 2011. Three genotyping methods, mip (macrophage infectivity potentiator) genotyping, SBT (sequence-based typing), and FAFLP (fluorescent amplified fragment length polymorphism analysis) were used to type these waterborne L. pneumophila isolates. The three methods were capable of typing all the 134 isolates and a reference strain of L. pneumophila (ATCC33153), with discriminatory indices of 0.7034, 0.9218, and 0.9376, for the mip, SBT, and FAFLP methods respectively. Among the 9 serotypes of the 134 isolates, 10, 50, and 34 molecular types were detected by the mip, SBT, and FAFLP methods respectively. The mip genotyping and SBT typing are more feasible for inter-laboratory results sharing and comparison of different types of L. pneumophila. The SBT and FAFLP typing methods were rapid with higher discriminatory abilities. Combinations of two or more of the typing methods enables more accurate typing of Legionella isolates for outbreak investigations and source tracking of Legionnaires.

View full text

# data[].attributes.attachment_link.title
The rise and fall of Legionnaires' disease in Nottingham

# data[].attributes.attachment_link.description
The annual number of confirmed cases of Legionnaires' disease in both Nottingham, and England and Wales, reached a peak in 1980 and has since declined...

# data[].attributes.headline
this article talks about a decline of legionnaires on 2 factors: the UK using thermostatic mixing valve in 1991 and the USA using fluoroquinolones and azithromycin this can be found in the community outbreak and prevention section

# data[].attributes.subject
this article talks about a decline of legionnaires on 2 factors: the UK using thermostatic mixing valve in 1991 and the USA using fluoroquinolones and azithromycin this can be found in the community outbreak and prevention section

# data[].attributes.attachment_link.readable_text
INTRODUCTION

Legionnaires' disease has a false but enduring status as an exotic plague. In reality, this disease is a common form of severe pneumonia, but these infections are infrequently diagnosed. Failure to diagnose Legionnaires' disease is largely due to a lack of clinical awareness. In addition, legionellae, the bacteria that cause this disease, are fastidious and not easily detected.

Legionellae are gram-negative bacteria found in freshwater environments. The first strains of Legionella were isolated in guinea pigs by using procedures for the isolation of Rickettsia ( 203). The first was isolated in 1943 by Tatlock, with another strain isolated in 1947 by Jackson et al. ( 268). In 1954, Drozanski isolated a bacterium that infected free-living amoebae from soil in Poland ( 68). This organism was classified as a species of Legionella in 1996 ( 143). The genus Legionella was established in 1979 after a large outbreak of pneumonia among members of the American Legion that had occurred 3 years earlier ( 38, 104) and was traced to a previously unrecognized bacterium, Legionella pneumophila ( 203). Legionellae are intracellular parasites of freshwater protozoa and use a similar mechanism to multiply within mammalian cells ( 91). These bacteria cause respiratory disease in humans when a susceptible host inhales aerosolized water containing the bacteria or aspirates water containing the bacteria.

Clinical Presentation

Legionellosis classically presents as two distinct clinical entities, Legionnaires' disease, a severe multisystem disease involving pneumonia ( 104), and Pontiac fever, a self-limited flu-like illness ( 114). Additionally, many persons who seroconvert to Legionella will be entirely asymptomatic ( 33).

It is not possible to clinically distinguish patients with Legionnaires' disease from patients with other types of pneumonia ( 76). Features of Legionnaires' disease include fever, nonproductive cough, headache, myalgias, rigors, dyspnea, diarrhea, and delirium ( 273). Although no chest X-ray pattern can separate this infection from other types of pneumonia, alveolar infiltrates are more common with Legionnaires' disease ( 188). The key to diagnosis is performing appropriate microbiologic testing when a patient is in a high-risk category.

There is some debate as to whether Legionnaires' disease presents as a clinical spectrum or whether the only manifestation of disease is pneumonia. In a study of persons exposed to L. pneumophila who did not develop pneumonia during a large outbreak of Legionnaires' disease in the Netherlands, there was no difference in the rate of respiratory symptoms between those who were seropositive and those who were seronegative. This would suggest that patients either develop pneumonia or are asymptomatic ( 33). Outbreaks of legionellosis have occurred in which some patients develop Legionnaires' disease and others Pontiac fever ( 21).

One species of Legionella, L. pneumophila, causes approximately 90% of all reported cases of legionellosis in the United States ( 199; R. E. Besser, unpublished data). This figure may be inflated because most diagnostic tests are specific for L. pneumophila. Currently, there are 48 species comprising 70 distinct serogroups in the genus Legionella ( 5, 23, 184, 185). Although there are now 15 serogroups of L. pneumophila, 79% of all culture-confirmed or urine antigen-confirmed cases are caused by L. pneumophila serogroup 1 ( 199; A. L. Benin and R. E. Besser, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. 873, 2001). Approximately one-half of the 48 species of legionellae have been associated with human disease. Since all legionellae are presumed to be capable of intracellular growth in some host cell ( 91), it is likely that most legionellae can cause human disease under the appropriate conditions ( 91). Infections due to the less common strains of Legionella are infrequently reported because of their rarity and because of the lack of diagnostic reagents ( 90).

Studies have estimated that between 8,000 and 18,000 persons are hospitalized with legionellosis annually in the United States ( 200). The disease is a major concern of public health professionals and individuals involved with maintaining building water systems. Legionellosis is generally considered a preventable illness because controlling or eliminating the bacterium in certain reservoirs will prevent cases of the disease. This concept of preventable illness has resulted in a number of guidelines and new control strategies aimed at reducing the risk of legionellosis in building water systems. The factors that lead to outbreaks or cases of Legionnaires' disease are not completely understood, but certain events are considered prerequisites for infection. These include the presence of the bacterium in an aquatic environment, amplification of the bacterium to an unknown infectious dose, and transmission of the bacteria via aerosol to a human host that is susceptible to infection ( 103).

Microbial Ecology

Water is the major reservoir for legionellae, and the bacteria are found in freshwater environments worldwide ( 98). Legionellae have been detected in as many as 40% of freshwater environments by culture and in up to 80% of freshwater sites by PCR ( 90). A single exception to this observation is Legionella longbeachae, a frequent isolate from potting soil ( 257). This species is the leading cause of legionellosis in Australia and occurs in gardeners and those exposed to commercial potting soil ( 244). The first U.S. cases of L. longbeachae infection associated with potting soil were reported in 2000 ( 45).

L. pneumophila multiplies at temperatures between 25 and 42°C, with an optimal growth temperature of 35°C ( 159). Most cases of legionellosis can be traced to human-made aquatic environments where the water temperature is higher than ambient temperature. Thermally altered aquatic environments can shift the balance between protozoa and bacteria, resulting in rapid multiplication of legionellae, which can translate into human disease. Legionellosis is a disease that has emerged in the last half of the 20th century because of human alteration of the environment. Left in their natural state, legionellae would be an extremely rare cause of human disease, as natural freshwater environments have not been implicated as reservoirs of outbreaks of legionellosis. Some outbreaks of legionellosis have been associated with construction, and it was originally believed that the bacteria could survive and be transmitted to humans via soil. However, L. pneumophila does not survive in dry environments, and these outbreaks are more likely the result of massive descalement of plumbing systems due to changes in water pressure during construction ( 159, 205).

Association with Amoebae

The presence of the bacteria in an aquatic environment and warm water temperature are two factors that can increase the risk of Legionnaires' disease. The third component is the presence of nutritional factors that allow the bacteria to amplify. These bacteria require a unique combination of nutrients in order to be grown in the laboratory. Initially, these unusual nutritional requirements appeared to contradict the widespread distribution of legionellae in freshwater environments. The levels of nutrients that legionellae require are rarely found in fresh water and, if present, would serve only to amplify faster-growing bacteria that would compete with the legionellae. However, these nutrients represent an intracellular environment, not soluble nutrients commonly found in fresh water.

Legionellae survive in aquatic and moist soil environments as intracellular parasites of free-living protozoa ( 91, 243). These bacteria have been shown to multiply in 14 species of amoebae, two species of ciliated protozoa, and one species of slime mold, while growth of legionellae in the absence of protozoa has been documented only on laboratory media ( 91, 123, 254). Protozoa naturally present in environments implicated as sources of Legionnaires' disease can support intracellular growth of legionellae in vitro ( 17). While protozoa are the natural hosts of legionellae, the infection of human phagocytic cells is opportunistic. Much of our understanding of the pathogenesis of legionellae has come from an analysis of the infection process in both protozoa and human host cells. Studies contrasting the role that virulence factors play in these two host populations allow speculation on the bacteria's transition from their obligatory relationship with protozoa to their opportunistic relationship with humans.

Association with Biofilms

Legionellae survive within biofilms in building water systems. The bacteria are more easily detected from swab samples of biofilm than from flowing water, suggesting that the majority of the legionellae are biofilm associated ( 235). A limited number of studies have attempted to characterize the bacteria's interaction within these complex ecosystems ( 236, 237, 282). These studies have evaluated the effect of temperature and surface materials on the growth of L. pneumophila as well as the effect of biocides on sessile legionellae. The use of biofilm models to evaluate biocide efficacy against L. pneumophila represents a vast improvement over previous studies, which primarily evaluated the susceptibility of agar-grown bacteria in sterile water ( 120, 174).

The majority of Legionella-biofilm studies that have been conducted employed naturally occurring microbial communities. Such studies have the advantage of representing a true and natural microbial community, but not all the organisms present have been identified and their contribution to the survival and multiplication of legionellae remains unknown. Biofilm matrices are known to provide shelter and a gradient of nutrients. The complex nutrients available with biofilms have led some researchers to propose that the biofilms support the survival and multiplication of legionellae outside a host cell ( 237). This concept is certainly plausible; most facultative intracellular bacteria are known to multiply extracellularly in some environments. If legionellae can multiply extracellularly within biofilms, the characterization of this phenomenon could have tremendous impact on control strategies for the prevention of legionellosis.

Investigators have attempted to detect extracellular growth of L. pneumophila by using a biofilm reactor and a defined bacterial biofilm grown on nonsupplemented potable water ( 211). The base biofilm was composed of Pseudomonas aeruginosa, Klebsiella pneumoniae, and the Flavobacterium-like organism isolated from a water sample containing legionellae. The addition of the amoeba Hartmannella vermiformis to the reactor resulted in a reproducible equilibrium between the amoeba and heterotrophic bacteria. L. pneumophila associated with and persisted in these biofilms with and without H. vermiformis. L. pneumophila cells did not appear to develop microcolonies, and growth measurement studies indicate that L. pneumophila did not multiply within this biofilm in the absence of amoebae. L. pneumophila did multiply in the biofilm and planktonic phase in the presence of H. vermiformis, and the majority of these bacteria appeared to be shed into the planktonic phase. These studies suggest that L. pneumophila may persist in biofilms in the absence of amoebae, but in the model, the amoebae were required for multiplication of the bacteria. This model biofilm was constructed of five preselected organisms and does not represent the many potentially diverse biofilms that may support the growth of legionellae in the environment. Additional studies are needed to determine if legionellae possess a means to multiply independent of a host cell within biofilms.

The control of biofilm-associated legionellae may lead to the most effective control measures to prevent legionellosis. Institutions that have experienced outbreaks of legionellosis are all too aware of how tenacious legionellae can be within building water system biofilms.

PATHOGENESIS

The Intracellular Cycle

Our understanding of the pathogenesis of Legionella spp. has grown tremendously in the past 10 years. Two major areas of accomplishment are characterization of the life cycle of legionellae and identification of virulence determinants by molecular techniques.

The primary feature of the pathogenesis of legionellae is their ability to multiply intracellularly. The life cycle of legionellae has been characterized in both protozoa and mammalian cells. Current understanding of this infectious cycle is outlined in Fig. 1. Studies of the infectious cycle are primarily based on microscopic observation by transmission and scanning electron microscopy and by fluorescent microscopy after labeling various bacterial and host cell components.

A series of classic experiments performed by Horwitz and associates in the 1980s outlined the basic pathway for L. pneumophila in human phagocytic cells. These studies determined that the bacteria enter the cells by coiling phagocytosis and that, once phagocytized, the bacteria reside within a unique phagosome that does not fuse with lysosomes or become highly acidic ( 145, 147, 148). Phagocytosis in human monocytes has been shown to be partly mediated by a three-component system composed of complement receptors CR1 and CR3, although the role of these receptors has never been determined ( 20, 223). Horwitz also described the interaction of the phagosome with mitochondria and ribosome-studded vesicles ( 144).

Rowbotham first demonstrated that L. pneumophila could infect amoebae and later characterized the life cycle of the bacterium in amoebae based entirely on observations made by light microscopy ( 240, 243). He noted that the bacteria would attach to extended trophozoites and enter the amoeba cell as several bacteria per vesicle. The bacteria initiated multiplication and became motile inside the host cell. Rowbotham also noted that the bacteria could either leave the host cell after lysis or be maintained within an encysted amoeba ( 240). Two growth phases were described for the bacterium. The multiplicative form was nonmotile and contained a rumpled wall and little or no β-hydroxybutyrate. The nonmultiplicative or infective forms were smaller, with smooth walls, motile, and contained numerous β-hydroxybutyrate inclusions.

Recently, these phases of the L. pneumophila life cycle have been characterized in greater detail, linking the expression of several traits with a particular phase of growth. Hammer and Swanson have proposed that host cell amino acid depletion causes the accumulation of 3′,5′-bispyrophosphate (ppGpp) ( 125). This would increase the amount of stationary-phase σ factor RpoS, resulting in the expression of stationary-phase genes. The stationary-phase proteins facilitate the infection of a new host cell and include sodium sensitivity, cytotoxicity, osmotic resistance, motility, and evasion of phagosome-lysosome fusion ( 263). Additional evidence for a precise cell cycle comes from the observation that expression of flagellin is coordinately regulated with the bacteria's ability to infect host cells. Pruckler et al. demonstrated a link between the loss of flagellar protein (Fla −) and loss of the ability to multiply in H. vermiformis and U937 cells ( 227). However, some Fla − strains retained their intracellular capability, demonstrating that the flagellar protein itself is not a virulence factor. These observations were confirmed by analysis of 30 laboratory-maintained strains of legionellae, showing that all strains possessing intact flagella were able to infect H. vermiformis, while strains that did not possess flagellar protein were unable to infect the amoebae ( 34).

There are striking similarities in the processes by which legionellae infect protozoa and mammalian phagocytic cells. Microscopically, the processes are virtually identical, although notable differences in the mechanism of entering and exiting the host cell do exist. The method of uptake of the bacteria has been described as coiling phagocytosis in both macrophages and amoebae ( 37, 146). Subsequently, studies have determined that L. pneumophila can enter host cells by conventional phagocytosis ( 50). The significance of coiling phagocytosis remains unclear, and this form of uptake has only been observed by transmission electron microscopy. L. pneumophila cells attach to small hair-like projections (filapodia) of the amoeba H. vermiformis ( 93). Filapodia are hair-like, naturally adherent structures, and the bacteria may bind to the projections by random encounter. Once attached to a Hartmannella host cell, the bacteria rapidly enter the cell by a nonphagocytic process.

Studies with the metabolic inhibitors methylamine and cytochalasin D have shown that L. pneumophila enter H. vermiformis by a form of receptor-mediated endocytosis ( 162). Actin polymerization, an essential component of phagocytosis, is not required for L. pneumophila to infect H. vermiformis, although polymerization is essential for the infection of human monocyte-like cells (U937). The attachment of L. pneumophila to H. vermiformis induces a time-dependent tyrosine dephosphorylation of multiple host proteins, including a 170-kDa protein homologue of the Entamoeba histolytica galactose- and N-acetylgalactosamine(Gal/GalNAc)-inhibitable lectin ( 277). This lectin is a putative receptor for the attachment of L. pneumophila to H. vermiformis.

Another difference between the uptake of L. pneumophila in human phagocytic cells and H. vermiformis is the role of host cell protein synthesis. Cycloheximide, an inhibitor of eukaryotic cell protein synthesis, inhibits the ability of L. pneumophila to enter H. vermiformis cells in a dose-dependent manner ( 2). L. pneumophila requires host cell protein synthesis to infect H. vermiformis but not to infect human U937 cells. The role of the proteins synthesized during bacterial cell uptake remains undefined.

Once an L. pneumophila cell has entered a host, the bacterium occupies a unique phagosome that does not follow the endosomal pathway. A number of studies have shown that the L. pneumophila phagosome does not possess markers or characteristics of conventional phagosomes. Swanson and Hammer have published an extensive review of these studies ( 263). Briefly, the early L. pneumophila phagosome lacks alkaline phosphatase, major histocompatibility complex (MHC) class I and II markers, transferrin receptors, Rab 7, LAMP-1, and cathepsin D. In addition, these vacuoles do not accumulate endocytic tracers such as Texas Red-ovalbumin, CM-DiI, or Alexa Fluor-streptavidin.

L. pneumophila occupies a vacuole that is independent of the endocytic pathway. Approximately 4 to 6 h after entering the host cell, the L. pneumophila phagosome is associated with ribosome-studded membranes that have been shown to be the host cell endoplasmic reticulum. Studies using antisera to detect the endoplasmic reticulum-specific protein BiP have shown that the protein colocalizes with the L. pneumophila phagosome in both protozoa and human host cells ( 1, 264). This capability is shared by Brucella abortus, another intracellular bacterial pathogen which multiplies within the host cell endoplasmic reticulum ( 60). L. pneumophila avoids lysosomes and the traditional endocytic pathway by utilizing a novel pathway into the host cell. It is not clear how much of this novel means of uptake is directed by the pathogen and what portion of this mechanism is under the control of the host cell.

The final stage of the infectious cycle is host cell death and release of the bacteria. L. pneumophila kills its host cell by either apoptosis or necrosis mediated by a pore-forming activity or both. In macrophages and alveolar epithelial cells, L. pneumophila induces apoptosis during the early stages of infection ( 107, 124). A second phase of necrosis induced by a pore-forming activity takes place in infected human phagocytes. In contrast, death of host amoeba cells has not been associated with apoptosis in studies utilizing Acanthamoeba castellani and Acanthamoeba polyphaga ( 108, 124). The pore-forming activity of L. pneumophila is required for killing and exiting A. polyphaga. These studies suggest that a different mechanism is used for killing and exiting mammalian and protozoan host cells.

Identification of Virulence Determinants by Molecular Techniques

A number of virulence factors have been described for L. pneumophila. This discussion will be limited to genes and gene products that play a role in the infection of mammalian and protozoan cells. The study of virulence factors in such diverse hosts has led to much speculation on the evolution of intracellular pathogens. This increasing body of research suggests that some intracellular pathogens of higher vertebrates acquired this ability in response to predation by protozoa ( 3, 91, 263). This interaction provides an excellent selective pressure for the acquisition of factors facilitating intracellular survival and, subsequently, infection.

In 1989, the first virulence-associated gene of L. pneumophila was detected by site-specific mutagenesis. This gene, designated mip for macrophage infectivity potentiator, encodes a 24-kDa surface protein (Mip) ( 48). Mip is a prokaryotic homolog of the FK506-binding proteins and exhibits peptidyl-prolyl- cis/trans isomerase activity ( 97). Subsequently, mip-like genes have been detected in other species of Legionella and other bacteria ( 47, 231). mip is required for efficient infection of guinea pigs, mammalian phagocytic cells, and protozoa, and this was the first gene shown to be involved in the pathogenesis of both mammalian and protozoan hosts ( 49). The mechanism of action for Mip remains unknown.

Genes within the loci encoding the type IV secretion system of L. pneumophila were the first factors detected that were essential for infection of the host cell. These loci comprise 24 genes in two separate regions of the Legionella chromosome and have been named Dot/Icm (defective for organelle trafficking/intracellular multiplication) ( 28, 196). This type IV secretion system encodes factors involved in the assembly and activation of conjugal transfer of plasmid DNA ( 263, 281). It is believed that L. pneumophila utilizes these operons to deliver virulence factors required for entering the host cell in a manner that initiates the infectious process. It is postulated that the system delivers a protein during phagocytosis that diverts the phagosome from the endocytic pathway. The only secreted substrate that has been identified for the Dot/Icm system is DotA, a polytopic membrane protein ( 213). A 19-amino-acid leader peptide is removed from DotA prior to secretion.

Genes encoding the loci for type II secretion systems are required for unrestricted intracellular growth of L. pneumophila. This type II secretion system is similar to the PilBCD piliation system in Pseudomonas aeruginosa ( 181). These genes were detected in L. pneumophila by analysis of mutants defective in type IV pilus formation ( 259). The two genes that have been analyzed the most extensively are pilE (pilin protein) and pilD (prepilin peptidase) ( 181, 259), The pilE gene/pilin protein is not required for intracellular growth but may be involved in attachment to the host cell. The pilD gene encodes the prepilin peptidase and is essential for pilus production and type II secretion of proteins. The ability of the pilD mutant strain to multiply within U937 cells, H. vermiformis, and guinea pigs is greatly impaired ( 180). In addition, this mutant showed reduced secretion of enzymatic activities ( 11, 12). The secreted protein that facilitates intracellular growth of L. pneumophila has not been identified. Recent studies have shown that loss of type II secretion explains the intracellular defect of the pilD mutant in amoeba, while a novel pilD-dependent mechanism may be involved in the infection of human cells ( 239).

Several other loci involved in the intracellular growth of L. pneumophila have been identified. These include mak (macrophage killing), mil (macrophage-specific infectivity loci), and pmi (protozoan and macrophage infectivity) ( 109, 110, 245). Defects in any of these loci result in either decreased intracellular multiplication of the organism or complete abolition of intracellular growth. The mechanism of action of these genes is not known.

Additional potential virulence factors include several cytotoxins, heat shock proteins, phospholipases, lipopolysaccharides, compounds associated with acquisition of iron, and metalloproteases ( 263). These factors will not be addressed in this review.

DIAGNOSIS

Taxonomy

The family Legionellaceae consists of the single genus Legionella. Some investigators have proposed placing the legionellae in three separate genera: Legionella, Fluoribacter, and Tatlockia ( 102, 112). However, recent studies using 16S rRNA analysis confirm the family Legionellaceae as a single monophyletic subgroup within the gamma-2 subdivision of the Proteobacteria ( 23, 106). Within the genus Legionella, the DNA relatedness between strains of a given species is at least 70%, whereas the DNA relatedness of one species to another is less than 70% ( 23). This conforms to the definition for a genus and species as defined by an ad hoc committee on reconciliation of approaches to bacterial systematics ( 23). Phylogenetically, the nearest relative to the Legionellaceae is Coxiella burnettii, the etiologic agent of Q fever ( 4, 263). These organisms have similar intracellular lifestyles and may utilize common genes to infect their host.

The number of species and serogroups of legionellae continues to increase. As previously mentioned, there are currently 48 species comprising 70 distinct serogroups in the genus Legionella (Table 1). There are 15 serogroups of L. pneumophila and two each in L. bozemanii, L. longbeachae, L. feeleii, L. hackeliae, L. sainthelensi, L. spiritensis, L. erythra, and L. quinlivanii, and a single serogroup in each of the remaining species ( 23). Some legionellae cannot be grown on routine Legionella media and have been termed Legionella-like amoebal pathogens (LLAPs) ( 242). These organisms have been isolated and maintained by cocultivating the bacteria with their protozoan hosts. One LLAP strain was isolated from the sputum of a pneumonia patient by enrichment in amoebae and is considered a human pathogen ( 242). Additional LLAP strains may be human pathogens, but proving this is difficult because they cannot be detected by conventional techniques used for legionellae. Recently, three LLAP strains were named Legionella species ( 5).

Legionellae are identified by growth on buffered charcoal yeast extract (BCYE), appropriate colonial morphology, and a requirement for the amino acid l-cysteine (excepting L. oakridgensis and L. spiritensis) ( 92). Isolates that react with specific antisera against known Legionella species are confirmed legionellae. When such isolates fail to react with specific antisera to all known Legionella species, they must be evaluated as potential new species within this genus. Species within the Legionellaceae can be distinguished by biochemical analysis, fatty acid profiles, protein banding patterns, serology, and nucleic acid analysis ( 23).

Biochemical data for legionellae other than L. pneumophila are limited. Legionellae are gram-negative, catalase-positive, motile rods with polar or lateral flagella ( 23). Most species produce beta-lactamase and liquefy gelatin. The oxidase reaction is variable, and reactions for nitrate reduction, urease, and carbohydrate utilization are negative. Amino acids are the carbon source for legionellae ( 224). Strains belonging to all serogroups of L. pneumophila except serogroups 4 and 15 strongly hydrolyze hippurate ( 133). Several laboratories have described methods for identifying putative Legionella isolates to the genus level and in some cases to the species level by using phenotypic characteristics ( 101, 129, 278).

All legionellae contain large amounts of branched-chain cellular fatty acids and contain ubiquinones with side chains of 9 to 14 isoprene units ( 208). The use of fatty acid and ubiquinone profiles allows all members of the Legionellaceae to be assigned to the genus. Although not all strains can be reliably identified to the species level, the narrowing of strains to groups facilitates further serologic testing and confirms additional tests.

Legionellae have been characterized based on analysis of soluble peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and protein profiling of native proteins by PAGE of soluble cytoplasmic protein banding patterns ( 89, 178). This method was shown to be complementary to analysis by fluorescent antibody, fatty acid, and ubiquinone analysis for identification of Legionella spp. prior to DNA-DNA hybridization.

Identification of Legionella species by serologic methods is the most frequently used technique. Antisera produced in rabbits have been prepared against all species and serogroups of Legionella and have been used in the Centers for Disease Control and Prevention (CDC) laboratory to identify most Legionella strains in a slide agglutination test ( 23, 270). It should be noted that these sera have been found to cross-react with new serogroups and species not previously identified. These antisera are not available commercially, and only a very limited number of laboratories have antisera to all species. Several commercial suppliers have produced antisera to a limited number of Legionella species and serogroups for use in identifying cultures by either direct fluorescent antibody or latex agglutination. Cross-reactions with non- Legionella bacteria have been reported for antisera produced against several Legionella species and serogroups ( 25, 80, 154). Monoclonal antibodies against genus-wide antigens have been produced to facilitate identification of isolates. Several investigators have produced monoclonal antibodies to the Legionella heat shock protein with various ranges of specificity ( 246, 258). Helbig et al. produced monoclonal antibodies against the Mip protein which reacted with 82 Legionella strains representing 34 species tested ( 138). A Legionella genus-wide antibody against flagella was produced by Bornstein et al. ( 32). No single antiserum is routinely used to identify all legionellae.

DNA-DNA hybridization is the definitive procedure for establishing the identity of a strain of Legionella or a new species. This procedure requires that DNA from the test strain be hybridized with DNA from all known species of Legionella. Therefore, only a very limited number of specialty laboratories are able to perform this procedure. Sequence analysis of specific genes has been employed for taxonomic analysis of legionellae. Analysis of 16S rRNA genes led to the designation of Legionella within the gamma-2 subdivision of the class Proteobacteria and has been used to show the phylogenetic relatedness of new species of this genus ( 106). Recently, a sequence-based classification scheme has been developed for legionellae which targets the mip gene ( 232). This procedure can unambiguously discriminate among the 39 species of Legionella tested. Many researchers are convinced that all taxonomic analysis will eventually become sequence-based in the near future.

Culture of Legionella

L. pneumophila was first isolated by using Mueller-Hinton agar supplemented with hemoglobin and IsoVitaleX (MH-IH) ( 88). The essential component in hemoglobin was found to be a soluble form of iron, and l-cysteine is the essential amino acid provided by the IsoVitaleX. These refinements led to the development of Feeley-Gorman agar, which provides better recovery of the organism from tissue ( 88). Later, starch was replaced with charcoal to detoxify the medium and the amino acid source was changed to yeast extract, resulting in charcoal yeast extract agar ( 87). Charcoal yeast extract agar is the base form for most media used to grow legionellae. The medium used for the culture of legionellae has been improved several times, eventually resulting in the medium currently used, buffered charcoal-yeast extract (BCYE) agar enriched with α-ketoglutarate with and without selective agents added ( 71, 73, 221). Culture requires the use of selective and nonselective media. These media can be prepared with or without indicator dyes, which impart a color specific for certain species of Legionella ( 279). Although the majority of Legionella spp. grow readily on BCYE agar, some require supplementation with bovine serum albumin to enhance growth ( 207).

Culture diagnosis remains the gold standard for diagnosis of legionellosis and is the most specific diagnostic procedure. Based upon culture of serologically positive (fourfold rise) patients, the sensitivity is near 60% and the specificity is near 100% ( 74, 192). However, a number of factors limit the sensitivity of culture. First, laboratories experienced in the isolation of legionellae are more likely to recover the organism. A survey by the College of American Pathologists indicated that as many as two-thirds of clinical microbiology laboratories in the United States are unable to grow a pure and heavy culture of L. pneumophila ( 76). Moreover, to improve the specificity of the diagnosis of pneumonia caused by pyogenic bacteria, hospital laboratories commonly reject sputum specimens containing many squamous epithelial cells or few polymorphonuclear leukocytes. However, some patients with Legionnaires' disease produce little or nonpurulent sputum, and sputum specimens that would be routinely discarded may contain culturable legionellae ( 150). The bacteria survive poorly in respiratory secretions, and immediate culture of these specimens is critical (R. F. Benson, unpublished data). The reported sensitivity of culture isolation from respiratory secretions ranges widely, from 20 to 80%, reflecting the perishable nature of these specimens and the expertise required ( 238).

Legionellae can be isolated from a number of specimens, including blood, lung tissue, lung biopsy specimens, respiratory secretions (sputum, bronchial alveolar lavage [BAL], and bronchial aspirates), and stool ( 74, 241). Respiratory secretions are considered the specimen of choice ( 74). In a recent review, Maiwald et al. suggested that sputum was less suitable than other respiratory secretions, especially in early disease, when few patients have a productive cough ( 192). When culturing sputum, it is best to pretreat the sample by either acidification or heat ( 40, 81). Legionella has also been cultured from a number of extrapulmonary sites such as bone marrow, prosthetic heart valves, and sternal wounds ( 67, 75). Several guidelines give detailed methods for the isolation of Legionella spp. from clinical samples ( 67, 260, 280, 285).

DFA Detection of Legionella

Microscopic examination of specimens using direct fluorescent antibody (DFA) staining was the first method used to detect legionellae in lung tissue (from autopsy or biopsy specimens) and respiratory secretions. Legionellae can be detected in respiratory secretions by DFA for several days after the start of antimicrobial therapy. DFA staining has also been used for serologic identification of Legionella isolates, as mentioned previously. A limited number of fluorescein-conjugated polyclonal sera are available for L. pneumophila serogroups and non- L. pneumophila species. The specificity and sensitivity of these reagents have not been evaluated, and cross-reactions with non-Legionella bacteria have been reported ( 25, 79, 154, 219). It has been recommended that polyclonal antisera to non- L. pneumophila species not be used for routine testing of clinical samples unless there is a high suspicion of disease due to non- L. pneumophila species or in epidemic investigations ( 192). The sensitivity of DFA testing of respiratory secretions for the diagnosis of Legionnaires' disease has ranged from 25 to 75%, and the specificity is >95% ( 74). While DFA provides a rapid method of identifying Legionella species, immunofluorescent microscopy is technically demanding and should be performed only by laboratory personnel experienced in the procedure. The specificity of polyvalent DFA reagents is probably lower than that of monoclonal reagents, leading some clinical microbiologists to recommend against routine use of polyvalent reagents ( 76).

A commercially available monoclonal antibody (Genetic Systems, Seattle, Wash.) which reacts with an outer membrane protein and detects all serogroups of L. pneumophila can be used to detect Legionella in clinical samples ( 117). This reagent is highly specific for L. pneumophila strains, making it possible to confirm isolates belonging to this species. It should be noted that a cross-reaction with Bacillus cereus spores from a strain isolated from water has been reported ( 99). However, this organism is easy to differentiate from Legionella in pure culture. The reagent's utility in detecting Legionella in clinical specimens has been evaluated and found to be comparable with polyvalent antisera ( 77). Genus-specific monoclonal antibodies have been produced to detect either a heat shock protein or the MIP protein, but these have not been shown to be useful for detection of Legionella in clinical samples ( 138, 139, 246, 258).

Serologic Diagnosis

A number of serologic tests have been developed to detect antibodies to Legionella spp. The use of the indirect immunofluorescence assay (IFA) was used to detect antibodies in patients from the Philadelphia outbreak and was instrumental in determining the cause of the illnesses. Enzyme immunoassay (EIA) and microagglutination have also been used to detect antibodies to the Legionnaires' disease bacterium ( 84, 85, 165). An indirect hemagglutination assay has been used for diagnosis of Legionnaires' disease due to L. pneumophila serogroups 1 to 4 ( 293). A counterimmunoelectrophoresis test and latex agglutination have been used to diagnose Legionnaires' disease ( 140- 142). In addition to the IFA test, a rapid microagglutination test is used extensively in Europe for diagnosis ( 130).

The rapid microagglutination test has some advantages over immunofluorescence, such as the ease of testing a large number of samples and the early appearance of agglutinating antibodies. The specificity is in the range of 97 to 99%, and the sensitivity is 80% ( 128). Cross-reactions due to antibodies to Pseudomonas aeruginosa in cystic fibrosis patients have been reported as well as cross-reactions between Legionella and Campylobacter spp. ( 35, 54). Klein reported cross-reactions in sera with elevated titers to Pseudomonas pseudomallei ( 164).

Of all these tests, the IFA has been evaluated extensively and is available commercially. The IFA originally developed by CDC used a progression of antigen preparations from live bacteria ( L. pneumophila serogroup 1, Philadelphia 1) grown in infected hen yolk sacs to ether-killed antigen grown on enriched Mueller-Hinton agar and finally to a heat-killed antigen suspension grown on various laboratory media ( 203, 289). Validation of this heat-killed antigen with epidemic sera yielded a sensitivity of 78 to 91% and a specificity of 99% ( 287). Subsequent CDC studies demonstrated that a heat-killed antigen grown on BCYE medium gave the same level of sensitivity and specificity as formalin-killed antigen from the same medium if a higher cutoff value for positive results was used ( 286).

Additional factors to consider in the serologic diagnosis of Legionnaires' disease are the use of an anti-human immunoglobulin which recognizes immunoglobulin G (IgG), IgM, and IgA ( 15, 288). These antibody responses may be serogroup specific or may react with an antigen common to L. pneumophila; therefore, it is not possible to reliably determine the serogroup or species causing infection ( 290). Early studies recommended the use of polyvalent antigen pools to screen sera and subsequent testing with the monovalent components ( 83, 290). Recent experience by several laboratories has indicated that 25% of seroconversions with polyvalent antigens could not be confirmed when retested with the monovalent antigen ( 72). Current recommendations suggest the use of L. pneumophila serogroup 1 antigen only and the avoidance of polyvalent antigen pools. It is extremely difficult to make a diagnosis based on results of IFA tests with polyvalent pools.

The IFA test used in Europe is slightly different from the test described above. The strain of L. pneumophila serogroup 1 used is Pontiac 1, which is grown in embryonated hens' eggs and formalin killed ( 127). Harrison et al. evaluated both the IFA and the rapid microagglutination test mentioned earlier for the diagnosis of Legionnaires' disease ( 126). The sensitivity of both assays was 80%, and additionally, 40% of patients had suggestive titers within the first week of hospital admission. Evaluation of the specificity of the IFA with sera from patients with Mycoplasma pneumoniae, Chlamydia psittaci, Coxiella burnetti, influenza A virus, and adenovirus showed no cross-reactions with the formalin yolk sac antigen ( 269).

Patients with nonlegionellosis pneumonia and bacteremia have been found to have false-positive titers to Legionella spp. These infections have included Bacteroides fragilis, C. psittaci, and Pseudomonas aeruginosa in cystic fibrosis patients, as well as mycobacteria, mycoplasmas, campylobacters, Citrobacter freundii, Pseudomonas pseudomallei, Haemophilus influenzae, Coxiella burnetti, Rickettsia typhi, and Proteus vulgaris OX19 ( 31, 36, 54, 69, 80, 118, 119, 164, 198, 204, 212, 218, 288). Cross-reactions occur more frequently with non- L. pneumophila species.

Despite the obvious utility of serologic diagnosis, these tests have a number of important limitations. Even in cases of culture-confirmed Legionnaires' disease, a fourfold rise in antibody by IFA can be documented for only 70 to 80% of patients, and seroconversion following legionellosis may not occur for up to 2 months after illness onset ( 72). Past case definitions for Legionnaires' disease included a presumptive diagnosis based on an elevated titer of ≥1:256. Recent studies of patients hospitalized with community-acquired pneumonia showed that a single titer of ≥1:256 did not distinguish between Legionnaires' disease and pneumonia due to other agents. Only 10% of 68 patients with legionellosis confirmed by culture or a fourfold rise in antibody had acute-phase titers of ≥256, compared with 6% of 636 patients with pneumonia caused by other agents ( 226).

Various studies have demonstrated the presence of L. pneumophila serogroup-specific antigens, L. pneumophila-specific antigens, and Legionellaceae-specific antigens ( 14, 52, 53). Several investigators have attempted to identify specific antigens that could be used to improve the specificity of antibody determinations. Sampson et al. ( 247), using immunoblot analysis, demonstrated antibody response to major protein antigens of L. pneumophila SG 1. Further studies with a purified 60-kDa protein antigen demonstrated the presence of Legionella-specific and nonspecific epitopes ( 225). This protein was later shown to be a heat shock protein which is a cross-reactive common antigen. The use of flagella in an EIA test was useful only for L. pneumophila serogroup 1 ( 216). Studies have been performed with whole bacteria ( 18), heat-killed soluble antigens ( 248), and sonicated bacteria ( 15). A lipopolysaccharide fraction for detecting IgG and IgM was the most specific, followed by an IgA assay with a sonicated antigen, which was the most sensitive ( 15).

Recently, two enzyme-linked immunosorbent assay (ELISA) test kits have been introduced. The Wampole Laboratories (Cranbury, N.J.) kit utilizes a heat-inactivated, solubilized pool of L. pneumophila serogroups 1 to 6 and detects IgG and IgA. The Zeus Scientific, Inc. (Raritan, N.J.) kit uses a formalin-inactivated, sonicated suspension of L. pneumophila serogroups 1 to 6 and detects IgG, IgM, and IgA. This test is also sold by Sigma Diagnostics (St. Louis, Mo.). These test systems have not been independently evaluated.

It is important to use reagents that detect all major immunoglobulin classes (IgG, IgM, and IgA) to increase test sensitivity ( 288). However, commercial kits for measuring antibody response do not use a secondary antibody proven to react with IgM and IgA ( 72). Studies have shown that many patients produce primarily IgM antibodies and that these are useful for early diagnosis of Legionnaires' disease ( 59, 214, 294). However, IgM may be present later in some confirmed cases, limiting the usefulness of the assay for early diagnosis in all patients. In a 2-year follow-up study of IgM in 35 patients, 13 had seroconversion to IgM within 7 days. Seven patients had titers of >64 at 2 years after the disease. The authors stated that persisting titers made it impossible to determine a reliable value of a single titer ( 157).

Serologic diagnosis of Legionnaires' disease was once the most frequently used diagnostic test. The need for testing of paired serum samples collected 3 to 6 weeks apart has diminished the use of serology, and urine antigen detection is now the most frequently used diagnostic test (A. L. Benin and R. E. Besser, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. 873, 2001). Perhaps the development of sensitive and specific assays for detecting IgM will increase the usefulness of serologic testing.

Urine Antigen Detection

Urinary antigen testing has led to the recognition of outbreaks of Legionnaires' disease and allowed a rapid public health response ( 96, 100, 179). In addition, urine antigen testing permits early diagnosis and initiation of appropriate antibiotic therapy ( 158). The capture antibody used in the majority of these assays is considered to be specific for L. pneumophila serogroup 1. Therefore, even though the majority of human disease is associated with L. pneumophila serogroup 1, total dependence on this diagnostic assay may miss as many as 40% of cases of legionellosis. Legionella antigen present in urine specimens appears to degrade upon prolonged storage ( 234). There is a single report of a false-positive urinary antigen assay on a specimen obtained from a renal transplant patient who had received rabbit serum with antibodies to thymocytes ( 57).

Changes in the use of diagnostic tests for Legionnaires' disease may have had an impact on the number of reported cases, the reported mortality, and the distribution of serotypes. In the United States, cases diagnosed by culture have declined, while cases diagnosed by urine antigen have increased. Since urine antigen testing allows earlier diagnosis, this may have allowed more appropriate antimicrobial therapy and improved the outcome of Legionnaires' disease. However, since urine antigen testing primarily detects serogroup 1 infections, cases of Legionnaires' disease caused by other serogroups and species may have been missed (A. L. Benin and R. E. Besser, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. 873, 2001).

Shortly after the outbreak of Legionnaires' disease at the American Legion Convention in Philadelphia, two investigators reported the ability to detect antigen in the urine of serologically confirmed Legionnaires' disease patients by ELISA ( 27, 271). Both studies tested a small number of patients but suggested the utility of a rapid technique for diagnosing Legionnaires' disease by using a clinical sample that is easily obtained. These studies were followed by experiments with a radioimmunoassay to detect antigen in nine patients confirmed by culture, direct fluorescent antibody, or serologic testing of specimens collected on or before erythromycin therapy. The sensitivity was 100%, and urine samples from 245 controls were negative, for a specificity of 100% ( 168). The antigen detected is a component of the lipopolysaccharide portion of the Legionella cell wall and is heat stable ( 168, 292). Antigens are generally detectable in urine within a few days of illness onset and can remain so for several weeks after initiation of appropriate antimicrobial therapy ( 167). Studies have shown that antigen is excreted as soon as 3 days after the onset of symptoms and can persist for >300 days ( 167).

A study relating the onset of symptoms and antigen detection indicated that antigen was detected in 88% of patients tested during days 1 to 3, 80% tested during days 4 to 7, 89% tested during days 8 to 14, and 100% tested after day 14 ( 167). In another study, multiple urine samples from 23 patients were analyzed after initiation of therapy to determine how long antigen continued to be excreted. In some patients, antigen was no longer detected within 4 days of therapy, but it persisted for >300 days in one patient. The duration of antigen excretion has implications for the diagnostic relevance of antigen detection in patients with recurrent pneumonia in the months following illness. Also, concentration of urine specimens can lead to an increased sensitivity for the urine antigen assay, although specificity is not affected ( 64, 166).

These early studies on antigen detection in legionellosis patients led to the development of a commercial radioimmunoassay (RIA) (Du Pont, Wilmington, Del.) for urine specimens, later manufactured and distributed by Binax, Inc. (Portland, Maine). An evaluation of the urinary antigen testing by RIA for the diagnosis of L. pneumophila serogroup 1 infection indicated a sensitivity of 93% and specificity of 100% ( 6). In studies comparing urinary antigen testing, DFA, and culture, the authors concluded that urinary antigen detection was the most useful test ( 230). Several studies assessing the overall sensitivity (all Legionella infections, not just L. pneumophila serogroup 1) of the Binax Equate Legionella Urinary Antigen kit determined this to range from 53 to 56% ( 29, 226). A major drawback of urinary antigen testing with the RIA is the difficulty involved in the handling and disposal of radioisotopes required to perform RIA. For this reason, the RIA test was replaced by an ELISA in the mid-1980s (Binax, Inc.). Resulting comparisons of these two assays showed that the ELISA format was positive for 67 to 88% of specimens that were positive with the RIA kit ( 65, 122, 160). Another study showed that the sensitivity of the EIA was significantly greater than that of the RIA, and if the cutoff was lowered to ≥2.5, the sensitivity increased to 90%, while the specificity remained 100% ( 46).

Currently, there are three commercially available ELISA urinary antigen tests: Wampole (formerly distributed by Binax, Inc.), Cranbury, N.J.; Trinity Biotech, Bray, Ireland; and Biotest AG, Dreieich, Germany. Two of these tests (Wampole and Trinity Biotech) are intended to be specific for L. pneumophila serogroup 1 and are not likely to detect the 20 to 30% of Legionnaires' disease cases that are caused by other serogroups and species ( 199). The Biotest ELISA is intended to detect antigen of other L. pneumophila serogroups and Legionella species. Domínguez et al. ( 63) compared the Binax (Wampole) and Biotest EIA kits with concentrated and nonconcentrated urine samples. There were no significant differences in sensitivity between the two tests, and concentrating urine samples improved the sensitivity of both assays. Both tests detected antigens from 14 serogroups of L. pneumophila and L. bozemanii, and both tests were negative for L. longbeachae serogroup 1.

The most recent method for detecting antigenuria is the immunochromatographic (ICT) membrane assay. The ICT assay is similar to a home pregnancy test and is commercially available (NOW Legionella urinary antigen test; Binax, Inc.). The test is simple to perform and does not require special laboratory equipment, and results can be obtained within 15 min. A comparison of the Binax NOW with the Binax EIA showed 98% overall agreement between the two tests and a specificity of 100% ( 62). The authors suggested using concentrated urine samples to increase test sensitivity. Another study determined that the sensitivity of the ICT assay for L. pneumophila serogroup 1 was 80% and the specificity was 97% ( 135).

There is still a need to develop antigen capture assays that can diagnose infections with all species and serogroups of Legionella. Development of a genus-wide urinary antigen test appears feasible and would provide a distinct diagnostic advantage ( 265, 266). Tang and Toma ( 266) developed a broad-spectrum ELISA that could detect soluble antigens from numerous L. pneumophila serogroups and species by using rabbit antisera from a combination of intradermal, intramuscular, and intravenous inoculations over a period of 8 months. The spectrum of serogroups and species detected with the broad-spectrum ELISA has since been reported to include L. pneumophila serogroup 12, L. sainthelensi, L. bozemanii, L. cincinnatiensis, L. hackeliae, L. maceachernii, and L. parisiensis ( 24, 111, 265, 267).

Another recent review of the impact of urine antigen testing on diagnosis and therapy and a more thorough review of urine antigen testing was written by Kashuba and Ballow ( 158).

Detection of Legionella Nucleic Acid

The first assay designed to detect the DNA of L. pneumophila was a radiolabeled ribosomal probe specific for all strains of Legionella spp. (Gen-Probe, San Diego, Calif.). Researchers reported varying sensitivity and specificity for this assay ( 61, 222, 291). The use of the probe at one hospital resulted in 13 false-positive cases, with no symptoms consistent with Legionnaires' disease in 13 of 23 patients ( 175). The assay was removed from the market soon after this pseudo-outbreak.

PCR represents one of the few diagnostic tests with the potential to detect infections caused by all of the known species of Legionella. Since most rapid tests only detect infections due to L. pneumophila serogroup 1, an accurate PCR test would greatly enhance the ability to diagnose these infections. Various PCR tests that have been developed for legionellae target either random DNA sequences for L. pneumophila ( 256), the 5S rRNA gene ( 187, 189), the 16S rRNA gene ( 156, 183, 206), or the mip gene ( 82, 189, 229). A summary of DNA targets for various PCR assays is presented in Table 2. The most widely used test was a commercially produced kit designed to detect legionellae in the environment (EnviroAmp kit; Perkin-Elmer, Inc., Norwalk, Conn.). This test was removed from the market in 1997. It simultaneously amplified two targets, a 5S rRNA target specific for the Legionella genus and a portion of the mip gene specific for L. pneumophila. The kit was designed for testing environmental samples, and the manufacturers did not attempt to have it approved for clinical use. Nevertheless, several researchers successfully used these kits to detect legionella DNA in human specimens ( 161, 201, 284).

A very limited number of laboratories test for legionellae by PCR at this time. The few studies which have been conducted indicate that the PCR detection of legionellae infections has a moderate sensitivity and a high specificity. Legionella DNA has been detected in respiratory secretions such as BAL, pharyngeal swabs, nasopharyngeal swabs, peripheral blood mononuclear cells, urine, and serum ( 136, 151, 156, 193, 202, 209, 210, 229, 253).

The PCR procedures described above have used either visualization of DNA amplicons by ethidium bromide staining in agarose gels or reverse dot blot hybridization to probes immobilized on nylon membranes with biotinylated primers. Other PCR assays targeting the 16S rDNA gene have been reported to be highly sensitive and specific ( 51). However, a number of false-positive results were obtained, revealing homology to Acinetobacter spp. or an unidentified proteobacterium. False-positive reactions have also occurred with the commercially available tests and when using primers for 16S rDNA, which reacted with Gemella spp. (R. Benson, personal communication).

Recently, several researchers have reported on the use of real-time PCR combined with the use of a hybridization probe to confirm the product identity for rapid detection of legionellae in clinical specimens ( 13, 131). This method reduces the risk of cross-contamination, reduces the time required to process samples, and eliminates the need for sample analysis after PCR. These assays promise increased sensitivity and specificity, although further validation is required. Limited studies and preliminary data suggest that PCR may add to the diagnostic repertoire, but this procedure may lack the sensitivity and specificity to provide anything other than supplemental data for the clinician.

Subtyping of Legionella spp.

L. pneumophila serogroup 1 comprises a fairly heterogeneous group of organisms that account for most of the cases of legionellosis in the United States. L. pneumophila serogroup 1 can be divided into a number of subtypes by various techniques. These procedures are used to match environmental isolates with patient isolates obtained from outbreak investigations of legionellosis. Because of the diversity within L. pneumophila serogroup 1, clinical and environment isolates must be matched by molecular techniques to adequately identify environmental sources of disease.

Initially, legionellae were identified to the serogroup level during investigations of legionellosis. This form of serologic subtyping uses polyvalent or monoclonal antisera and may be adequate for identifying reservoirs of some of the more uncommon legionellae causing disease. The variety of strains and distribution of L. pneumophila serogroup 1 necessitate more elaborate subtyping procedures to discriminate among these bacteria. Several research groups have developed monoclonal antibodies for this purpose ( 220, 272, 283). An international panel of seven monoclonal antibodies (MAb) was proposed in 1986 ( 155). Although much information has been gained through the use of this panel, several of the cell lines have been lost, and most of these reagents are no longer available. Use of these monoclonal antibodies has identified 12 "type" strains within L. pneumophila serogroup 1 ( 16). Monoclonal antibodies produced by Helbig et al. and labeled the Dresden Legionella LPS MAb, along with MAb 3 described by Joly et al., allow the subtyping of L. pneumophila serogroup 1 into 15 phenons ( 137, 155). Recent investigations have shown that the use of monoclonal antibodies may be inadequate for discriminating between disease-causing strains and other environmental isolates of L. pneumophila serogroup 1 ( 228).

Newer molecular techniques such as pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) are able to discriminate within monoclonal subtypes of L. pneumophila serogroup 1 and identify sources of disease-causing strains ( 116). These techniques appear to complement the use of monoclonal antibodies and are the most recent of several techniques that separate strains based on DNA polymorphism ( 251). Researchers have used a variety of other techniques to discriminate between isolates of legionellae. The use of electrophoretic alloenzyme typing (multilocus enzyme typing) is able to subtype L. pneumophila into more than 40 subtypes ( 252). This technique was used to subdivide the Philadelphia 1 monoclonal subgroup of L. pneumophila serogroup 1 into five electrophoretic types ( 252).

Plasmid analysis has been used to subtype legionellae, but results have shown this technique to have limited use. In one investigation, the clinical strains were plasmidless and the environmental strains contained plasmids ( 39, 190, 191, 260). Restriction fragment length polymorphism (RFLP) has been used to subtype Legionella either alone or in combination with rRNA or DNA probing of the DNA digest (ribotyping) ( 56, 116, 249, 250, 275). The RFLP procedure has been further modified to use PCR amplification of a specific locus, such as the 16S-23S spacer region, followed by restriction enzyme digestion, with the resultant products separated by agarose gel electrophoresis ( 275).

Other procedures used to subtype L. pneumophila include detection of repetitive elements within DNA, amplified fragment length polymorphism, and sequence-based typing ( gyrA gene) ( 56, 86, 113, 116, 176, 177, 197, 274, 276).

A comparison of AP-PCR and PFGE with strains from seven outbreaks of legionellosis indicated that PFGE provided slightly better discrimination ( 228). Both techniques offer better discrimination and are less labor intensive than other techniques such as SDS-PAGE, ribotyping, and multilocus enzyme electrophoresis. Discrimination is improved when AP-PCR and PFGE are used in conjunction with monoclonal antibody typing. Even so, caution must be used when interpreting results of subtyping for the identification of an outbreak reservoir. A recent study determined that strains isolated from non-outbreak-related sources were identical to the clinical isolates by four subtyping procedures (AP-PCR, PFGE, monoclonal antibody, and multilocus enzyme electrophoresis) ( 171).

Amplified fragment length polymorphism (AFLP) represents the current state-of-the-art subtyping procedure ( 274). The technique has two variations: one uses a single restriction enzyme and a selective primer, and the other employs two restriction enzymes and corresponding primers. The latter method has been modified and termed infrequent-restriction-site PCR ( 233). The DNA is digested with a restriction enzyme(s), and the restriction fragments are ligated to specially constructed adapters. Subsequent PCR with primers specific for the ligated adapters allows the amplification of a subset of the digested DNA. The primers can be labeled with a fluorescent tag to allow analysis of the fragments with an automated sequencer, or the fragments can be separated by gel electrophoresis and stained with ethidium bromide. A comparison of PFGE and AFLP on 48 unrelated and epidemiologically related strains of L. pneumophila serogroup 1 indicated that the method was rapid, versatile, and reproducible, provided useful discrimination, and was easier to perform than PFGE ( 233). An intercenter study by the European Working Group on Legionella Infections comprising 13 laboratories using a standardized protocol demonstrated that the method was highly reproducible and epidemiologically concordant with good discrimination. The method is to be adopted as the first standardized typing method for the investigation of travel-associated Legionnaires' disease in Europe ( 105).

EPIDEMIOLOGIC TRENDS

Incidence

Accurate data are not available to assess whether any trends have occurred in the incidence of Legionnaires' disease. In the United States between 1980 and 1998, an average of 356 cases were reported to CDC each year, with no trend (Fig. 2) (A. L. Benin and R. E. Besser, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. 873, 2001). This is a fraction of the 8,000 to 18,000 cases estimated to occur each year ( 200). There are many reasons why reporting does not reflect the true incidence of disease. Clinicians may fail to perform testing for Legionnaires' disease. As empirical therapy for community-acquired pneumonia has changed to cover a wider array of infectious agents, the use of diagnostic testing to determine pneumonia etiology may have declined. For Legionnaires' disease, this is a problem not only for the individual patient who is denied the benefit of targeted therapy, but for the community as a whole, since each case of Legionnaires' disease may represent a sentinel event heralding an outbreak.

Cases of Legionnaires' disease that are diagnosed in hospitals may not get reported to state and local officials. This may be due to a lack of awareness that Legionnaires' disease is reportable in all states (except Oregon and West Virginia). It may also be due to concerns that cases of nosocomial Legionnaires' disease frequently prompt litigation. States may in turn not report to CDC. Faced with competing priorities, health departments may not have personnel to complete case report forms and submit in a timely fashion or at all.

The vast majority of cases of Legionnaires' disease reported in the United States are sporadic. During the 1980s, 11% of all cases were associated with an outbreak: 37% of possible nosocomial cases and 4% of community-acquired cases ( 199). In studies assessing the etiology of hospitalized cases of community-acquired pneumonia, the proportion due to Legionnaires' disease has ranged from 2 to 15% ( 41). Risk factors for Legionnaires' disease include increasing age, smoking, male sex, chronic lung disease, hematologic malignancies, end-stage renal disease, lung cancer, immunosuppression, and diabetes ( 199). CDC surveillance data indicate that persons with human immunodeficiency virus (HIV) infection are at greater risk of Legionnaires' disease than the general population ( 199); however, legionellae are rarely detected in studies of pulmonary disease among cohorts of HIV patients ( 30, 121). This may be due to prophylaxis of HIV-infected patients with trimethoprim-sulfamethoxazole, an antimicrobial agent with activity against Legionella species.

Data from a population-based study investigating the etiology of cases of pneumonia requiring hospitalization demonstrated that cases occur throughout the year, with no clear seasonal predilection ( 200). However, reports to CDC increase twofold in the summer ( 199), possibly because of increased testing ( 199). Outbreaks in the United States associated with cooling towers frequently occur in the summer and fall ( 26). Nosocomial cases occur year-round, with no seasonal pattern.

Treatment

Empirical therapy for persons hospitalized with community-acquired pneumonia should include coverage for Legionnaires' disease ( 19). Delay in starting appropriate therapy has been associated with increased mortality ( 132). Overall, mortality for Legionnaires' disease in the United States has been declining during the 1990s, possibly due to changes in empirical therapy for community-acquired pneumonia (A. L. Benin and R. E. Besser, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. 873, 2001). Historically, erythromycin has been the drug of choice for Legionnaires' disease. Because of the difficulty in amassing enough cases of Legionnaires' disease in one institution and the cost and complexity of multicenter studies, it is unlikely that clinical trials will ever be conducted to determine whether other agents are better. In vitro data suggest that azithromycin and many fluoroquinolone agents have superior activity against Legionella species. Additionally, these agents have fewer side effects than erythromycin ( 70). Azithromycin and levofloxacin are licensed by the Food and Drug Administration for the treatment of Legionnaires' disease and are considered preferable to erythromycin. There is debate as to whether rifampin provides additional benefit to patients with Legionnaires' disease ( 70, 261). The use of rifampin in addition to the newer regimens is not supported by in vitro data ( 70) and is not recommended by the Infectious Diseases Society of America ( 19).

Travel-Related Legionnaires' Disease in the United States

Travel is an underappreciated factor in the acquisition of Legionnaires' disease in the community. This is surprising given that the largest outbreak in U.S. history and the one for which this disease was named was travel associated ( 104). The 1976 outbreak among American Legion members was not typical of more recent travel-associated clusters in that it was explosive and easily recognizable.

A more typical travel-related outbreak occurred in Georgia in 1999 ( 21). A 64-year-old woman was diagnosed with Legionnaires' disease in New York after returning from a wedding in Georgia. Five of her extended family members in Massachusetts developed Pontiac fever after returning from the same wedding. Extensive case finding revealed one other case of Legionnaires' disease and six cases of Pontiac fever. A cohort study implicated the hotel whirlpool spa as the source of the outbreak. The investigation allowed measures to be taken that may have prevented additional cases from occurring.

This outbreak demonstrates many of the characteristics of typical travel-related Legionnaires' disease outbreaks that make detection very difficult: low attack rates, long incubation periods, dispersal of persons away from the source of the infection, and inadequate surveillance. Discovery of the outbreak was serendipitous; cases occurred within one family from two states, with no other common exposures. If one of the clinicians caring for the two persons who developed Legionnaires' disease had not tested for the disease, the outbreak would not have been detected. At the time of diagnosis, none of the patients was still in Georgia. If the Georgia Division of Public Health had not been contacted, the outbreak would have been missed. And if the state health department had not contacted CDC, the outbreak would not have been investigated.

Although there are two national surveillance systems for Legionnaires' disease in the United States, neither is able to detect clusters of travel-associated Legionnaires' disease. The National Electronic Telecommunications System for Surveillance, an electronic system that collects data on Legionnaires' disease in addition to all reportable diseases, is timely but does not collect information on travel. The Legionnaires' Disease Reporting System, a paper-based system that collects detailed information on travel as well as other risk factors for Legionnaires' disease, is very insensitive and is used mainly for tracking major epidemiologic trends.

The true burden of travel-related legionellosis in the United States is not clear. Although 21% of cases reported to CDC through the paper-based Legionnaires' disease reporting system indicate an out-of-home stay during the incubation period (CDC, unpublished data), a case-control study of risk factors for sporadic Legionnaires' disease in Ohio found that the rate of travel was similar between patients and controls ( 262). Unfortunately, this study did not look in detail at travel-related activities to determine whether particular exposures when traveling or returning home were associated with Legionnaires' disease. Further studies are needed to determine the proportion of disease attributable to travel and to design appropriate prevention strategies.

Detection of travel-related outbreaks offers the potential for providing new prevention opportunities. The European Working Group on Legionella Infections conducts surveillance for travel-related Legionnaires' disease in Europe ( 149) and has been quite successful at identifying clusters of travel-associated Legionnaires' disease ( 149). In 1999, the European Working Group on Legionella Infections detected 29 clusters among 63 travelers in 16 countries (F. Lever, E. Slaymaker, C. Joseph, and C. Bartlett, 5th International Conference on Legionella, abstr. 40, 2000). Twelve of these clusters would not have been detected without this surveillance scheme in place because they involved two travelers from different countries.

In response to threats of bioterrorism and emerging infectious diseases, CDC is in the process of developing a system of a national electronic surveillance that will be rapid and efficient ( 152). The National Electronic Disease Surveillance System, when implemented, will be a logical platform for conducting travel-related surveillance for Legionnaires' disease.

Community Outbreaks and Prevention

During the past 3 years, the epidemiology of Legionnaires' disease has been dominated by the occurrence of several large outbreaks, two linked to cooling towers and one linked to a whirlpool spa. In April 2000, a large outbreak of Legionnaires' disease occurred among persons visiting the newly constructed aquarium in Melbourne, Australia ( 9). By June, 119 persons were confirmed to have Legionnaires' disease, and four (3.6%) persons died. This outbreak was due to a new cooling tower which had recently come on line. It demonstrated that even new cooling tower systems pose a risk when coming on line and that decontamination procedures should be followed. The American Society of Heating, Refrigeration, and Air-Conditioning Engineers (ASHRAE) has issued guidelines for the control of Legionella in buildings; they describe maintenance procedures that should be performed when bringing cooling towers back on line ( 8).

In 1999, an outbreak in the Netherlands among attendees at a flower show resulted in 133 confirmed and 55 probable cases of Legionnaires' disease ( 58). Eleven percent of cases died (C. Navarro, A. Garcia-Fulgueiras, J. Kool, C. Joseph, J. Lee, C. Pelaz, and O. Tello, update on the outbreak of Legionnaires' disease in Murcia, Spain, Eurosurveillance Wkly., http://www.eurosurv.org/update/ ). This outbreak was due to a contaminated whirlpool spa on display at a consumer product fair attached to the flower show ( 33). Whirlpool spas have been implicated previously in outbreaks of Legionnaires' disease ( 21, 22, 153) and Pontiac fever ( 115, 186, 195, 255). These outbreaks involved spas that were in use. In-store whirlpool spa displays have also been implicated in the past, but never with an outbreak of this magnitude ( 22). This outbreak demonstrates the importance of biocide treatment for all spas that will be in use, whether or not they will be occupied by bathers ( 8, 43).

Recently, a large community-wide outbreak of Legionnaires' disease that affected more than 750 persons was reported from Spain. Of these, 310 were confirmed to have legionellosis, making this the largest Legionnaires' disease outbreak ever reported. Remarkably, only one death has been reported (C. Navarro, A. Garcia-Fulgueiras, J. Kool, C. Joseph, J. Lee, C. Pelaz, and O. Tello, update on the outbreak of Legionnaires' disease in Murcia, Spain, Eurosurveillance Wkly., http://www.eurosurv.org/update/ ). A cooling tower is suspected as the source of the outbreak.

The outbreaks in Australia and Spain had very low case fatality rates compared with the outbreak in the Netherlands. It is possible that differences in strain virulence, host factors, or case ascertainment account for this difference in mortality. Another intriguing possibility is that diagnostic and treatment practices in the various countries played a role. It has been reported that the choice of antibiotics for community-acquired pneumonia differs between these countries, with agents active against Legionella being used empirically in Spain and Australia and beta-lactam antibiotics being used empirically in the Netherlands (C. Navarro, A. Garcia-Fulgueiras, J. Kool, C. Joseph, J. Lee, C. Pelaz, and O. Tello, update on the outbreak of Legionnaires' disease in Murcia, Spain, Eurosurveillance Wkly., http://www.eurosurv.org/update/ ). In the United States, case fatality rates for reported cases of Legionnaires' disease have declined during the 1990s, a time when empirical use of fluoroquinolones and azithromycin increased for patients hospitalized with pneumonia and when cases were diagnosed earlier because of use of the urine antigen test (A. L. Benin and R. E. Besser, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. 873, 2001).

The occurrence of large community outbreaks of Legionnaires' disease can be reduced with the adoption of guidelines for the proper maintenance of cooling towers and other aerosol-generating devices ( 8, 43, 55). The prevention of sporadic cases is more difficult and will require increased understanding of the route of transmission. A first step, however, is the identification of cases of sporadic disease through the use of proper diagnostic tests. For patients being hospitalized with community-acquired pneumonia who have risk factors for Legionnaires' disease, this should include sputum culture and urine antigen testing. Urine antigen testing alone will miss cases due to non- L. pneumophila serogroup 1 strains; from 1980 to 1989, these cases represented 28% of reported disease ( 199).

Nosocomial Outbreaks and Prevention

Health care-associated Legionnaires' disease continues to present a significant public health problem. Although the magnitude of the problem is difficult to measure, reports of outbreaks continue to abound. Hospitals represent ideal locations for Legionnaires' disease transmission: at-risk individuals are present in large numbers; plumbing systems are frequently old and complex, favoring amplification of the organism; and water temperatures are often reduced to prevent scalding of patients.

To reduce the likelihood of Legionnaires' disease transmission in health care facilities, CDC recommends a strategy focusing on proper maintenance of water systems, universal testing of patients with nosocomial pneumonia with appropriate tests, and investigation of situations in which transmission has been shown to occur ( 44). At present, prevention efforts are inadequately implemented in the majority of health care facilities for a variety of reasons.

In 2000, ASHRAE issued guidelines for maintaining water systems to minimize the likelihood of Legionnaires' disease transmission ( 8). These guidelines recommend maintaining water systems at temperatures unfavorable for the amplification of legionellae. However, many states have regulations that limit the water temperature in health care facilities as a means of reducing scalding injuries ( 194). In Great Britain, elevated water temperatures were recommended as the primary means of preventing legionellosis in hospitals in 1991 ( 55). Thermostatic mixing valves are used to reduce the likelihood of scalding. Adoption of these guidelines has resulted in a dramatic decline in nosocomial Legionnaires' disease without increased reports of scalding. Adoption of the ASHRAE guidelines could dramatically reduce the likelihood that legionellae will be amplified in a water system, thereby diminishing the risk of transmission.

The second part of prevention involves clinical surveillance. Currently, a minority of institutions in the United States perform universal Legionella testing of at-risk patients with nosocomial pneumonia ( 95). Infection control personnel should educate medical staff to heighten suspicion for health care-associated Legionnaires' disease, especially in high-risk patients, and to implement routine testing policies. Appropriate diagnostic testing should be available on site in institutions with high-risk patients and should include Legionella culturing and urine antigen testing. Introduction of routine appropriate testing has led to the identification of previously undetected transmission ( 173, 179). Only when transmission is recognized can appropriate steps be taken to prevent additional cases.

When cases of Legionnaires' disease are identified, epidemiologic and environmental investigations should be undertaken to determine the means of transmission. Detailed reviews of the procedures for conducting these investigations have been published previously ( 44).

There has been some debate in the literature regarding the role of environmental culturing for Legionella in the absence of disease transmission ( 7, 42, 44; Report of the Maryland Scientific Working Group to Study Legionella in Water Systems in Healthcare Institutions, Maryland State Dept. Health Hygiene UR, http://www.dhmh.state.md.us/html/legionella.htm ). While opinions differ as to the role of routine periodic environmental monitoring, there is agreement that all health care facilities should have a control strategy in place and that the strategy adopted should vary based on a risk assessment. This recommendation has recently been adopted by the Joint Council on Accreditation of Healthcare Organizations (Standard EC.1.7, www.jcaho.org/standard/stds2002_mpfrm.html ). The risk assessment should take into account the types of patients treated, the age and complexity of the potable water system, previous occurrence of Legionnaires' disease, and type of biocide being used.

Potential Impact of Monochloramine

An increasing volume of epidemiologic and laboratory data have begun to accumulate which suggest that the use of monochloramine as a biocide in municipal water systems is more effective than chlorine in preventing transmission of Legionella and has the potential to greatly reduce the incidence of Legionnaires' disease in health care facilities. If these results are reproduced with prospective studies, a new era in Legionnaires' disease prevention may arrive.

In 1999, Kool et al. reported a case-control study assessing the risk of a hospital having a drinking water-associated outbreak of Legionnaires' disease, based on the type of disinfectant used by the municipality ( 172). Hospitals that had experienced an outbreak were nine times more likely to be in municipalities that used chlorine rather than monochloramine as the drinking water disinfectant. These results suggest that ≈90% of these outbreaks could be prevented by a change at the water treatment plant.

Another study by Kool et al. looked at risk factors for Legionella colonization in hospitals in three Texas counties ( 170). Once again, they found that hospitals in municipalities that used chlorine as the residual disinfectant were significantly more likely to have legionellae isolated from the water supply.

To assess whether municipal disinfection with monochloramine was associated with a lower occurrence of health care-associated Legionnaires' disease, Heffelfinger et al. conducted a survey of hospital epidemiologists (J. D. Heffelfinger, J. L. Kool, S. K. Fridkin, V. J. Fraser, J. C. Carpenter, J. Hageman, B. A. Kupronis, and W. C. Gaines, Program Abstr. 4th Decennial Conf. Hospital Infections, Atlanta, Ga., 2000). They collected information on the occurrence of outbreaks of Legionnaires' disease, hospital characteristics, including the presence of a transplant unit, and type of hospital and municipal water disinfection. Hospitals using chlorine as the residual disinfectant were significantly more likely to have cases of health care-associated Legionnaires' disease. Based on these data, more than 70% of health care-associated Legionnaires' disease cases might not have occurred if monochloramine had been used instead of free chlorine for residual disinfection.

What is the mechanism by which monochloramine might achieve this effect? Monochloramine is more stable than chlorine and may provide a better residual disinfectant over long distribution systems ( 163). It also appears to penetrate the biofilms where Legionella reside more effectively than chlorine. This property has been demonstrated in studies by Donlan et al. comparing the effects of monochloramine and chlorine on H. vermiformis-associated L. pneumophila in a laboratory-grown potable-water biofilm ( 66). While both chlorine and monochloramine were effective at eradicating planktonic Legionella, monochloramine was significantly more effective than chlorine at killing Legionella in the biofilm.

Many municipalities are switching to monochloramine as the biocide of choice for their public water distribution systems. Currently, nearly 25% of municipalities use monochloramine, motivated not by Legionnaires' disease prevention but rather by a desire to minimize some of the disinfection by-products produced by chlorine. The U.S. Environmental Protection Agency issued regulations that require reductions in the levels of certain disinfection by-products that have been associated with cancer in laboratory animals ( 217). Monochloramine is attractive for municipal water systems because it results in reduced production of these regulated disinfection by-products.

If, in fact, many sporadic cases of Legionnaires' disease are transmitted through potable-water systems, it is plausible that the use of monochloramine could have an impact on community-acquired cases as well. Given that many municipalities are already planning to convert from chlorine to monochloramine disinfection in response to the Environmental Protection Agency regulations, there should be many opportunities to study the impact of this intervention prospectively.

FUTURE DIRECTIONS AND CONCLUSIONS

Over the past 25 years, we have learned a great deal about legionellae and legionellosis. Researchers are on the brink of fully characterizing the novel mechanism by which these bacteria infect eukaryotic cells. Understanding of this means of entry into a host cell may have unforeseen benefits. Currently, we know that legionellae can infect very diverse hosts, ranging from slime molds to protozoa to mammalian cells. There may be additional hosts yet to be discovered for these diverse intracellular organisms.

The convergence of studies on microbial biofilms and the cell-cell signaling that occurs within these communities appear particularly relevant to legionellae. We have only begun to characterize the interaction of legionellae with the microbial community. While it seems clear that legionellae are common inhabitants of biofilms, the significance of biofilms to the multiplication and distribution of legionellae remains undefined. Reducing legionellae in the biofilms that form in building water systems is a primary goal in strategies to prevent Legionnaires' disease. Application of advances in cell signaling to the study of legionellae in biofilms will lead to a better understanding of the ecology of these bacteria and to more efficient mechanisms to control this disease.

We continue to make progress in understanding the epidemiology of Legionnaires' disease. Many opportunities now exist to improve the prevention of legionellosis, especially in the United States. Computer-based reporting systems may one day provide a means of conducting timely surveillance for previously undetected outbreaks of travel-related Legionnaires' disease. New rapid diagnostic tests can improve case detection, but at a public health cost if these new methodologies replace bacterial cultures. Municipal disinfection with monochloramine may provide a means of reducing the incidence of both health care-associated and community-acquired Legionnaires' disease.

FIG. 1. Life cycle of L. pneumophila in protozoa and human macrophages.

FIG. 2. Legionnaires' disease, 1980 to 1998; annual number of cases reported.

TABLE 1. Legionella species and serogroupsa

Species

No. of serogroups

No. associated with disease

18. L. erythra

2

1b

Species are listed in chronological order based on the date of isolation or identification.

Serogroup 2 of L. erythra has been associated with human disease.

TABLE 2. Detection of Legionella nucleic acid by gene probe and PCR in clinical samples

Clinical sample tested

Gene target(s)

Results

Reference

Respiratory secretions (BAL, sputum)

16S rRNA

77 samples, 2 culture-positive, both PCR-positive (LightCycler)

Information & Contributors

Information

Published In

Clinical Microbiology Reviews

Volume 15 * Number 3 * July 2002

History

Published online: 1 July 2002

Permissions

Request permissions for this article.

Contributors

Authors

# data[].attributes.attachment_link.title
Legionella and Legionnaires' Disease: 25 Years of Investigation | Clinical Microbiology Reviews

# data[].attributes.attachment_link.description
SUMMARY There is still a low level of clinical awareness regarding Legionnaires' disease 25 years after it was first detected. The causative agents, legionellae, are freshwater bacteria with a fascinating ecology. These bacteria are intracellular pathogens ...

# data[].attributes.headline
decrease in LD cases n the Netherlands due to diagnostic testing

# data[].attributes.subject
decrease in LD cases n the Netherlands due to diagnostic testing

# data[].attributes.attachment_link.readable_text
Introduction

Legionnaires' disease (LD) is an acute pneumonia caused by the inhalation or aspiration of aerosols contaminated with the Legionella bacteria. It was named after a point-source outbreak in a hotel that hosted the convention of the American Legion in 1976 [ 1, 2]. Legionnaires' disease is characterized by an acute pneumonia, a low attack rate (0.1-5%), and an average incubation time of 2-10 days, although it may extend to even longer than 10 days [ 1- 4]. The disease proves fatal in about 6% of cases [ 5].

In the Netherlands, LD has been a notifiable disease since 1987. Treating physicians report cases of LD to the Municipal Health Services, who then report them to the Centre for Infectious Disease Control, where a national database is situated [ 6]. Around 100-300 LD cases per year were reported between 1999 and 2008 (with an unexpected peak of 436 reported cases in 2006) [ 7- 10]. In 2009, the number of reported LD cases dropped to 251, which was the lowest number in the previous 5 years of surveillance [ 9]. This sudden drop gave rise to several hypotheses that might explain the decrease of reported LD cases, ranging from the influence of weather patterns, to the effect of the concurrent occurrence of the influenza A (H1N1) pandemic and the Q fever outbreak in 2009 [ 11- 14]. Several studies have suggested that specific meteorological variables like relative humidity and temperature are related to the LD incidence, as these factors might influence the outdoor survival of the Legionella bacteria [ 15- 17]. Weather conditions in 2009 may, therefore, have affected the LD incidence. Furthermore, the influenza A (H1N1) pandemic was spread around the Netherlands from April 2009 onwards [ 11, 12] and competed for the daily headlines with the Q fever outbreak that started in the southern part of the country and peaked in the spring of 2009 [ 13, 14]. The nationwide increased attention for these two respiratory infectious diseases may have diminished the interest of both the public and health care employees for LD. As a consequence, changes in laboratory practices with respect to LD diagnostics could have resulted in a diagnostic bias and an underreported number of LD cases in 2009.

The aim of this study was to investigate if the number of requests for Legionella diagnostics in the Netherlands between 2007 and 2009 could explain the number of reported LD cases in these years. We, therefore, analyzed the Legionella diagnostics data from three, large, microbiological laboratories from different geographical regions in the Netherlands, between 2007 and 2009.

Methods

Patients

Legionnaires' disease has been notifiable in the Netherlands since 1987. Treating physicians are required to report cases of LD to a public health physician at one of the 28 Municipal Health Services within 24 h of diagnosis. The public health physicians are then required to report all confirmed and probable cases of LD to the Ministry of Health and, more recently, to the Centre for Infectious Disease Control within 24 h. The LD cases diagnosed in this present study were defined according to the standardized case definitions of the European Legionnaires' Disease Surveillance Network (ELDSNet) [ 18]. A confirmed case of LD is a patient presenting clinical and/or radiological signs of pneumonia with at least one of the following laboratory criteria: (1) isolation of Legionella spp. from respiratory secretions or any normally sterile site; (2) detection of L. pneumophila antigen in urine; or (3) L. pneumophila serogroup 1 specific antibody response. A probable case of LD is defined as a patient presenting clinical and/or radiological signs of pneumonia with at least one of the following laboratory criteria: (1) detection of L. pneumophila antigen in respiratory secretions or lung tissue, e.g., by direct fluorescent antibody staining; (2) detection of Legionella spp. nucleic acid in a clinical specimen; (3) L. pneumophila non-serogroup 1 or other Legionella spp. specific antibody response; or (4) a single high titer in specific antibody response for L. pneumophila serogroup 1, other serogroups, or other Legionella spp.

Laboratories

Three, large, microbiological laboratories in the Netherlands participated in this study and provided data on all requested diagnostic LD tests in 2007, 2008, and 2009. The Izore Centre for Infectious Diseases Friesland is situated in the city of Leeuwarden in the north of the Netherlands, and performs LD diagnostic tests for the 650,000 inhabitants of its adherence region. The Laboratory of Medical Microbiology and Immunology of the St. Elisabeth Hospital in Tilburg is situated in the southern part of the Netherlands, and performs LD diagnostic tests for the 800,000 inhabitants of its adherence region. The Regional Public Health Laboratory Kennemerland in Haarlem is situated in the western part of the country, and has an adherence region of about 700,000 inhabitants.

Diagnostic tests

Each laboratory performed a spectrum of different tests on the available patient materials for which Legionella diagnosis was requested by the treating physician of the patient.

Urinary antigen test

In all three laboratories, a commercial urinary antigen test (BinaxNOW, Portland, ME, USA) was used to test urine samples for the presence of L. pneumophila antigens. The BinaxNOW test has been recommended as a rapid specific test for LD caused by L. pneumophila serogroup 1, with several technical advantages over a conventional enzyme immunoassay test: there is no need for expensive laboratory equipment and the processing speed is higher [ 19]. However, the detection of non- L. pneumophila serogroup 1 cases is higher for the Binax EIA [ 19, 20].

Serological investigation

All laboratories used a commercial enzyme-linked immunosorbent assay (ELISA) to detect IgM and IgG serotype 1-7 antibodies to L. pneumophila in the acute- and (when available) in the convalescent-phase sera of patients (Serion ELISA; Institut Virion/Serion GmbH, Würzburg, Germany).

Culture

In all laboratories, available respiratory secretion, bronchoalveolar lavage specimen, or lung tissue of patients were used to culture Legionella spp. The available specimen was cultured on two media at 35°C, with increased humidity. In Haarlem and Tilburg, the two media used were buffered charcoal yeast extract supplemented with α-ketoglutarate (BCYE-α) and (1) the antibiotics polymyxin B, cefazolin, and pimaricin or (2) the antibiotics polymyxin B, anisomysin, and vanomycin (Oxoid Ltd., Hampshire, UK). In Leeuwarden, the available specimen (bronchoalveolar lavage specimen or respiratory secretion) was cultured on two media at 35°C, with increased humidity. The two media used were buffered charcoal yeast extract (1) without antibiotics and (2) with the antibiotics polymyxin B and cefazolin (Mediaproducts BV, Groningen, the Netherlands).

Polymerase chain reaction

In the laboratories of Haarlem [ 21, 22] and Leeuwarden [ 23], a polymerase chain reaction (PCR) assay that targeted the 16S ribosomal DNA gene was used to detect Legionella nucleic acid in the available patient materials. In Tilburg, samples were tested for Legionella spp. DNA in a 16S rRNA-based PCR and in an mip gene-based PCR for L. pneumophila [ 24].

Data collection

For each laboratory, we first calculated the total number of patients for whom one or more Legionella diagnostic tests were requested in 2007, 2008, and 2009. Additionally, the number of different diagnostic tests that were performed was calculated for the three laboratories over the same period.

Statistical analyses

The trend over time of the incidence rate of LD cases (between 1999 and 2008) was assessed by the use of the Cochran-Armitage trend test (R Foundation for Statistical Computing, Vienna, Austria) [ 25]. Incidence rates were compared between different years, using a two-tailed χ ²-test (PASW Statistics release 18.0, SPSS Inc., Chicago, IL, USA). The trend over time of the number of patients for whom a diagnostic test for Legionella was performed between 2007 and 2009 was assessed by the use of linear regression analysis (PASW Statistics release 18.0, SPSS Inc., Chicago, IL, USA). The trend over time (between 2007 and 2009) of the patients with a positive diagnostic test for Legionella as a proportion of the total number of patients with a diagnostic test was assessed by the use of the Cochran-Armitage trend test [ 25].

Results

From 1987 to 2009, a total of 3,393 LD cases (probable or confirmed) were reported in the Netherlands, which corresponds to an average annual incidence rate of 0.94 per 100,000 population (Fig. 1) [ 7- 10]. Between 1999 and 2008, there has been a significant increase in the incidence rate ( p < 0.001, Cochran-Armitage trend test), with two peaks, one in 2002, with an incidence rate (95% confidence interval [CI]) of 1.79 (1.64-1.94), and one in 2006, with an incidence rate (95% CI) of 2.67 (2.52-2.82). In 2009, the lowest incidence rate was reported in 5 years of surveillance: 1.52 per 100,000 population (95% CI: 1.32-1.73). This rate was significantly lower ( p = 0.001, χ ² test) compared to the other two years of the study period 2007-2009: in 2007, the incidence rate (95% CI) was 1.97 (1.76-2.17), and in 2008, the incidence rate was 2.05 (1.85-2.26).

Legionnaires' disease (LD) incidence rate and number of patients in the Netherlands 1987-2009. The bars represent the annual number of reported LD patients. The line represents the incidence rate per 100,000 population. The p-value reflects the trend of the incidence rate over time between 1999 and 2008 (Cochran-Armitage trend test)

Figure 2a shows the total number of patients for whom one or more diagnostic tests for Legionella were performed between 2007 and 2009 in the three laboratories. Overall, the number of patients for whom one or more tests were performed increased between 2007 and 2009: 3914 patients in 2007, 4484 patients in 2008, and 4978 patients in 2009 ( p = 0.026, linear regression). Figure 2a shows the data for the three laboratories separately.

Diagnostic tests for Legionella between 2007 and 2009. The top panel ( a) shows the total number of patients for whom one or more diagnostic tests for Legionella were performed between 2007 and 2009 in the three participating laboratories. The p-values reflect the trend over time (linear regression analysis). The lower panel ( b) shows the proportion of patients with a positive diagnostic test for Legionella between 2007 and 2009. The p-values reflect the trend over time (Cochran-Armitage trend test)

Figure 2b shows the patients with a positive diagnostic test for Legionella as a proportion of the total number of patients with a diagnostic test. In total, there were 197 cases (96 probable; 101 confirmed) reported by the three laboratories between 2007 and 2009: 64 cases (32 probable; 32 confirmed) in 2007, 80 cases (35 probable; 45 confirmed) in 2008, and 53 cases (29 probable; 24 confirmed) in 2009. The corresponding incidence rates (95% CI) for the three laboratories were 2.98 (2.24-3.72) per 100,000 population of the adherence region in 2007, 3.72 (2.98-4.46) per 100,000 population in 2008, and 2.47 (1.73-3.20) per 100,000 population in 2009. The data from all three laboratories taken together showed that the proportion of patients with a positive diagnostic test (95% CI) decreased between 2007 and 2009: 1.64 (1.26-2.01) in 2007 (64 cases in 3,914 tested patients), 1.78 (1.43-2.14) in 2008 (80 cases in 4,484 tested patients), and 1.06 (0.73-1.39) in 2009 (53 cases in 4,978 tested patients) ( p  = 0.019, Cochran-Armitage trend test). The data from Tilburg showed a slight increase in the proportion of patients with a positive test between 2007 and 2008, which was followed by a decrease between 2008 and 2009.

In Table 1, the data are shown for the number of different diagnostic tests that were performed in the three laboratories between 2007 and 2009. Several patients were diagnosed by more than one diagnostic method, which is reflected by the higher total number of tests that were annually performed compared to the total number of tested patients (Fig. 2a). In all three laboratories, the majority of diagnostic tests consisted of either a serological test or a urinary antigen test. Culture was the third most common test, followed by PCR. The increase between 2007 and 2009 in the total number of diagnostic tests that were performed in Leeuwarden and Tilburg was mostly due to the increase in serological tests (both laboratories) and in urinary antigen tests (Tilburg only), but remained the same when only the urinary antigen tests were considered: in total, 1,461 urinary antigen tests were performed in 2007, 2,004 tests in 2008, and 2,558 tests in 2009 (Table 1).

Table 1 Diagnostic tests performed between 2007 and 2009

Discussion

Our data show that there was no decrease in the number of patients for whom a diagnostic test for Legionella was performed in three large microbiological laboratories from different geographical regions in the Netherlands between 2007 and 2009. On the contrary, the number of patients with a diagnostic test increased in two of the three laboratories in this period. There was an overall decrease in the proportion of patients with a positive diagnostic test for Legionella.

One of the strengths of our study was the large amount of diagnostic data provided by the three different laboratories. Their large adherence regions and the location of the laboratories in three different geographical regions in the Netherlands strengthens our belief that the available data on diagnostic tests formed a representative sample of the total number of performed diagnostic tests in the Netherlands.

The results are not in agreement with our hypothesis that the decrease in the number of reported Legionella pneumonia patients in 2009 could be due to a decrease in the number of patients for whom a diagnostic test was performed. We, therefore, conclude that it is more likely that other factors might explain the sudden drop in reported Legionella pneumonia patients in 2009 compared to the previous years, and it would be interesting to investigate this for the period described in this current study.

# data[].attributes.attachment_link.title
Diagnostic testing for Legionnaires' disease in the Netherlands between 2007 and 2009: a possible cause for the decline in reported Legionnaires' disease patients - European Journal of Clinical Microbiology & Infectious Diseases

# data[].attributes.attachment_link.description
Legionnaires' disease (LD) is an acute pneumonia caused by the inhalation or aspiration of aerosols contaminated with the Legionella bacteria. In the Netherlands, around 300 LD cases per year were reported between 2000 and 2008, but in 2009, the number dropped to 251, which was the lowest number in the previous 5 years of surveillance.

# data[].attributes.headline
this article talks about the decline of mortality from LD

# data[].attributes.subject
this article talks about the decline of mortality from LD

# data[].attributes.headline
the effect of Monochloramine on reducing LDs colonization of hot water water supplys 

# data[].attributes.subject
the effect of Monochloramine on reducing LDs colonization of hot water water supplys 

# data[].attributes.attachment_link.readable_text
Brendan Flannery, * Lisa B. Gelling, † Duc J. Vugia, ‡ June M. Weintraub, § James J. Salerno, ¶ Michael J. Conroy, ¶ Valerie A. Stevens,* Charles E. Rose,* Matthew R. Moore,* Barry S. Fields,* and Richard E. Besser*

Monochloramine reduced colonization in building hot water systems.

Keywords: Legionella, Legionnaires' disease, water supply, disinfection, chloramines, research

Abstract

Monochloramine disinfection of municipal water supplies is associated with decreased risk for Legionnaires' disease. We conducted a 2-year, prospective, environmental study to evaluate whether converting from chlorine to monochloramine for water disinfection would decrease Legionella colonization of hot water systems. Water and biofilm samples from 53 buildings were collected for Legionella culture during 6 intervals. Prevalence ratios (PRs) comparing Legionella colonization before and after monochloramine disinfection were adjusted for water system characteristics. Legionella colonized 60% of the hot water systems before monochloramine versus 4% after conversion (PR 0.07, 95% confidence interval 0.03-0.16). The median number of colonized sites per building decreased with monochloramine disinfection. Increased prevalence of Legionella colonization was associated with water heater temperatures <50°C, buildings taller than 10 stories, and interruptions in water service. Increasing use of monochloramine in water supplies throughout the United States may reduce Legionella transmission and incidence of Legionnaires' disease.

Keywords: Legionella, Legionnaires' disease, water supply, disinfection, chloramines, research

Legionnaires' disease, named after an outbreak of severe pneumonia at a legionnaires' convention in 1976, is a form of community-acquired and nosocomial pneumonia. It is caused by inhalation of aerosols or microaspiration of water containing Legionella bacteria. Legionella spp. are ubiquitous in fresh water and occur naturally as intracellular parasites of amebae ( 1). Potable hot water systems provide environments for amplification of Legionella pneumophila, the most common species isolated from patients with Legionnaires' disease. L. pneumophila grows optimally at 35°C and multiplies between 25°C and 42°C. Investigations of outbreaks of Legionnaires' disease in hospitals and other community settings have implicated potable hot water systems as sources of transmission ( 2- 5).

No strategies have been proven to prevent community-acquired Legionnaires' disease. Prevention of transmission within healthcare facilities focuses primarily on preventing or limiting Legionella colonization of plumbing systems through temperature control or use of biocides ( 6). Healthcare facilities are of special concern because of increased susceptibility to and a high case-fatality ratio of Legionnaires' disease among immunocompromised patients and those with underlying illnesses ( 5, 7). Because colonized water distribution systems are often implicated in Legionella transmission ( 2, 5, 8, 9), effective water disinfection strategies could provide the best measure to prevent Legionnaires' disease.

Chloramination is a method of drinking water disinfection that provides a lasting residual disinfectant in the distribution system. The process involves adding ammonia to chlorinated water; aqueous chlorine reacts with ammonia to form inorganic chloramines ( 10). Monochloramine is the most active compound and forms preferentially at certain ratios of ammonia to chlorine. Approximately 55% of 11.8 million people living in the 25 largest cities in California currently receive water disinfected with monochloramine (unpub. data). A survey in 2004 of municipal water utilities in the United States found that 30% used monochloramine for residual disinfection ( 11). The Environmental Protection Agency estimates that municipal water utilities using surface water sources will increasingly convert to monochloramine to meet federal regulations that limit disinfection byproducts in drinking water ( 12).

Use of monochloramine for residual disinfection compared with chlorine was associated with a lower prevalence of Legionella colonization in plumbing systems ( 13) and decreased risk of nosocomial outbreaks of Legionnaires' disease in cross-sectional and retrospective case-control studies ( 14, 15). The planned conversion to monochloramine for municipal drinking water disinfection in San Francisco, California, provided an opportunity to prospectively investigate the effect of chloramination on Legionella colonization in potable hot water systems. We report here on the results of a 2-year environmental study.

Methods

Study Site

The San Francisco Public Utilities Commission provides an average of 250 million gallons (950 million liters) of water per day to ≈2.4 million residents in northern California, including 750,000 in the city and county of San Francisco. Surface water makes up >99% of the water supply. Chlorine was added to kill microorganisms present in source water (primary disinfection) throughout the study period. Chlorine concentrations are monitored at several locations throughout the distribution system. Chlorine used for residual (or secondary) disinfection was replaced with monochloramine on February 2, 2004.

Buildings with >3 stories in San Francisco were identified from lists of commercial customers of the San Francisco Public Utilities Commission and real property owned by the city and county of San Francisco. Building managers and owners gave permission for sample collection inside the buildings for the duration of the study. Results of Legionella cultures were provided only at the completion of the study. Standardized questionnaires were administered to building engineers and facilities managers to obtain information on the age of the building, capacity of water heaters and hot water storage tanks, type of water heating system (boiler, heat exchanger, or instantaneous heaters), and type of pipe material used throughout most of the building. At the completion of the study, building engineers were surveyed about routine maintenance plans for the potable hot water system, standard procedures for flushing outlets after a disruption of water service, and knowledge of industry guidelines for controlling Legionella growth in building water systems ( 16).

Environmental Sampling

Samples from each building were collected 6 times during the 2-year period, 3 times before and 3 times after conversion to monochloramine disinfection. Each round of sampling lasted 8-10 weeks. Preconversion and postconversion rounds of sampling were conducted at corresponding seasonal intervals.

Nine samples were collected from each building during sampling rounds, including a 1-L water sample from a water heater or heat exchanger, four 1-L samples of hot water, and 4 swabs of biofilm at point-of-use outlets (faucets or shower heads). Water samples were collected in sterile, 1-L plastic bottles (Nalge Nunc International, Rochester, NY, USA) containing 0.5 mL 0.1 N sodium thiosulfate solution to neutralize free chlorine and chloramines. Water heater samples were drawn from the drain valve, pressure relief valve, or from the closest outlet to heat exchangers. Point-of-use outlets were selected at farthest points from water heaters when possible. Biofilm samples were collected from shower outlets and faucets by inserting a sterile, polyester-tipped applicator swab (Falcon, Becton Dickinson and Company, Sparks, MD, USA) and rotating it firmly against the interior surface. Biofilm swabs were placed in sterile, screw-capped test tubes containing 0.1 mL sodium thiosulfate solution in 5 mL of water from the same site. Hot water taps were run until the temperature reached a maximum for collection of water samples. The same locations were sampled in each round. When sampling could not be performed at the selected site, the nearest substitute site was sampled; however, only samples collected from the same site before and after monochloramine conversion were included in analyses.

Water temperature, pH, and free (disassociated) and total chlorine concentrations were measured in a separate sample bottle. Total chlorine includes free chlorine plus monochloramine. Temperature was measured with a hand-held thermometer. pH was measured with a digital meter (pHep 3, Hanna Instruments, Leighton Buzzard, UK). Free and total chlorine residuals were measured by using the N, N-diethyl-p-phenylenediamine method with a colorimeter and test kit (Model DR/890, Hach Chemical Co., Loveland, CO, USA). Building engineers were asked about any interruptions in water service affecting the building or specific sites in the 3 months preceding the sampling date.

Laboratory Procedures

All culturing for Legionella species and amebae was performed in the Legionella Laboratory at the Centers for Disease Control and Prevention in Atlanta, Georgia, following standard procedures ( 17). Legionella organisms were speciated or serogrouped by macroscopic slide agglutination with a panel of polyclonal rabbit antisera against Legionella species and L. pneumophila serogroups ( 18). Laboratorians were blinded to the identity of buildings from which samples were obtained, and buildings were assigned different identification numbers in each round. Samples were transported at ambient temperature and processed a mean (± standard deviation) of 3 (±2) days after collection. Water samples from point-of-use outlets were concentrated 100-fold by filtration through a 0.2-μm polycarbonate filter (Nucleopore, Pleasanton, CA, USA). Biofilm swab samples were placed on a lawn of Escherichia coli for detection of ameba and treated with diluted acid (0.1 mol/L KCl, 0.005 mol/L HCl) to reduce the number of non- Legionella bacteria before plating.

Concentrations of Legionella spp. in water samples are expressed as CFU/mL based on plate counts of Legionella colonies grown from a known volume of original sample. Concentrations determined by this method are approximate. The upper and lower limits of detection were 0.05 and 25 CFU/mL for point-of-use outlets and 10 and 5,000 CFU/mL for water heaters. Plate counts were not determined for samples overgrown with non- Legionella organisms.

Surveillance for Legionnaires' Disease

Active, laboratory-based surveillance for culture-confirmed Legionella infections in San Francisco residents was conducted from January 1, 2003, through December 31, 2004, through the Active Bacterial Core surveillance activity of the California Emerging Infections Program ( 19). We reviewed legionellosis case report forms from the national passive surveillance system for cases among San Francisco residents or persons with a history of travel to San Francisco during the incubation period. Surveys were sent to infection control departments at all San Francisco hospitals to identify cases of probable or confirmed Legionnaires' disease during 2003 and 2004. Information was solicited from hospitals about environmental testing for Legionella spp. in water systems, and measures taken to reduce microbial contamination of water systems during 2003 and 2004.

Statistical Analysis

Data were entered into Access version 2002 (Microsoft, Redmond, WA, USA) and analyzed by using SAS for Windows version 9.0 (SAS Institute, Cary, NC, USA). We conducted building- and site-specific analyses of the prevalence of Legionella colonization. A building was considered colonized at a timepoint if Legionella spp. were cultured from any site. We considered a point-of-use outlet colonized if Legionella spp. were cultured from either a water sample or biofilm swab. Wilcoxon rank sum test was used to analyze differences in the proportions of positive sites or concentrations of Legionella. We also calculated adjusted prevalence ratios (PRs) and 95% confidence intervals (CIs) or p values by using PROC GENMOD (SAS Institute) for the clustered nature of sites within buildings. Preconversion and postconversion sampling rounds were considered repeated measures. Multivariable models investigated associations between Legionella colonization and water measurements or building characteristics.

Results

Effects of Conversion to Monochloramine on Water Distribution System

The conversion to monochloramine provided higher concentrations of total chlorine (which includes both free chlorine and monochloramine) and lower concentrations of trihalomethane compounds, the principal disinfection byproducts in treated water entering the distribution system ( Table 1 ). The conversion to monochloramine also resulted in an ≈10-fold increase in total chlorine concentrations measured in building hot water systems. Average temperature and pH measured in building water samples did not change significantly.

Table 1

Measurement (unit)*

2003, mean (range)

2004,† mean (range)

Reference value‡

Treated water

Water sampled from water heaters in 53 buildings¶

Environmental Sampling

Prospective Legionella testing was performed in 53 buildings, including 24 public and 29 commercial buildings. When chlorine was the residual disinfectant in municipal drinking water, Legionella spp. were cultured from building water systems on 96 (60%) of 159 occasions, and 37 (70%) of 53 buildings were colonized with Legionella spp. in >1 of the 3 sampling rounds ( Table 2 ). After conversion to monochloramine, Legionella spp. were found on 7 (4%) of 159 occasions in 5 (9%) of 53 buildings. These 5 buildings had been colonized at multiple sites before disinfection with monochloramine. Conversion to monochloramine resulted in a 93% reduction in the prevalence of Legionella colonization in building water systems (PR 0.07, 95% CI 0.03-0.16). Colonized water systems were no more likely than Legionella-free systems to include hot water storage tanks or material other than copper for hot water plumbing, although sample size limited building-level analyses. Legionella spp. were recovered from 12 (60%) of 20 buildings for which engineers reported maintaining water systems according to standard practices, such as maintaining backflow prevention and flushing outlets after interruption of water service, versus 18 (75%) of 24 buildings for which no standard maintenance of water systems was reported (p = 0.28).

Table 2

Water source

Chlorine (2003)

Chloramine (2004)

Jan-May

Jun-Jul

Oct-Dec

Mar-May

Jun-Aug

Sep-Dec

p value†

Building water systems (n = 53)

No. colonized with Legionella spp. (%)

27 (51)

34 (64)

35 (66)

5 (9)

0

2 (4)

<0.001

Median no. Legionella-positive samples in colonized buildings (range)‡

3 (1-9)

3 (1-8)

4 (1-8)

1 (1-4)

-

1 (1-2)

0.007

Water heaters (n = 53)

No. colonized with Legionella spp. (%)

12 (23)

16 (30)

17 (33)

1 (2)

0

0

<0.001

Median Legionella concentration in colonized water heaters, CFU/mL (range)‡

200 (40-400)

40 (10-500)

20 (10-5,000)

10

-

-

0.17

Point-of-use outlets (n = 212)

No. colonized with Legionella spp. (%)

75 (37)

85 (41)

86 (41)

7 (3)

0

2 (1)

<0.001

Median Legionella concentration in colonized sites, CFU/mL (range)‡

2.5 (0.05-25.0)

1.5 (0.05-25.0)

1.0 (0.05-25.0)

0.05 (0.05-0.40)

-

0.18 (0.10-0.25)

<0.001

A total of 364 (13%) of 2,822 water and biofilm samples yielded Legionella spp: 352 (25%) of 1,405 samples collected before conversion and 12 (<1%) of 1,417 samples collected after conversion to monochloramine. Five Legionella species and 7 serogroups of L. pneumophila were identified ( Figure ). L. pneumophila serogroup 1 accounted for >60% of all Legionella organisms. The same species of Legionella and serogroups of L. pneumophila were repeatedly cultured from individual sites ( Figure ).

Legionella spp. were cultured from 46 (15%) of 316 water samples from building water heaters: 45 (29%) of 157 samples collected before conversion versus 1 (<1%) of 159 after conversion to monochloramine (p<0.001). When we controlled for water heater temperature, building height, and interruptions in water service, monochloramine use decreased the prevalence of Legionella colonization in water heaters by 96% ( Table 3 ). Colonization of water heaters was more prevalent in buildings with >10 stories and in which water service had been interrupted in the past 3 months. Water temperatures >50°C were associated with the lowest prevalence of colonization, and Legionella spp. were not detected when the temperature exceeded 60°C (140°F). Over the 2-year study period, temperatures of water in building water heaters were >50°C at 88 (28%) of 318 sampling timepoints. In buildings in which engineers reported familiarity with industry guidelines for controlling Legionella growth in water systems, water heater temperatures were >50°C on 26 (21%) of 125 occasions versus 46 (32%) of 144 occasions in buildings in which engineers were not familiar with industry guidelines (p = 0.04).

Table 3

Factor

No. samples

% Legionella colonization

Adjusted prevalence ratio (95% CI)*

p value

Residual disinfectant

Water heater temperature, °C

Building height (stories)

Disruption in service in last 3 mo

At point-of-use outlets, Legionella spp. were cultured from 247 (20%) of 1,252 water samples and 70 (6%) of 1,254 biofilm swab samples. Combining culture results from the water samples and biofilm swabs from each site, Legionella spp. were cultured from 246 (39%) of 624 paired samples before conversion to monochloramine versus 9 (1%) of 622 paired samples after conversion (p<0.001). Median concentrations of Legionella spp. at colonized outlets were significantly lower after conversion to monochloramine ( Table 2 ). Legionella were cultured from both the water and biofilm samples on 59 (24%) of 246 occasions before conversion versus 2 (22%) of 9 occasions after conversion. The same Legionella species and serogroup was cultured from biofilm swabs and water samples on 56 (92%) of 61 occasions when both were positive. When we controlled for Legionella spp. in the sampled water heater, water temperature at the point of use, building height, and interruptions in water service, monochloramine use decreased the prevalence of Legionella colonization at point-of-use outlets by 96% ( Table 4 ). Legionella colonization at point-of-use outlets was independently associated with Legionella spp. in the sampled water heater, building height, and interruptions in water service. Legionella spp. were cultured only from point-of-use outlets and not from water heater samples on 72 (61%) of 118 occasions when building water systems were colonized, including 6 (86%) of 7 occasions after monochloramine conversion.

Table 4

Factor

No. samples

% Legionella colonization

Adjusted prevalence ratio (95% CI)*

p value

Residual disinfectant

Water heater colonized with Legionella spp.

Temperature of water at point-of-use outlet, °C

Building height (stories)

Disruption in service in last 3 mo

Amebae at sampled sites were associated with Legionella spp. colonization only when chlorine was used for residual disinfection. Legionella spp. were cultured from 61 (36%) of 169 samples in which amebae were present versus 291 (24%) of 1,236 samples without amebae (p = 0.01). After conversion to monochloramine, Legionella were found in 1 (1%) of 78 samples containing amebae and 8 (1%) of 866 samples without amebae (p = 0.75). During disinfection with chlorine, Legionella concentration was higher in samples containing amebae (median 9.0 CFU/mL, range 0.1-25.0) compared with those without amebae (median 1.5 CFU/mL, range 0.05-25.0, p<0.001). The prevalence of amebae decreased from 169 (12%) of 1,405 samples when chlorine was the residual disinfectant to 78 (8%) of 944 samples collected in the first 2 rounds after conversion to monochloramine (p = 0.006). Results of ameba cultures from the final round of sampling were discarded after amebae were found in negative control water samples.

Surveillance for Legionnaires' Disease

Active, population-based surveillance for Legionella infections identified 1 confirmed case of Legionnaires' disease in a San Francisco resident in November 2004, who traveled to Mexico within 2-10 days of symptom onset. Infection control departments in 7 (70%) of 10 hospitals in San Francisco, including the 3 largest hospitals, reported no hospitalized patients meeting the definition of a probable or confirmed case of Legionnaires' disease ( 20) during the study period. Review of case report forms from the national passive surveillance system did not identify any cases of Legionnaires' disease in persons with history of travel to San Francisco during the incubation period of their illness.

No environmental testing for Legionella spp. in hospital water systems was conducted during 2003 and 2004 in the San Francisco hospitals that responded to the survey. Two hospitals added supplemental chlorine to their water system to prevent microbial contamination before monochloramine conversion; supplemental chlorination was discontinued after monochloramine was added to municipal drinking water.

Discussion

This is the largest study to prospectively evaluate the effect of monochloramine disinfection on Legionella colonization in a water distribution system. Legionella spp. were prevalent and stable in building water systems over 3 rounds of sampling when chlorine was used for residual disinfection of drinking water. Monochloramine disinfection of the water supply reduced Legionella colonization in hot water systems. Our findings suggest that monochloramine in drinking water provides better control of Legionella growth in building plumbing systems than chlorine. This study supports the biologic plausibility of decreased risk of nosocomial outbreaks of Legionnaires' disease associated with chloraminated water compared with chlorinated water ( 14, 15).

The conversion from chlorine to monochloramine for residual disinfection resulted in lower concentrations of trihalomethane compounds in drinking water, which met the objectives of the municipal water supplier. Increased stability of monochloramine resulted in higher disinfectant concentrations in potable hot water systems because chlorine dissipates rapidly at higher temperatures. Higher concentrations of disinfectant and the ability of monochloramine to penetrate biofilms were likely responsible for the effect on Legionella spp. In model systems, monochloramine eliminates 99.9% of biofilm-associated Legionella spp ( 21), and Legionella spp. are cleared rapidly after addition of monochloramine ( 22). Although amebae in model systems protect Legionella spp. from the short-term effects of monochloramine ( 21), we found no evidence of this protective effect in the buildings we sampled.

The results of this study are relevant for strategies to control Legionnaires' disease in hospitals. Several strategies are currently used by hospitals to control Legionella growth in water systems and prevent nosocomial transmission of Legionnaires' disease ( 23). Thermal eradication (superheating water followed by flushing point-of-use outlets) and hyperchlorination were among the earliest methods effective at controlling Legionella growth ( 23, 24). However, superheating increases the risk of scalding injuries and hyperchlorination is associated with increased corrosion of plumbing. Copper-silver ionization has also been used with mixed success ( 25- 27). Monochloramine use for drinking water disinfection has been associated with lower prevalence of Legionella spp. in plumbing systems of hospitals ( 13). Our study demonstrated that Legionella colonization in a plumbing system was effectively eliminated by monochloramine. Hospitals or other facilities colonized with Legionella spp. might control Legionella growth and prevent disease transmission by adding monochloramine to their potable water system. The potential use of supplemental monochloramine in hospitals to prevent nosocomial Legionnaires' disease needs to be evaluated.

The results of our study are striking considering that we observed few cases of Legionnaires' disease despite evidence that Legionella spp. colonized most of the San Francisco buildings tested before use of monochloramine. Some cases of Legionnaires' disease may have gone undetected because patients with community-acquired pneumonia are increasingly treated empirically with antimicrobial drugs without microbiologic confirmation ( 28). Although we sampled 4 point-of-use outlets in each building, exposures to aerosols produced by these outlets may have been minimal. Persons exposed to any Legionella-containing aerosols may have been at low risk for Legionnaires' disease. Alternatively, the Legionella organisms present, even though some were L. pneumophila serogroup 1, might lack virulence factors needed to cause human disease ( 29).

Routine maintenance programs for plumbing systems were not effective in preventing colonization with Legionella spp., which is consistent with a previous study of hospital water systems ( 30). However, our findings suggest that existing guidelines were not fully implemented in the buildings sampled. Although nearly half of building engineers reported knowledge of industry guidelines for preventing Legionella colonization of potable water systems, only 13% of sampled water heaters were set at the recommended temperature of >60°C (140°F) ( 16). Legionella spp. were not found in water heaters set at the recommended temperature. Maintaining the recommended temperatures in water heaters could help prevent Legionella growth in hot water systems. Investigations of legionellosis outbreaks have consistently demonstrated that temperatures of 25°C to 42°C facilitate the growth and amplification of Legionella spp. to high concentrations ( 1).

The repeated measurement of Legionella colonization at the same sites over time represents a strength of this study. Colonization was stable during the first 3 sampling rounds and no seasonal effect on the prevalence of colonization was observed before conversion to monochloramine. Collection of samples at multiple point-of-use outlets in each building, in addition to water heater samples, increased detection of colonization within buildings. In an outbreak setting, widespread sampling, including sampling of sites that served as likely exposures for cases, is an important step in identifying possible sources of transmission. Filter concentration of water samples from point-of-use outlets increased the yield of positive cultures and provided additional information about the distribution of Legionella spp.

This study was not designed to analyze effects of conversion from chlorine to monochloramine on outcomes other than Legionella colonization in building water systems. Few data exist on the health effects of ingestion of monochloramine despite a long history of its use in water disinfection ( 31). Since monochloramine eliminates Legionella spp., other organisms may colonize water distribution systems ( 32). Our findings may be specific to characteristics of the water or distribution system in San Francisco, although they are consistent with results of a similar study in Pinellas County, Florida ( 33). Because monochloramine was added continuously to the municipal water supply after conversion and concentrations were maintained within specified ranges, effects on Legionella spp. at different monochloramine concentrations may vary.

Monochloramine disinfection of municipal water supplies is the only community-based intervention associated with reduced risk of Legionnaire's disease ( 14, 15). Control of Legionnaires' disease is unlikely to be a major factor in a water utility's decision to convert to monochloramine for residual disinfection. However, if water suppliers increasingly convert to monochloramine to reduce concentrations of disinfection byproducts, control of the growth of Legionella spp. in potable water systems may be an additional health benefit.

Acknowledgments

We thank Jon Rosenberg for his assistance with Legionnaires' disease surveillance; Gretchen Rothrock, José Beltrán, and Paul Gladden for study coordination, interviews of building managers, and sample collection; Ronald Jetke, Roselle Ferrer, Steve Francies, and Fernando Jimenez for technical expertise and sample collection; Ben Christmann, Claressa Lucas, and Ellen Brown for culturing Legionella spp. and amebae; and Carolyn Wright for review of legionellosis case report forms. We also thank the owners and managers of the San Francisco buildings for permission to collect water samples for the duration of the study, building engineers for providing information about building water systems and facilitating sampling, and hospital infection control practitioners who provided information about Legionnaires' disease and environmental testing.

This research was supported by the Centers for Disease Control and Prevention's Emerging Infections Program, the California Department of Health Services, and the US Environmental Protection Agency.

Biography

Dr Flannery is an epidemiologist at the Centers for Disease Control and Prevention. His work focuses on surveillance, prevention, and control of bacterial pneumonia.

Footnotes

Suggested citation for this article: Flannery B, Gelling LB, Vugia DJ, Weintraub JM, Salerno JJ, Conroy MJ, et al. Reducing Legionella colonization of water systems with monochloramine. Emerg Infect Dis [serial on the Internet]. 2006 Apr [ date cited]. http://dx.doi.org/10.3201/eid1204.051101

References

1. Fields BS, Benson RF, Besser RE. [[ Legionella and Legionnaires' disease: 25 years of investigation. Clin Microbiol Rev. 2002;15:506-26. 10.1128/CMR.15.3.506-526.2002 PubMed] [ PMC free article] [ CrossRef] Google Scholar]

3. Breiman R. Modes of transmission in epidemic and nonepidemic [ Legionella infection: directions for further study. In: Barbaree J, Breiman R, Dufour A, eds. Legionella: current status and emerging perspectives. Washington: American Society for Microbiology; 1993. p. 30-5. Google Scholar]

10. Environmental Protection Agency. Alternative disinfectants and oxidants guidance manual. Washington: US Environmental Protection Agency Office of Water. EPA 815-R-99-014; 1999. [ Google Scholar]

11. Seidel CJ, McGuire MJ, Summers RS, Via S. Have utilities switched to chloramines? J Am Water Works Assoc. 2005;97:87-97. [ Google Scholar]

12. Environmental Protection Agency. National primary drinking water regulations. Stage 2 disinfectants and disinfection byproducts rule; national primary and secondary drinking water regulations: approval of analytical methods for chemical contaminants. Fed Regist. 2003;68:49547-96. [ Google Scholar]

16. Minimizing the risk of legionellosis associated with building water systems. Atlanta (GA): American Society of Heating, Refrigerating and Air-Conditioning Engineers, Inc.; 2000. p 16. [ Google Scholar]

17. Centers for Disease Control and Prevention. Procedures for the recovery of [ Legionella from the environment. Atlanta: US Department of Health and Human Services; 1994. Google Scholar]

18. Fields BS. Legionellae and Legionnaire's disease. In: Hurst CJ, Crawford RL, Knudsen GR, McInerney MJ, Stetzenbach LD, editors. Manual of environmental microbiology. 2nd ed. Washington: American Society for Microbiology; 2002. p. 860-70. [ Google Scholar]

19. Schuchat A, Hilger T, Zell E, Farley MM, Reingold A, Harrison L, et al. Active bacterial core surveillance of the emerging infections program network. Emerg Infect Dis. 2001;7:92-9. 10.3201/eid0701.010114 [[ PubMed] [ CrossRef] PMC free article] [ Google Scholar]

21. Donlan RM, Forster T, Murga R, Brown E, Lucas C, Carpenter J, et al. [ Legionella pneumophila associated with the protozoan Hartmannella vermiformis in a model multi-species biofilm has reduced susceptibility to disinfectants. Biofouling. 2005;21:1-7. 10.1080/08927010500044286 [ PubMed] [ CrossRef] Google Scholar]

23. Lin YS, Stout JE, Yu VL, Vidic RD. Disinfection of water distribution systems for [ Legionella. Semin Respir Infect. 1998;13:147-59. [ PubMed] Google Scholar]

26. Lin YS, Vidic RD, Stout JE, Yu VL. Negative effect of high pH on biocidal efficacy of copper and silver ions in controlling [[ Legionella pneumophila. Appl Environ Microbiol. 2002;68:2711-5. 10.1128/AEM.68.6.2711-2715.2002 PubMed] [ PMC free article] [ CrossRef] Google Scholar]

30. Vickers RM, Yu VL, Hanna SS, Muraca P, Diven W, Carmen N, et al. Determinants of [ Legionella pneumophila contamination of water distribution systems: 15-hospital prospective study. Infect Control. 1987;8:357-63. [ PubMed] Google Scholar]

33. Moore MR, Pryor M, Fields B, Lucas C, Phelan M, Besser RE. Introduction of monochloramine into a municipal water system: impact on colonization of buildings by [[ Legionella spp. Appl Environ Microbiol. 2006;72:378-83. 10.1128/AEM.72.1.378-383.2006 PubMed] [ PMC free article] [ CrossRef] Google Scholar]

Articles from Emerging Infectious Diseases are provided here courtesy of Centers for Disease Control and Prevention

# data[].attributes.attachment_link.title
Reducing Legionella Colonization of Water Systems with Monochloramine

# data[].attributes.attachment_link.description
Monochloramine reduced colonization in building hot water systems.Monochloramine disinfection of municipal water supplies is associated with decreased risk for Legionnaires' disease. We conducted a 2-year, prospective, environmental study to evaluate ...

# data[].attributes.headline
A case of a sterilizing antibody against YF with immunoglobulin

# data[].attributes.subject
A case of a sterilizing antibody against YF with immunoglobulin

# data[].attributes.attachment_link.readable_text
You do not currently have access to this article.

Purchase

Short-term Access

Don't already have a personal account? Register

NEUTRALIZING ANTIBODY RESPONSES IN THE MAJOR IMMUNOGLOBULIN CLASSES TO YELLOW FEVER 17D VACCINATION OF HUMANS - 24 Hours access

# data[].attributes.attachment_link.title
NEUTRALIZING ANTIBODY RESPONSES IN THE MAJOR IMMUNOGLOBULIN CLASSES TO YELLOW FEVER 17D VACCINATION OF HUMANS

# data[].attributes.attachment_link.description
Abstract. Monath, T. P. C. (CDC, Atlanta, Ga. 30333). Neutralizing antibody responses in the major immunoglobulin classes to yellow fever 17O vaccination of hum

# data[].attributes.headline
on page 114 there is a mention of the 1970 YF epidemic declining rapidly 

# data[].attributes.subject
on page 114 there is a mention of the 1970 YF epidemic declining rapidly 

# data[].attributes.attachment_link.title
The 1970 yellow fever epidemic in Okwoga District, Benue Plateau State, Nigeria: 1. Epide miological observations*

# data[].attributes.attachment_link.description
A focal epidemic of yellow fever occurred in late 1970 in southern Benue Plateau State, Nigeria, 1 year after a much larger outbreak in northern Nigeria. Like its predecessor, the 1970 epidemic was associated with virus transmission by wild-breeding…

# data[].attributes.headline
the decline of Yellow fever in 1987 nigeria

# data[].attributes.subject
the decline of Yellow fever in 1987 nigeria

# data[].attributes.headline
this article shows that bacteria simulates the hatching of yellow-fever carrying mosquitos

# data[].attributes.subject
this article shows that bacteria simulates the hatching of yellow-fever carrying mosquitos

# data[].attributes.attachment_link.title
Bacteria Stimulate Hatching of Yellow Fever Mosquito Eggs

# data[].attributes.attachment_link.description
Background Aedes aegypti Linnaeus is a peridomestic mosquito that lays desiccation-resistant eggs in water-filled human-made containers. Previous investigations connected egg hatching with declining dissolved oxygen (DO) that is associated with bacterial growth. However, past studies failed to uncou…

# data[].attributes.attachment_link.title
Genetic selection of a flavivirus-refractory strain of the yellow fever mosquito Aedes aegypti.

# data[].attributes.attachment_link.description
Genetic selection of a flavivirus-refractory strain of the yellow fever mosquito Aedes aegypti.

# data[].attributes.headline
this article mentions the vaccines and how they improved YF in the 1940s 

# data[].attributes.subject
this article mentions the vaccines and how they improved YF in the 1940s 

# data[].attributes.attachment_link.readable_text
Yellow fever (YF) is caused by the prototype member of the genus Flavivirus (family Flaviviridae), which contains approximately 70 positive-strand, single-strand RNA viruses, the majority of which are transmitted by arthropods (mosquitoes and ticks). Yellow fever is endemic in tropical regions of Africa and South America, and many review articles describe its epidemiology in the two continents [1], [2], [3]. A recent analysis of country-by-country geographic risk re-defined the borders of the YF endemic zone, but emphasized the fluid nature of virus activity and the occurrence of periodic expansions and retractions [4]. The virus has a relatively narrow host range for productive infection, and is maintained in nature by transmission between non-human primates and blood-feeding mosquitoes mainly belonging to the genera Haemagogus and Aedes ( Stegomyia) in South America and Africa, respectively, and by transovarial transmission in these vectors. Humans are infected sporadically when bitten by sylvatic mosquitoes that previously fed on a viremic monkey (so-called jungle yellow fever), but may also serve as the viremic host for inter-human transmission, mainly by Aedes aegypti, a species that breeds in water-containing vessels inside dwellings or in close proximity to them (so-called urban yellow fever). The epidemiology of YF in Africa is often mixed, involving both sylvatic and domestic vector species in inter-human transmission. Consequently, the force of infection in Africa is higher (generally 20-30×) than in South America, the consequence being large epidemics. In recent years, new efforts have been made to vaccinate the populations of high-risk countries in West Africa; the long-term consequences of this effort will be a reduction in major epidemics.

Yellow fever was a major threat to human health from the 18th Century to the early 20th Century, with repeated epidemics following introductions to coastal towns and cities distant from endemic areas in North America, the Caribbean and Europe. The identification in 1900 of A. aegypti mosquitoes as the agency whereby YFV was transmitted, and subsequent efforts to control the vector, resulted in a decline in yellow fever outside the tropical, endemic zone. The development of two live, attenuated YF vaccines in the 1930s, and their wide deployment in the 1940s, led to a further decline of the disease. Subsequently, there have been periodic upsurges of YF activity in endemic regions without routine immunization programs.

The annual reporting rate of officially reported cases in South America and Africa shown in Fig. 1, but these rely on passive surveillance and thus significantly underestimate the true incidence. The latter remains unknown, except in some discrete epidemics that have been actively investigated. In non-epidemic periods, estimates of 200,000 cases derived from serosurvey data and the rate of inapparent to apparent cases (7-12:1) have been cited from paper to paper for over 15 years, without supporting evidence. Since 2008, more active laboratory-based surveillance activities suggest that, of several thousand suspect clinical cases in Africa investigated, only 1-2% have laboratory evidence for YF. The World Health Organization (WHO) appropriately treats the detection of a case of YF as an emergency, since it reflects transmission of the virus and a risk of a further spread; if the affected region is an area of low vaccination coverage, a regional mass vaccination campaign is generally conducted in response. Between 2007 and 2010, 57 million people were vaccinated against YF in 10 countries at risk in Africa, and during the same period, 17 million people were protected through emergency vaccination [5]. In the 1990s, despite 50 years of use and over 500 million doses distributed, new safety concerns about the live attenuated YF 17D vaccine have come to light, revealing that in rare circumstances the vaccine can cause a disease similar to parental wild-type virus. This fact has modified vaccine policy and regulations in some circumstances, as will be discussed later on.

Despite the availability of vaccines since the 1940s, large epidemics occurred in areas without a background of naturally acquired or artificial immunity. Dramatic upsurges in YFV activity occurred in Africa in the 1960s and the late 1980s each involving >100,000 cases, and recent outbreaks affected southern Brazil, Paraguay and Argentina (2007-2009), Uganda (2010), and Sudan and Ethiopia (2012-2013). Although the absence of an immune barrier in the human population is a key factor, the underlying reasons for virus amplification remain unclear, and are multifactorial, involving deterministic (vector density and competence, viral virulence), and stochastic factors. Expansions of YFV activity have sometimes been associated with the emergence of a new virus lineage [6], but the lack of information about biological correlates of genetic change, make it difficult to assign causality. Perturbations of weather, particularly prolonged increases in rainfall and high temperatures have been associated with outbreaks of YF in Africa and South America.

In humans, YF is a severe acute illness with fever, nausea, vomiting, epigastric pain, hepatitis with jaundice, renal failure, hemorrhage, shock and death in 20-60% of cases. Yellow fever is the prototypical viral hemorrhagic fever, and shares many pathophysiological features with unrelated diseases associated with a similar syndrome, except that the severity of hepatic dysfunction is generally greater in YF patients. The lower case fatality in Africa (∼20%) than in South America (40-60%) [7], suggests that genetic factors determine lethality of the infection, a subject that deserves further study. Interestingly, the neutralizing antibody response to YF 17D vaccine is statistically higher in Caucasians than in African-Americans [8] possibly indicating genetic resistance to YF in the latter. Some New World monkeys, notably Alouatta (howling monkeys), are also susceptible to lethal infections, and epizootics associated with monkey deaths may precede the occurrence of human cases, a useful surveillance tool [9]. In contrast, almost all African nonhuman primates have inapparent, viremic infections. This reflects the origin of yellow fever virus (YFV) in Africa several thousand years ago, and a balanced co-evolution of virus and hosts. Based on these factors and genetic analyses, YFV was introduced into the Americas from West Africa during the slave trade about 400 years ago [10], and rapidly invaded a new ecological niche involving local hosts and vectors, much like another flavivirus, West Nile, did after its more recent introduction into the Americas.

A serious concern for the future is whether YFV could be introduced by a viremic air traveler to A. aegypti-infected areas outside the endemic zone, and particularly India and Southeast Asia. The recent spread of another virus transmitted in a human- Aedes cycle-Chikungunya-in islands of the Indian Ocean, India, southern Europe, and the Caribbean illustrates the threat. Although the WHO maintains an emergency YF 17D vaccine stockpile, an extensive outbreak could create a significant shortfall in vaccine supply.

# data[].attributes.attachment_link.description
Yellow fever, a mosquito-borne flavivirus disease occurs in tropical areas of South America and Africa. It is a disease of major historical importance...

# data[].attributes.headline
this article is about the end of the yellow fever outbreak in Ghana

# data[].attributes.subject
this article is about the end of the yellow fever outbreak in Ghana

# data[].attributes.attachment_link.readable_text
Ghana Health Service announced this week the end of the Lassa fever outbreak which began in February.

On Friday, 24 February 2023, the Greater Accra Regional Health Directorate was notified of two confirmed cases of Lassa fever at the Korle Bu Teaching Hospital in Accra. No new case has been recorded since the 1st of March, 2023. A total of 27 confirmed cases of Lassa fever and one death (CFR: 3 %) were reported.
Support Outbreak News Today with a donation
The maximum follow-up period of 42 days has elapsed since the last confirmed case of Lassa fever was discharged on March 10. The Ghana Health Service declared the end of the outbreak in line with the WHO-recommended mandatory 42 days post-surveillance countdown period from when the last case was discharged.
Measles on the rise in Ethiopia: A number of factors involved

# data[].attributes.attachment_link.title
Ghana declares end of Lassa Fever outbreak - Outbreak News Today

# data[].attributes.attachment_link.description
NewsDesk @bactiman63 Ghana Health Service announced this week the end of the Lassa fever outbreak which began in February. On Friday, 24 February 2023, the Greater Accra Regional Health Directorate was notified of two confirmed cases of Lassa fever at the Korle Bu Teaching Hospital in Accra.

# data[].attributes.headline
this article talks about yellows fever vaccine and  mentions a decline in infection and transmission 

# data[].attributes.subject
this article talks about yellows fever vaccine and  mentions a decline in infection and transmission 

# data[].attributes.attachment_link.readable_text
Ana Carolina Campi-Azevedo, Vanessa Peruhype-Magalhāes, Jordana Grazziela Coelho-dos-Reis, Lis Ribeiro Antonelli, Christiane Costa-Pereira, Elaine Speziali, Laise Rodrigues Reis, Jandira Aparecida Lemos, José Geraldo Leite Ribeiro, Luiz Antônio Bastos Camacho, Maria de Lourdes de Sousa Maia, Sheila Maria Barbosa de Lima, Marisol Simões, Reinaldo de Menezes Martins, Akira Homma, Luiz Cosme Cota Malaquias, Pedro Luiz Tauil, Pedro Fernando Costa Vasconcelos, Alessandro Pecego Martins Romano, Carla Magda Domingues, Andréa Teixeira-Carvalho, Olindo Assis Martins-Filho, and Collaborative Group for Studies of Yellow Fever Vaccine

Abstract

We evaluated the duration of neutralizing antibodies and the status of 17DD vaccine-specific T- and B-cell memory following primary and revaccination regimens for yellow fever (YF) in Brazil. We observed progressive decline of plaque-reduction neutralization test (PRNT) seropositivity and of the levels of effector memory CD4+ and CD8+ T cells, as well as interferon-γ+CD8+ T cells, 10 years after primary vaccination. Revaccination restored PRNT seropositivity as well as the levels of effector memory CD4+, CD8+, and interferon-γ+CD8+ T cells. Moreover, secondary or multiple vaccinations guarantee long-term persistence of PRNT positivity and cell-mediated memory 10 years after booster vaccination. These findings support the relevance of booster doses to heighten the 17DD-YF-specific immune response to guarantee the long-term persistence of memory components. Secondary or multiple vaccinations improved the correlates of protection triggered by 17DD-YF primary vaccination, indicating that booster regimens are needed to achieve efficient immunity in areas with high risk for virus transmission.

yellow fever, 17DD vaccine, neutralizing antibodies, memory CD8 Keywords: ⁺ T-cells, vector-borne infections, viruses, Brazil

Yellow fever (YF) vaccination is recommended for persons living in YF-endemic areas as the most effective strategy to reduce the risk for infection (1). The 17D and 17DD live attenuated vaccines are considered similarly safe and immunogenic, regardless of the minor differences in their nucleotide sequences (1). The progressive expansion of areas with YF viral circulation in YF-endemic countries has required extensive vaccination campaigns that reduced the international vaccine stockpile and brought to light the discussion about the need for booster doses to guarantee long-term cell memory in populations living in YF-endemic countries. Outbreaks of YF occur occasionally in areas of Africa and South America (2-7).

In 2013, the World Health Organization (WHO) stated that a single dose of YF vaccine sufficed to provide lifelong protection and that no booster dose was required to guarantee protection against the disease (1,8). However, time-dependent loss of protective immunity has been reported (9-11). The levels of YF-neutralizing antibodies decrease significantly 10 years after vaccination; ≈25%-30% of primary vaccinees lack protective antibodies (10,11). In addition, the polyfunctional cellular immune responses elicited by YF vaccination that contribute to protection also displayed a time-dependent decline following primary vaccination (11,12). In light of this information, the single-dose regimen for YF vaccine has been questioned, especially in YF-endemic countries where the proportion of persons exposed to potential risks should be considered against the primary-vaccine failure rate and time-dependent decline of protective immunity.

The goal of this study was to evaluate the proxies of protection elicited by primary, secondary, and multiple vaccinations and verify the duration of neutralizing antibodies and 17DD-specific T- and B-cell memory following these distinct vaccination regimens. We sought to clarify the importance of 17DD-YF booster vaccination to heighten the immune response of those primary vaccinees living in endemic areas whose immunity declines to nonprotective levels.

Materials and Methods

Study Population

We conducted this investigation during May 12, 2014-December 16, 2016, simultaneously sampling from Rio de Janeiro and 2 municipalities of Minas Gerais state (Alfenas and Ribeirão das Neves), Brazil. We assigned participants to groups on the basis of official vaccination records. The study included 421 samples collected from 326 healthy adults 18-77 years of age, initially categorized into 3 arms: primary vaccination, secondary vaccination, and multiple vaccination ( Figure 1 ). We designed the primary vaccination and secondary vaccination arms as 2 complementary independent approaches, each including a longitudinal (95 paired samples) and a cross-sectional investigation (231 unpaired samples). Study groups were coded to indicate participants' vaccination status (NV for nonvaccinated persons, PV for those who had had primary YF vaccination only, RV for those who had been revaccinated) and time since last vaccination, given in days or years (e.g, d0 for day zero).

Samples and Tests

We collected whole blood samples from each participant. We used samples of 5 mL without anticoagulant for plaque-reduction neutralization test (PRNT) and samples of 20 mL in heparin for 17DD-YF phenotypic and functional analyses.

PRNT

We used serum samples to quantify the PRNT levels to the 17DD-YF virus by the micro-PRNT50 test, as described previously by Simões et al. (13). We performed assays at Laboratório de Tecnologia Virológica (LATEV), Bio-Manguinhos, and expressed results from replicates as the reciprocal of sample dilution, considering seropositivity of PRNT titers >1:50 serum dilution.

Dengue IgG Indirect ELISA

We performed serologic tests for dengue virus (DENV) IgG using a Panbio dengue IgG indirect ELISA kit ( https://www.alere.com). Tests were performed at Laboratório de Flavivírus, Instituto Osvaldo Cruz, as previously reported (14).

Phenotypic and Functional Memory Biomarkers

We performed in vitro 17DD-YF-specific peripheral blood lymph proliferative assay as previously reported by Costa-Pereira et al. (12). In brief, we incubated replicates of PBMC suspension (1.0 × 10 ⁶/well) for 144 hours at 37°C in 5% CO ₂. We harvested cells from control (CC) and 17DD-YF antigen-stimulated (17DD-YF Ag) cultures, labeled them with live/dead dye (Life Technologies, https://www.thermofisher.com), and used a cocktail of monoclonal antibodies (mAbs) to quantify the phenotypic memory status of T cells and B cells. For T cells we used anti-CD4/(RPA-T4)/FITC, anti-CD8/(SK1)/PerCP-Cy5.5, anti-CD27/(M-T271)/PE, anti-CD45RO/(UCHL1)/PE-Cy7, and anti-CD3/(SK7)/APC-Cy7; for B cells, we used anti-CD19/(HIB19)/PerCP, anti-CD27/(M-T271)/PE, and anti-IgD/(IA6-2)/FITC. We obtained all mAbs from BD Pharmingen ( https://bdbiosciences.com).

In parallel, we stained cultured PBMC aliquots with live/dead dye and a mix of mAbs to quantify the functional memory status of T and B cells: anti-CD3/(UCHT1)/Qdot605 (Invitrogen, https://www.thermofisher.com); anti-CD4/(GK1.5)/APCe-Fluor780 (eBioscience, https://www.thermofisher.com); anti-CD8/(SK1)/PerCP (BD Biosciences, https://bdbiosciences.com); and anti-CD19/(HIB19)/Alexa-Fluor700 (eBioscience). After a fix/permeabilize procedure, we incubated cells with a mAbs cocktail of anti-TNF-α/(clone MAb11)/PE-Cy7, anti-interferon (IFN)-γ/(clone B27)/Alexa-Fluor488), anti-interleukin (IL)-5/(JES1-39D10)/PE, and anti-IL-10/(JES3-19F1)/APC, all from BD Biosciences. We fixed the stained cells and stored them at 4°C for <24 hours before acquisition on a BD LSR Fortessa Flow Cytometer (BD Biosciences).

We acquired a total of 100,000 lymphocytes from each sample. We used FlowJo version 9.3.2 software (Tree Star, https://www.flowjo.com) to quantify the memory T-cell and B-cell subsets, as well as the percentage of cytokine-producing T and B cells. We expressed the results as 17DD-YF Ag/CC Index, calculated as the ratio of cells observed in the 17DD-YF Ag cultures divided by the respective control culture.

Data Analysis

This study was composed of 2 independent but complementary approaches: a longitudinal investigation and a cross-sectional investigation. We performed statistical analyses for the longitudinal investigation using paired t-test to compare NV(d0) versus PV(d30-45) groups for primary vaccination, as well as #PV(y>10) (primary vaccination >10 years ago) versus RV(d30-45) for secondary vaccination. For the cross-sectional design, we made the transversal comparisons among groups using analysis of variance adjusted to multiple comparisons and set statistical significance at p<0.05. (We did not highlight nonsignificant differences in the figures.) We used the χ ² test to compare seropositivity rates between NV and PV groups and also between #PV and RV groups.

We performed biomarker signature analysis as described previously by Luiza-Silva et al. (15). In brief, we calculated the global median value of 17DD-YF Ag/CC Index for each phenotypic and functional biomarker and used that value as the cutoff to identify each biomarker as low index (below global median) or high index (above global median). We considered only biomarkers observed in >50% of study participants for comparative analysis among groups.

We conducted Venn diagram analysis ( http://bioinformatics.psb.ugent.be/webtools/Venn) to select common biomarkers among subgroups. We overlaid biomarker signatures for comparative analysis of time-dependent changes of biomarker sets observed after vaccination.

Results

Booster Vaccination and PRNT

Data analysis demonstrated that primary vaccination triggered significant levels of 17DD-YF-specific neutralizing antibodies (p<0.0001), reaching a seropositivity rate of 96% ( Figure 2 ). Of note, we observed progressive decrease in PRNT levels (p<0.0001) and in the PRNT seropositivity rates along the time compared with PV(d30-45). The seropositivity rate declined to ≈71% by 10 years after primary vaccination ( Figure 2 , panel B). Booster vaccination significantly increased the PRNT levels (p<0.0001) and raised the seropositivity rate from 69% to 100% ( Figure 2 , panels A, B). The secondary vaccination was accompanied by higher seropositivity rate after 10 years upon booster dose, regardless of the decrease in PRNT levels (p<0.0001) observed over time compared with #PV(d30-45) ( Figure 2 , panel B).

Secondary Booster Vaccination and YF-specific Cell-Mediated Memory

Comparative analysis of NV(d0) versus PV(d30-45) after 1 or 2 doses of 17DD-YF vaccine demonstrated that primary vaccination is followed by an increase of memory T cells, including eEfCD4 (p<0.05), EMCD4 (p<0.05), and EMCD8 (p = 0.0006), and all B-cell subsets evaluated, NCD19 (p = 0.01), nCMCD19 (p = 0.001), and CMCD19 (p = 0.001). We also observed a decrease of NCD8 (p = 0.02), eEfCD8 (p = 0.02), and CMCD8 (p = 0.001). The results showed that cellular immunity, eEfCD4 (p = 0.01), EMCD4 (p = 0.01), and EMCD8 (p = 0.009); and all B-cell subsets, NCD19 (p = 0.003), nCMCD19 (p = 0.02), and CMCD19 (p = 0.0002), clearly wane over 10 years, compared with 30-45 days after primary vaccination ( Figure 3 ).

Secondary vaccination with 17DD-YF was able not only to increase the level of memory T-cell subsets, eEfCD4 (p<0.05), EMCD4 (p<0.05), and EMCD8 (p = 0.04) at 30-45 days after the second dose but also to sustain the maintenance of eEfCD4 and EMCD8 levels even after >10 years upon booster vaccination compared with 30-45 days after secondary vaccination. We observed no substantial changes in memory B-cell subsets upon secondary vaccination ( Figure 3 ).

Revaccination and IFN-γ-Mediated T-Cell Memory

Data analysis for in vitro 17DD-YF antigen recall revealed that primary vaccination induced significant increases of functional memory biomarkers in CD4+ (tumor necrosis factor [TNF]-α/p = 0.04, IFN-γ/p = 0.04 and IL-5/p = 0.0001) and CD8+ T cells (TNF-α/p = 0.0003, IFN-γ/p = 0.008 and IL-5/p = 0.0002) as well as in B cells (TNF-α/p<0.05 and IL-5/p = 0.016) ( Figure 4 ). We observed a decrease of IL-10+CD4+ T cells (p = 0.04) at 30-45 days after primary vaccination and a clear decrease of TNF-α, IFN-γ, and IL-5 produced by CD4+ and CD8+ T cells. We also saw a decrease in TNF-α and IL-5 from B cells over time, particularly at >10 years, compared with 30-45 days after primary vaccination (p<0.05 in all cases). Conversely, we detected an increase of IL-10+ B cells over time after primary vaccination ( Figure 4 ).

Secondary vaccination was able to restore T cell functional memory mediated by IFN-γ and B-cell functional memory by TNF-α. We observed sustained production of IFN-γ by CD8+ T cells even >10 years after booster vaccination ( Figure 4 ).

Booster Vaccination and Long-lasting Persistence of Effector Memory

We constructed overlays of biomarker signatures of NV(d0) versus PV(d30-45) in the primary-vaccination arm of the study as well as #PV(y>10) versus RV(d30-45) in the secondary-vaccination arm to select those attributes eligible as universal memory-related biomarkers ( Appendix Figure 1). Venn diagram analysis revealed 3 common attributes (EMCD4, EMCD8, and IFNCD8) that we tagged for follow-up analysis after primary, secondary, or multiple 17DD-YF vaccination ( Appendix Figure 1).

After we selected the universal set of follow-up attributes, we compared biomarker signatures over time after 17DD-YF primary or secondary vaccination ( Figure 5 ). Data analysis demonstrated that all 3 biomarkers were observed in PV(d30-45) and PV(y1-5). EMCD8 was maintained in PV(y>5-9) but not in PV(y>10). Comparative analysis between #PV(y>10) and RV(d30-45) demonstrated restoration of universal memory-related biomarkers (EMCD4, EMCD8, and IFNCD8) upon revaccination. Moreover, we identified EMCD8 in a high proportion of secondary vaccinees across the time periods after booster dose ( Figure 5 ).

Comparison of 17DD-YF Primary and Booster Vaccination Effects on Cell-Mediated Memory

We used individual PRNT and EMCD8 profiles to assemble a memory matrix and calculated the resultant YF-specific memory. We classified each volunteer by positive results above the cutoff threshold as EMCD8, PRNT, none, or both ( Figure 6 ). Data analysis demonstrated that primary vaccination leads to a resultant memory in 97% of volunteers, with 3% of failure in the PV(d30-45) group. However, an increase in the proportion of primary vaccinees with "none" positive attributes, neither PRNT nor EMCD8 biomarkers, was observed, reaching a critical value of 30% at ≥10 years after primary vaccination ( Figure 6 , panel A).

The comparison between #PV(y>10) and RV(d30-45) demonstrated that secondary vaccination restored the resultant memory in 100% of the volunteers, which was different from primary vaccination results. All secondary vaccines evaluated simultaneously for PRNT and EMCD8 profile presented a preserved resultant memory. In particular, at >10 years after secondary vaccination, 100% of vaccinees had 1 or both biomarkers detectable ( Figure 6 , panel B).

Multiple Vaccination and the Overall Profile of Protection

We analyzed 17DD-YF memory status triggered by multiple vaccinations ( Figure 7 ). The results demonstrated that all vaccinees who received multiple shots had PRNT levels above the cutoff limit ( Figure 7 , panel A). The proportion of vaccinees with EMCD8 above the cutoff limit was higher for RV(y1-5) and RV2+(y1-5) than for PV(y1-5) ( Figure 7 , panel B). Likewise, the overall resultant memory for RV(y1-5) and RV2+(y1-5) was higher than that for PV(y1-5) - ( Figure 7 , panel C).

PRNT Seronegativity before Revaccination and Cell-Mediated Memory Response

The volunteers from the #PV(y≥10) group were further categorized into subgroups, PRNT- and PRNT+, according to their PRNT results before revaccination. The levels of humoral and cellular biomarkers, as well as the magnitude of baseline fold changes in neutralizing antibodies, were higher in PRNT- than in PRNT+ vaccines ( Figures 8 , ​,9). 9 ). An increase in PRNT titer by a factor >4 at follow-up further demonstrated the relevance of booster doses to restore the immunological memory of these subjects ( Figure 8 ). Furthermore, the booster dose had higher impact on cellular immunity and biomarker signature of PRNT- than PRNT+ primary vaccinees, and both groups restored the EMCD8 biomarker compared with the #PV(y>10) group ( Figure 9 ).

Dengue Virus Seropositivity

We analyzed the results to determine whether DENV seropositivity influences the humoral and cellular memory after secondary vaccination ( Appendix Figures 2 , ​,3). 3 ). We found DENV seropositivity in 28% of volunteers in the secondary vaccination arm. The results demonstrated that DENV seropositivity did not influence PRNT seropositivity over time after secondary vaccination ( Appendix Figure 2 ). Moreover, DENV seropositivity did not affect cellular immunity (EMCD8), which remained above the cutoff limit in all subgroups of secondary vaccinees ( Appendix Figure 3 ).

Discussion

YF vaccination induces an efficient immunity and represents one of the most effective strategies to reduce the risk for infection in YF-endemic countries. The vaccine is highly immunogenic, eliciting a strong antibody response together with a broad and complex innate (16-22) and adaptive immunity (23-28). Although the YF vaccine has been considered a benchmark among vaccines because of its ability to induce long-lasting immune response, the duration of humoral and cellular immunity following YF vaccination is still controversial. Some studies demonstrate that neutralizing antibodies and YF-specific CD8+ T cells after primary vaccination are suggestive imprints compatible with long-lived memory (8,26,29-31); other studies emphasize that the immunity to YF vaccine wanes over time, suggesting the need for booster doses to guarantee long-lasting, efficient immunity memory (9-12,32-36).

We could not assess protective immunity to YF in humans by challenge with live wild-type YF virus. Therefore, the protective or nonprotective immunity status to YF virus in humans is based on laboratory methods, known as correlates of protection; PRNT level has been considered the standard for measuring postvaccination immunity to YF. Pinpointing cellular immunity biomarkers is relevant in studies that pose the question of whether PRNT seronegativity necessarily means absence of protective immunity (34). Our investigation proposes that, in addition to PRNT seropositivity, YF-specific cellular immunity may be a useful tool for monitoring the duration of YF vaccine-induced memory over time. However, upon closer examination, our findings indicate the decline of YF-specific immune response over time, emphasizing that the YF-specific immunity wanes shortly after primary vaccination and that a substantial proportion of primary vaccines (14%-30%) do not present sufficient levels of neutralizing antibodies or CD8+ T-cell memory within 5-10 years after primary vaccination.

Some studies have investigated the relevance of booster doses to heighten the YF-specific immune response in primary vaccinees whose immunity has declined to nonprotective levels. Weiten et al. (31) postulated that booster vaccination did not increase the titers of YF-specific antibodies nor induce or alter the phenotypes of CD8+ T cells. Conversely, Kongsgaard et al. (37) have demonstrated that, although most vaccinees responded to a booster vaccination, the antibody titers and the cellular immune responses observed following revaccination were lower than for primary vaccination responses. In this study, we have demonstrated that the booster dose was able to upregulate the levels of neutralizing antibodies and heighten the cellular immunity signature and restore the proportion of vaccinated participants with high levels of effector memory CD8+ T cells. We strongly believe that the differences we observed among the published studies could be attributed to differences in the populations under scrutiny.

The magnitude of increase in the neutralizing antibody titers and YF-specific CD8+ T-cell response achieved with a booster dose may be closely related to the baseline immune activation, suggesting that an activated immune microenvironment before revaccination impairs the response to the YF vaccine. Muyanja et al. (38) showed that 17D-204 vaccinees in Africa displayed decreased levels of YF-specific immunity compared with vaccinees in Europe. It is possible that the immune microenvironment affects the quantitative and qualitative response, as well as the vaccine efficacy. Whereas vaccinees in Europe displayed persistent PRNT levels even at 10 years after vaccination, those in Africa exhibited reduced persistence of YF immunological memory. Constant exposure to infectious diseases, as well as diet and gut microbiota, may lead to a state of immune hyperactivation and exhaustion that can contribute to reduced magnitude of cellular and humoral responses and impair the YF vaccine memory. Of note, the impaired YF vaccine-induced memory was boosted by a second vaccination (38). Our results corroborate this hypothesis, showing the relevance of a booster dose to improve the immune response among primary vaccinees. This recommendation should be considered to restore the YF protective immunity in those primary vaccinees whose correlates of protection fade over time, reaching nonprotective levels.

The loss of immunity in a subpopulation of vaccine recipients should be taken into consideration, and a booster dose should be administered. Previous studies have demonstrated that, on average, approximately 1 in 5 persons from non-YF-endemic areas and 3 in 10 persons living in YF-endemic areas may lose measurable antibody responses within 10 years after primary vaccination. In this sense, it is true that the 17DD-YF vaccination elicits long-lasting immunity; however, lifelong immunity is not observed in every vaccinated individual. Although PRNT is the standard assay to monitor YF protective immunity, it is not a feasible method available in local laboratories to identify participants who should receive a booster vaccination.

The expansion of risk areas for YF worldwide has contributed to the depletion of YF vaccine stockpile and the need for new strategies to reverse the imminent shortage of vaccine. WHO has considered measures to improve YF vaccine supply, including a dose-sparing strategy as a short-term measure. However, the dose sparing is not proposed for routine immunization. WHO also considered advising a single lifetime dose of YF vaccine. In YF outbreaks or periods of limited vaccine production and reduced stockpiles, the YF primary vaccination should be the priority, as recommended by the Strategic Advisory Group of Experts on Immunization. However, once the YF vaccine supply has normalized and no outbreaks are reported, revaccination should be suggested. On the basis of our findings, >1 booster dose at 10 years after primary vaccination is suggested for travelers entering higher-risk areas and required for residents of YF-endemic countries who are at risk for infection (34,39).

Altogether, our findings emphasize the relevance of booster doses to heighten the 17DD-YF specific immune response to achieve efficient immunity. Secondary vaccination improved the correlates of protection that had waned over time after 17DD-YF primary vaccination and lowered the loss of protection over time. However, multiple vaccination seems to better support long-lasting protection in all vaccinees, which suggests that >3 booster doses at 10-year intervals should be recommended, especially in areas with high risk for YF transmission. Whether the waning of immune markers observed over time is associated with loss of YF vaccine effectiveness and increased risk for breakthrough YF infection remains to be seen.

In summary, we evaluated the evidence for benefits and risks associated with YF vaccine booster doses using the Grading of Recommendations, Assessment, Development, and Evaluation framework (40). A primary dose of YF vaccine is well known to be highly safe and effective, with few vaccine failures. However, a critical wane of correlates of protection has been documented by our group and others, particularly at ≥10 years postvaccination. Few reports of serious adverse events have been observed following booster doses of the vaccine. We recommend the booster dose to prevent this serious disease that has no treatment and substantial mortality rates.

Appendix:
Additional information about yellow fever revaccination and long-term cell memory, Brazil.

Acknowledgments

We thank the Program for Technological Development in Tools for Health-RPT-FIOCRUZ for the use of the flow cytometry facility.

This study was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Bio-Manguinhos/FIOCRUZ, PROEP/CPqRR/FIOCRUZ, Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (grant nos. 444417/2014-1 and 458134/2014-7), and Programa Nacional de Imunizações, Secretaria de Vigilância em Saúde-Ministério da Saúde, Brazil (grant no. TC 277/2013). O.A.M.-F., A.T.-C., and L.R.A. received research fellowships from CNPq.

Author contributions: study design, A.C.C.-A., L.A.B.C., M.L.S.M., R.M.M., A.H., P.L.T., P.F.C.V., A.P.M.R., C.M.D., A.T.-C., O.A.M.-F.; funding acquisition, L.A.B.C., O.A.M.-F., A.P.M.R., C.M.D.; experiments: A.C.C.-A., V.P.-M., J.G.C.-R., L.R.A., C.C.-P., E.S., L.R.R., S.M.B.L., M.S., A.T.-C., O.A.M.-F.; field study: J.A.L., J.G.L.R., L.A.B.C., L.C.C.M.; data acquisition: A.C.C.-A., V.P.-M., J.G.C.-R., L.R.A., C.C.-P., E.S., S.M.B.L., M.S.; data analysis: A.C.C.-A., A.T.-C., O.A.M.-F.; validation: P.L.T., P.F.C.V.; advisory committee: R.M.M., A.H., P.L.T., P.F.C.V.; manuscript: A.C.C.-A., J.G.C.-R., C.C.-P., L.R.R., A.T.-C., O.A.M.-F.

Biography

Dr. Campi-Azevedo is a cellular and molecular immunologist working in the Biomarker Research Group at the René Rachou Institute, Oswaldo Cruz Foundation (FIOCRUZ-Minas), Belo Horizonte, Brazil. Her primary research interests include vaccine immunity; diagnosis and management of infectious/parasitic diseases and biomarkers for posttherapeutic follow-up; bioprospection in experimental therapeutics; and biomarkers in oncology.

Footnotes

Suggested citation for this article: Campi-Azevedo AC, Peruhype-Magalhāes V, Coelho-dos-Reis JG, Antonelli LR, Costa-Pereira C, Speziali E, et al. 17DD yellow fever revaccination and heightened long-term immunity in populations of disease-endemic areas, Brazil. Emerg Infect Dis. 2019 Aug [ date cited]. https://doi.org/10.3201/eid2508.181432

References

1. World Health Organization. Vaccines and vaccination against yellow fever. WHO position paper - June 2013. Wkly Epidemiol Rec. 2013;88:269-83. [[ PubMed] Google Scholar]

4. de Almeida MA, Dos Santos E, da Cruz Cardoso J, da Fonseca DF, Noll CA, Silveira VR, et al. Yellow fever outbreak affecting [ Alouatta populations in southern Brazil (Rio Grande do Sul State), 2008-2009. Am J Primatol. 2012;74:68-76. 10.1002/ajp.21010 [ PubMed] [ CrossRef] Google Scholar]

8. World Health Organization. Background paper on yellow fever vaccine. In: Vaccine position papers. Geneva: SAGE Working Group; 2013. [ Google Scholar]

14. Nogueira RMR. Diagnóstico Laboratorial Específico e Diagnóstico Imunológico da Dengue [Specific laboratory diagnosis and immunodiagnosis of dengue]. Dengue Diagnóstico, Tratamento e Prevenção [Dengue diagnosis, treatment and prevention]. 2nd ed. Rio de Janeiro (Brazil): Editora Rubio Ltda.; 2008. p. 97-108. [ Google Scholar]

29. Poland JD, Calisher CH, Monath TP, Downs WG, Murphy K. Persistence of neutralizing antibody 30-35 years after immunization with 17D yellow fever vaccine. Bull World Health Organ. 1981;59:895-900. [[ PubMed] PMC free article] [ Google Scholar]

36. Estofolete CF, Nogueira ML. Is a dose of 17D vaccine in the current context of yellow fever enough? Braz J Microbiol. 2018;S1517-8382(18)30196-5. [ PMC free article] [ PubMed]

39. Staples JE, Bocchini JA Jr, Rubin L, Fischer M; Centers for Disease Control and Prevention (CDC). Yellow fever vaccine booster doses: recommendations of the Advisory Committee on Immunization Practices, 2015. MMWR Morb Mortal Wkly Rep. 2015;64:647-50. [[ PubMed] PMC free article] [ Google Scholar]

Articles from Emerging Infectious Diseases are provided here courtesy of Centers for Disease Control and Prevention

# data[].attributes.attachment_link.title
17DD Yellow Fever Revaccination and Heightened Long-Term Immunity in Populations of Disease-Endemic Areas, Brazil

# data[].attributes.attachment_link.description
We evaluated the duration of neutralizing antibodies and the status of 17DD vaccine-specific T- and B-cell memory following primary and revaccination regimens for yellow fever (YF) in Brazil. We observed progressive decline of plaque-reduction ...

# data[].attributes.headline
this article shows the benefits of the yellow fever vaccine

# data[].attributes.subject
this article shows the benefits of the yellow fever vaccine

# data[].attributes.attachment_link.title
Dynamics and turnover of memory CD8 T cell responses following yellow fever vaccination

# data[].attributes.attachment_link.description
Author summary Immunological memory, generated in response to infection or vaccination, may provide complete or partial protection from antigenically similar infections for the lifetime. Memory CD8 T cells are important players in protection from secondary viral infections but quantitative understan…

# data[].attributes.headline
According to this Article this is about a vaccine trial which was successful 

# data[].attributes.subject
According to this Article this is about a vaccine trial which was successful 

# data[].attributes.headline
According to this report Lassa fever declines when there is relative humidity and/or the temperature is between 28 C and 35 C

# data[].attributes.subject
According to this report Lassa fever declines when there is relative humidity and/or the temperature is between 28 C and 35 C

# data[].attributes.headline
on may 10th 2018 Nigeria announced that it had ended the Emergency phase of the outbreak due to a decline in cases

# data[].attributes.subject
on may 10th 2018 Nigeria announced that it had ended the Emergency phase of the outbreak due to a decline in cases

# data[].attributes.attachment_link.readable_text
Elsie A. Ilori, 1 Yuki Furuse, 1 Oladipupo B. Ipadeola, 1 Chioma C. Dan-Nwafor, 1 Anwar Abubakar, Oboma E. Womi-Eteng, Ephraim Ogbaini-Emovon, Sylvanus Okogbenin, Uche Unigwe, Emeka Ogah, Olufemi Ayodeji, Chukwuyem Abejegah, Ahmed A. Liasu, Emmanuel O. Musa, Solomon F. Woldetsadik, Clement L.P. Lasuba, Wondimagegnehu Alemu, Chikwe Ihekweazu, and Nigeria Lassa Fever National Response Team 2

The outbreak, which resulted in 423 confirmed cases and 106 deaths, was the largest recorded Lassa fever outbreak.

Keywords: Lassa fever, Lassa virus, Nigeria, outbreak, epidemiology, ribavirin, viruses

Abstract

Lassa fever (LF) is endemic to Nigeria, where the disease causes substantial rates of illness and death. In this article, we report an analysis of the epidemiologic and clinical aspects of the LF outbreak that occurred in Nigeria during January 1-May 6, 2018. A total of 1,893 cases were reported; 423 were laboratory-confirmed cases, among which 106 deaths were recorded (case-fatality rate 25.1%). Among all confirmed cases, 37 occurred in healthcare workers. The secondary attack rate among 5,001 contacts was 0.56%. Most (80.6%) confirmed cases were reported from 3 states (Edo, Ondo, and Ebonyi). Fatal outcomes were significantly associated with being elderly; no administration of ribavirin; and the presence of a cough, hemorrhaging, and unconsciousness. The findings in this study should lead to further LF research and provide guidance to those preparing to respond to future outbreaks.

Keywords: Lassa fever, Lassa virus, Nigeria, outbreak, epidemiology, ribavirin, viruses

Lassa fever (LF) is a febrile infectious disease caused by Lassa virus. The disease was first described in Nigeria in 1969 (1). Rodents, particularly Mastomys natalensis, are considered the natural hosts of the virus (2). The disease is mainly spread to humans through contamination with the urine or feces of infected rats (3,4). Human-to-human transmission can occur through contact with the body fluids of infected persons; therefore, healthcare workers (HCWs) are at high risk for infection when the standard precautions for infection prevention and control are inadequate (5,6). The incubation period of the disease is 3-21 days. The clinical manifestation of the disease is nonspecific and includes fever, fatigue, hemorrhaging, gastrointestinal symptoms (vomiting, diarrhea, and stomachache), respiratory symptoms (cough, chest pain, and dyspnea), and neurologic symptoms (disorientation, seizures, and unconsciousness) (3). The observed case-fatality rate (CFR) among patients hospitalized for severe LF is 15%-50% (7,8). However, ≈80% of infections are considered to cause mild or no symptoms in humans and are undiagnosed (8).

In Nigeria, laboratory-confirmed LF patients are treated in isolation units, according to national guidelines, to prevent community and nosocomial human-to-human infections (9). The country has 3 main LF treatment centers: the Irrua Specialist Teaching Hospital (Edo State), the Federal Medical Centre Owo (Ondo State), and the Federal Teaching Hospital Abakaliki (Ebonyi State) (10). Isolation units are also located in tertiary-care centers in other states. Ribavirin has been shown to reduce the CFR for LF (11); Nigeria national guidelines recommend that parenteral ribavirin be administered over a 10-day period for patients with confirmed LF (9).

Lassa fever is endemic to the West Africa countries of Benin, Ghana, Guinea, Liberia, Mali, Sierra Leone, and Nigeria (7); an estimated 300,000 LF cases occur each year in this region, resulting in ≈5,000 deaths (8). The annual peak of LF cases in Nigeria is observed in the dry season (December-April), and the number decreases around May (3). The increased likelihood of humans encountering Mastomys rodents and their excreta inside houses during the dry season (when these animals are seeking food) is thought to play a role in the seasonality of disease incidence (12). Transmission risk might be exacerbated by enhanced survival of the virus at decreased relative humidity (13).

During 2014-2016, around 100 laboratory-confirmed LF cases were reported annually in Nigeria (14,15). From the end of 2017 through May 2018, the country experienced its largest recorded LF outbreak. On January 22, 2018, the Nigeria Centre for Disease Control (NCDC) activated its Emergency Operations Centre to coordinate the outbreak response (16). During January 1-May 6, 2018, a total of 423 laboratory-confirmed cases were reported (17). On May 10, 2018, NCDC announced the end of the emergency phase of the outbreak because the LF case count had consistently declined in the preceding 6 weeks and had dropped below levels considered to be a national emergency, based on historical trends in LF incidence (10). Here we describe the epidemiologic and clinical aspects of this LF outbreak.

Methods

Ethics Considerations

This investigation was performed as a part of the LF public health response in Nigeria in 2018. The investigation was not considered to be research on human subjects, as per the US Department of Health and Human Services' Federal Policy for the Protection of Human Subjects (18).

Case Definition and Laboratory Confirmation

A suspected case of LF was defined as illness meeting 1 of the following criteria: 1) >1 signs/symptoms (e.g., malaise, fever, headache, sore throat, cough, nausea, vomiting, diarrhea, myalgia, central chest pain or retrosternal pain, and hearing loss) and a history of contact with excreta or urine of rodents; 2) >1 signs/symptoms and a history of contact with a person with probable or confirmed LF within 21 days of symptom onset; or 3) inexplicable bleeding or hemorrhaging (9). Probable LF cases were defined as any suspected case in a patient who died without the collection of a specimen for laboratory testing (9). Confirmed LF cases were defined as any suspected case with a laboratory confirmation (positive for IgM antibody, reverse transcription PCR [RT-PCR], or virus isolation) (9).

During the study period, blood samples from patients with suspected cases were sent to 1 of 4 laboratories: the Central Research Laboratory at College of Medicine of the University of Lagos-Lagos University Teaching Hospital (Lagos State), the Federal Teaching Hospital Abakaliki (Ebonyi State), the Institute of Lassa Fever Research and Control at the Irrua Specialist Teaching Hospital (Edo State), or the National Reference Laboratory (Federal Capital Territory). Laboratory confirmation was performed by using RT-PCR by means of the RealStar Lassa Virus RT-PCR Kit (Altona Diagnostics, https://www.altona-diagnostics.com), the LF diagnosis protocol developed by Nikisins et al. (19), or both. More than 95% of samples were tested by using both protocols to ensure greater sensitivity for the heterogeneous Lassa virus in Nigeria (20). A positive result in either or both of the protocols was regarded as positive for LF.

Contact Tracing

Persons who had contact with patients with confirmed LF were recorded and followed up daily for 21 days by Disease Surveillance and Notification Officers (DSNOs). If contacts had symptoms, blood samples were collected and tested for LF as described.

Data Collection and Report

All suspected LF cases were immediately reported to the DSNO for each Local Government Area and the State Epidemiologist for each state by using a surveillance reporting form developed for integrated disease surveillance and response in Nigeria (21). Samples were collected and tested for all suspected cases as long as the case-patient was alive (9). If the test was positive, detailed demographic (age, sex, and residential address), clinical (symptoms, outcome, and administration of ribavirin), and epidemiologic (occupation, onset date, and exposure history) information were collected by using a national case investigation form (CIF). All suspected, probable, and confirmed cases were line listed, and the information in the CIFs was submitted weekly by the state epidemiologists to NCDC. A summary of the figures was published weekly in a situation report (17); the compiled reports of the outbreak are provided in the Appendix. The projected population figures of each state were obtained from a report based on data from the National Population Commission of Nigeria and the National Bureau of Statistics (22). Anonymized clinical and epidemiologic data of case-patients are available by request, contingent on the recipient agreeing to appropriate guidelines for their use.

Statistical Analyses

We conducted binomial logistic regression analyses to determine the age- and sex-adjusted odds ratio (aOR) among survived and deceased patients with laboratory-confirmed LF. Likewise, we used aORs to compare the presence of each symptom and the administration of ribavirin among these cases, adjusted for age and sex. We conducted the Mantel-Haenszel test to see the statistical trend of CFR through the outbreak. We conducted chi-square tests to detect the statistical difference in exposure history between HCWs and non-HCWs.

We performed statistical tests using SPSS version 24 (IBM, https://www.ibm.com). We computed 95% CIs and p values to test statistical significance and adjusted p values by the Bonferroni correction for multiple comparisons. A p value of <0.05 was considered statistically significant.

Results

During the study period (January 1-May 6, 2018), a total of 1,893 suspected LF cases were reported, including 10 probable cases and 423 laboratory-confirmed cases. The laboratory-confirmed cases were reported from 20 states and the Federal Capital Territory. Most (80.6%) of the laboratory-confirmed cases were reported from the 3 states with a dedicated LF treatment center: Edo (178 cases), Ondo (99 cases), and Ebonyi (64 cases) ( Figure 1 , panel A). LF incidence was highest in these 3 states ( Table 1 ). Edo and Ondo also had the largest number of laboratory-negative suspected cases. The positive rate (i.e., the proportion of the number of laboratory-confirmed cases among all persons with suspected cases tested) was 22.5% (423/1,883) nationally, ranging from 3.4% (Lagos) to 70.0% (Delta). Among the 3 states with the highest number of cases, the positive rates were 16.5% (Edo), 31.6% (Ondo), and 69.6% (Ebonyi).

Table 1

State or territory

Population, ×1,000*

No. confirmed cases (deaths)

Case-fatality rate, %

No. confirmed cases/100,000 population

No. laboratory-negative suspected cases

No. probable cases

Positive rate, %

Taraba

3,067

19 (5)

26.3

0.620

41

0

31.7

CFR among the laboratory-confirmed cases was 25.1% (106/423 [95% CI 20.9%-29.2%]). Among the 3 most affected states, CFR was 14.6% (Edo), 24.2% (Ondo), and 23.4% (Ebonyi) ( Table 1 ). Among the 423 cases, a total of 414 CIFs with detailed information, including demographic information, onset date, symptoms, exposure history, and ribavirin administration, were collected (collection rate 97.9%). However, the data in some fields of the CIFs were incomplete. For example, the onset date was unknown in 2.4% (10/414) of cases, and symptom information was missing in 12.8% (53/414). An epidemic curve based on the onset date for laboratory-confirmed cases peaked at epidemiologic week 5 in 2018 ( Figure 1 , panel B). No statistically significant change was observed in the national CFR throughout the outbreak (p value for trend = 0.41).

We analyzed the age and sex distribution of the 414 patients with laboratory-confirmed cases ( Table 2 ). Median age was 32 years (interquartile range 20-44 years); 157 (37.9%) were female and 257 (62.1%) male. CFR was lowest in children <10 years of age (11.1%) and highest in adults >61 years of age (38.2%); the aOR of fatal outcomes in the elderly group was 4.9 (95% CI 1.5-15.6) compared with children <10 years of age. Adults 41-60 years of age also had statistically higher CFRs compared with children <10 years of age ( Table 2 ). CFR was higher for male patients (26.6%) than female patients (21.8%) but was not significantly different; the aOR of fatal outcomes in male patients compared with female patients was 1.3 (95% CI 0.81-2.1).

Table 2

Characteristic

No. girls and women (deaths)

No. boys and men (deaths)

Total no. (deaths)

Case-fatality rate, %

aOR (95% CI)

p value

Age group, y

>61

12 (5)

22 (8)

34 (13)

38.2

4.9 (1.5-15.6)

0.0074

The most common signs and symptoms among patients with laboratory-confirmed LF were fever (96.4%, 348/361), headache (58.7%, 210/358), vomiting (49.4%, 177/358), fatigue (43.3%, 155/358), and abdominal pain (40.2%, 144/358) ( Table 3 ). Hemorrhaging was observed in 17.0% (61/358) of these patients. Cough (p = 0.0050), hemorrhaging (p<0.001), and unconsciousness (p = 0.0018) were significantly more prevalent in fatal than nonfatal cases.

Table 3

Sign/symptom

Cases, % (no. positive/no. with data available)

aOR (95% CI)

p value

All cases

Fatal cases

Nonfatal cases

During the 3 weeks before symptom onset, 17.7% (62/350) of case-patients reported contact with patients who had known suspected or confirmed LF, 17.0% (56/330) reported contact with rodents or their urine or feces, and 2.9% (9/315) reported attendance at a burial ceremony ( Figure 2 , panel A). During the study period, 5,012 persons were determined to have had contact with confirmed case-patients; follow-up was conducted for 5,001 of them. During the 21-day follow-up period, 81 contacts experienced onset of symptoms, and 28 were found to have laboratory-confirmed LF. The positive rate among symptomatic contacts was 34.6% (28/81), and the secondary attack rate was 0.56% (28/5,001; 95% CI 0.35%-0.77%).

During the study period, 37 HCWs were infected, resulting in 8 deaths (CFR 21.6%). The incidence of HCW infections was distributed throughout the outbreak period ( Figure 1 , panel B). A significantly high proportion of HCWs (55.9%, 19/34) reported contact with patients with known suspected or confirmed LF compared with non-HCWs (p<0.001) ( Figure 2 , panel A).

Ribavirin was administered to 94.1% (334/355) of the patients with laboratory-confirmed cases. CFR for patients who received ribavirin was 20.7% (69/334), compared with 71.4% (15/21) for patients who did not receive ribavirin ( Figure 2 , panel B). We also analyzed the subset of patients who survived >7 days after symptom onset to account for the possible effect of the difference in clinical conditions. We further divided the patients who received ribavirin into 2 groups: patients who received the drug within 7 days after symptom onset and patients who received the drug after that point. Among case-patients who survived >7 days, CFR was significantly higher for patients who did not receive any ribavirin (66.7%, 12/18) compared with patients who received the drug (p<0.01), whether receipt of the drug occurred within 7 days of symptom onset or >7 days after onset ( Figure 2 , panel C). CFR was lower among patients who received the drug within 7 days of symptom onset (12.5%, 15/120) compared with patients who received the drug after that point (20.1%, 38/189), although this difference was not significant (p = 0.095). Because this reduction in CFR might have been attributable to not only ribavirin but also the other supportive treatments provided, the time between symptom onset date and the patient's arrival at a health facility was included in addition to age and administration of ribavirin as a covariable in the binomial logistic regression analysis for fatal outcomes. Although absence of ribavirin administration (p<0.001) and advanced age (p = 0.025) remained significant factors in fatal outcomes, delay in visiting health facility did not (p = 0.19).

Discussion

We describe the epidemiologic and clinical features of the LF outbreak in Nigeria during January 1-May 6, 2018. A total of 423 laboratory-confirmed cases were reported during the study period. Most of the laboratory-confirmed cases were reported from the 3 states (Edo, Ondo, and Ebonyi) where dedicated LF treatment centers are located; disease incidence was also highest in these areas. However, the positive rate among suspected cases was not especially high for these 3 states. In addition to the high prevalence of LF in the areas, HCWs in these 3 states had a high suspicion of LF in patients with high-grade fevers, which might have led to increased testing. Conversely, suspicion of LF might be low in some areas other than these 3 states. Lack of LF expertise and diagnostic capacity in these other areas might have discouraged active detection of LF patients, leading to underreporting of the disease. Also, LF incidence and prevalence might actually be lower in some areas for epidemiologic and environmental reasons, such as low prevalence of the virus in rodents or good hygiene practices that help reduce contact between humans and rodents. To clarify different LF prevalence by areas, sensitization and strengthening of the surveillance system to detect suspected cases and obtain test samples are required, particularly in states other than Edo, Ondo, and Ebonyi. In addition, seroprevalence surveys and ecologic studies of LF in humans and rodents should give further insight on the actual burden of the disease.

We used 2 protocols for molecular diagnosis of LF to cover the heterogeneous Lassa virus in Nigeria (20), and we tested >95% of the samples by using both protocols during the outbreak. Further study is needed to reveal sensitivity and specificity of each protocol in this country. That information would also give us further insight on genetic diversity of the virus in natural hosts in the country.

Although NCDC situation reports showed the peak of the outbreak at week 7 (17), our study found that the outbreak peaked at week 5. This difference occurred because the epidemic curve from the situation reports was based on the reporting date, whereas the epidemic curve in our study was based on symptom onset date. The difference can be explained by the time lag between symptom onset to health facility presentation and subsequent diagnosis and reporting. The median of the time lag between symptom onset to suspicion of LF was 7 days (interquartile range 4-11 days), and several additional days were required for a health facility to report through the DSNO for each Local Government Area and for the state epidemiologist in each state to report NCDC. Further strengthening of the surveillance system is required to shorten this time gap.

Girls and women accounted for a lower proportion of the laboratory-confirmed LF cases than boys and men (37.9% vs. 62.1%). Past studies have shown no or little difference in LF incidence between male and female patients (23-25). It is unclear whether the difference in this study came because men and boys were at higher risk for infection or more susceptible to the disease than women and girls or because ascertainment of cases in women and girls was low during this outbreak.

CFR among laboratory-confirmed cases during the study period was 25.1% and did not change substantially throughout the outbreak. CFR can reach 50% in hospitalized patients during epidemics (8), whereas the observed CFR among patients hospitalized with severe LF is 15% (7). CFR in this outbreak was especially high among elderly patients. Such difference in CFR among age groups was not observed in previous studies (26,27). The large number of cases in this outbreak might have increased the statistical power to detect the difference.

Gastroenteric symptoms, including vomiting, abdominal pain, and anorexia, were frequently observed among patients with laboratory-confirmed cases (>30%), whereas hemorrhaging was only observed in 17.0% of case-patients ( Table 3 ). This observation is consistent with previous reports (28,29). Because some symptoms, such as cough, hemorrhaging, and unconsciousness, were more frequently observed for fatal cases than nonfatal cases, these symptoms might be predictors for fatal outcomes. Patients with such symptoms would require more attention to achieve better clinical outcomes.

Lassa virus is primarily transmitted to humans from rodents. The virus is also occasionally transmitted through the body fluids of infected persons (3). In this outbreak, ≈17% of patients reported a history of contact with rodents. A similar percentage reported contact with patients with suspected or confirmed LF. Because this information was obtained by patient interview, recall bias might have influenced the accuracy of exposure history. Although the secondary attack rate was as low as 0.56%, the positive rate for LF among person with suspected cases was higher for those who had contact with confirmed case-patients than that in the general population (34.6% vs. 22.5%). Contact tracing did not account for 34 LF patients, although they had reported contact with other confirmed or suspected case-patients. Some case-patients might have been targeted in the contact tracing whereas others were not because they reported contact with persons with suspected (but not confirmed) cases. Strengthening and expanding contact tracing is required but posed a challenge in resource-limited settings during the outbreak. A high rate of contact with suspected or confirmed LF case-patients among HCWs suggests the possibility of nosocomial infections, although we could not rule out another source of infection, such as rodents (30). Nevertheless, good infection prevention and control practices and readily available personal protective equipment are important to protect HCWs from infection with the virus (5,6).

The findings in this study support the effectiveness of ribavirin in reducing mortality rates from LF. CFR was lowest among patients who received treatment with ribavirin. Patients in severe conditions might have not received ribavirin because they died before reaching healthcare facilities where treatment was available; that is, the difference in CFRs between patients who did or did not receive ribavirin might be attributable not only to the ribavirin treatment but also to the patient's clinical condition before ribavirin administration (6,28). To explore this issue further, we analyzed a subset of patients who survived >7 days after symptom onset. The highest CFR was still observed among patients who did not receive ribavirin; CFR was lower even if provision of ribavirin was deferred to >7 days after symptom onset. Furthermore, the early commencement of ribavirin treatment reduced CFR compared with deferred administration of the drug, although this difference was not statistically significant. Therefore, the reduction in CFR was more likely attributable to ribavirin than to the other supportive treatments provided. These findings support Nigeria's national guidelines for clinical management of the disease, which advise that patient outcomes are more favorable when ribavirin treatment is commenced earlier (9).

This study has several limitations. Although we collected CIFs from 414 of the 423 patients with laboratory-confirmed cases, the data in some fields were incomplete. Availability of CIFs for patients with suspected cases whose laboratory tests were negative would have enabled us to conduct a case-control study to better determine the risk factors for the disease; however, collection of this information for 1,460 laboratory-negative suspected cases would have been burdensome to outbreak response staff. Also, our study did not provide any insights into why the 2018 LF outbreak was larger than those in previous years. Studies using viral genomic data have suggested that the outbreak was not caused by ongoing human-to-human transmission from a single source but by multiple environmental sources (31). Better surveillance through increased availability of laboratory testing of suspected cases might have played a role in the larger number of laboratory-confirmed cases during this outbreak. Localized clusters, which were not investigated as part of this study, might have contributed to the inflation of case numbers in some areas.

LF is endemic in Nigeria. The LF surveillance data from Nigeria, even with their limitations, are arguably the best longitudinal data collected on LF globally. Although these data mostly relate to the 2018 outbreak, data collection has continued, and all aspects of surveillance are being continuously improved (e.g., educating HCWs on the case definition, increasing ease and efficiency of the transport of samples, and increasing capacity and quality of diagnosis). In this study, we described the epidemiologic and clinical features of the largest recorded LF outbreak, which had a high CFR. The investigation also revealed several risk factors for fatal outcomes and the contribution of early treatment in reduction of CFR. These findings should lead to further investigation of the disease. Our study also highlights the need for specific incidence and seroprevalence surveys to determine the actual burden of disease in Nigeria and West Africa. Although the emergency phase of this outbreak was declared over on May 10, 2018, a small number of cases in some areas continued to be reported thereafter (10). Ceaseless efforts to improve risk communication, surveillance, laboratory diagnosis, clinical management, infection prevention and control practices, logistics, and coordination could mitigate the impact of future outbreaks.

Appendix:
Compilation of situation reports issued by the Nigeria Centre for Disease Control during a Lassa fever outbreak, Nigeria, January 1-May 6, 2018.

Acknowledgments

We thank all the staff working at the national and international organizations who fought against this outbreak, including personnel at the healthcare facilities, LF diagnostic laboratories, Nigeria Centre for Disease Control, World Health Organization, Alliance for International Medical Action, Médecins Sans Frontières, Robert Koch Institute, African Field Epidemiology Network, Public Health England, US Centers for Disease Control and Prevention, and numerous other partners. We also express our sincerest condolences to the family and friends of those who died during the outbreak.

Biography

Ms. Ilori is an incident manager at the Nigeria Centre for Disease Control in Abuja, Nigeria. Her primary research interests include outbreak preparedness and response.

Footnotes

Suggested citation for this article: Ilori EA, Furuse Y, Ipadeola OB, Dan-Nwafor CC, Abubakar A, Womi-Eteng OE, et al. Epidemiologic and clinical features of Lassa fever outbreak in Nigeria, January 1-May 6, 2018. Emerg Infect Dis. 2019 Jun [ date cited]. https://doi.org/10.3201/eid2506.181035

¹ These first authors contributed equally to this article.

² Other members of the Nigeria Lassa Fever National Response Team are listed at the end of this article.

References

2. Monath TP, Newhouse VF, Kemp GE, Setzer HW, Cacciapuoti A. Lassa virus isolation from [ Mastomys natalensis rodents during an epidemic in Sierra Leone. Science. 1974;185:263-5. 10.1126/science.185.4147.263 [ PubMed] [ CrossRef] Google Scholar]

4. Keenlyside RA, McCormick JB, Webb PA, Smith E, Elliott L, Johnson KM. Case-control study of [ Mastomys natalensis and humans in Lassa virus-infected households in Sierra Leone. Am J Trop Med Hyg. 1983;32:829-37. 10.4269/ajtmh.1983.32.829 [ PubMed] [ CrossRef] Google Scholar]

13. Fichet-Calvet E, Lecompte E, Koivogui L, Soropogui B, Doré A, Kourouma F, et al. Fluctuation of abundance and Lassa virus prevalence in [ Mastomys natalensis in Guinea, West Africa. Vector Borne Zoonotic Dis. 2007;7:119-28. 10.1089/vbz.2006.0520 [ PubMed] [ CrossRef] Google Scholar]

26. Yinka-Ogunleye A, Ipadeola O, Saleh M, Ilori E, Dan-Nwafor C, Nwachukwu W, et al. 2016/2017 Lassa fever outbreak in Nigeria: age and sex have no effect on treatment outcome. Pan Afr Med J. 2018;8:3. [ Google Scholar]

Articles from Emerging Infectious Diseases are provided here courtesy of Centers for Disease Control and Prevention

# data[].attributes.attachment_link.title
Epidemiologic and Clinical Features of Lassa Fever Outbreak in Nigeria, January 1-May 6, 2018

# data[].attributes.attachment_link.description
The outbreak, which resulted in 423 confirmed cases and 106 deaths, was the largest recorded Lassa fever outbreak.Lassa fever (LF) is endemic to Nigeria, where the disease causes substantial rates of illness and death. In this article, we report an analysis ...

# data[].attributes.headline
a mathematical model which can help the patterns of a LF epidemic

# data[].attributes.subject
a mathematical model which can help the patterns of a LF epidemic

# data[].attributes.attachment_link.title
Modeling, analyzing and simulating the dynamics of Lassa fever in Nigeria - Journal of the Egyptian Mathematical Society

# data[].attributes.attachment_link.description
Lassa fever is an infectious and zoonotic disease with incidence ranging between a hundred to three hundred thousand cases, with approximately five thousand deaths reported yearly in West Africa. This disease has become endemic in the Lassa belt of Sub-Saharan Africa, thus increasing the health burden in these regions including Nigeria.

# data[].attributes.attachment_link.readable_text
2022, International Journal of Infectious Diseases

Similar to ribavirin, favipiravir should be avoided in pregnant and breastfeeding women because of teratogenicity and embryotoxicity in animal models, along with excretion in breast milk (Nagata et al., 2015). Convalescent plasma has shown some benefit in improving survival from LF in animal models, including NHPs, but success was dependent on the dose used and the match of neutralizing antibodies to the LASV strain causing infection (Jahrling et al., 1985a; Jahrling P B and Peters C J, 1984; Jahrling et al., 1984). Neutralizing antibodies against LASV often take several months to develop after natural infection, indicating that recently recovered individuals may not be suitable for convalescent plasma donation (Jahrling et al., 1985a).

This summary on Lassa virus (LASV) infection and Lassa fever disease (LF) was developed from a clinical perspective to provide clinicians with a condensed, accessible understanding of the current literature. The information provided highlights pathogenesis, clinical features, and diagnostics emphasizing therapies and vaccines that have demonstrated potential value for use in clinical or research environments.

We conducted an integrative literature review on the clinical and pathological features, vaccines, and treatments for LASV infection, focusing on recent studies and in vivo evidence from humans and/or non-human primates (NHPs), when available.

Two antiviral medications with potential benefit for the treatment of LASV infection and 1 for post-exposure prophylaxis were identified, although a larger number of therapeutic candidates are currently being evaluated. Multiple vaccine platforms are in pre-clinical development for LASV prevention, but data from human clinical trials are not yet available.

We provide succinct summaries of medical countermeasures against LASV to give the busy clinician a rapid reference. Although there are no approved drugs or vaccines for LF, we provide condensed information from a literature review for measures that can be taken when faced with a suspected infection, including investigational treatment options and hospital engineering controls.

2019, Journal of Virological Methods

LASV antigen can be detected by ELISA using LASV-speciﬁc antibodies (Bausch et al., 2000, Niklasson et al., 1984, Jahrling et al., 1985). However, this method has a relatively low sensitivity (ranging from 102-105 PFU/ml) (Jahrling et al., 1985). This peculiarity limits its usage, particularly at an early stage of LFD because of the low viral load in the biological fluids of patients.

Lassa fever is a severe viral hemorrhagic illness caused by Lassa virus. Based on estimates, the number of LASV infections ranges from 300,000 to 500,000 cases in endemic areas with a fatality rate of 1%. Development of fast and sensitive tools for the control and prevention of Lassa virus infection as well as for clinical diagnostics of Lassa fever are crucial.

Here we reported development and evaluation of a one-step quantitative RT-qPCR assay for the Lassa virus detection - LASV-Fl. This assay is suitable for the detection of lineages I-IV of Lassa virus. The limit of detection of the assay ranged from 10 ³ copies/ml to 10 ⁵ copies/ml and has 96.4% diagnostic sensitivity, whereas analytical and diagnostic specificities both were 100%. Serum, whole blood and tissue are suitable for use with the assay. The assay contains all the necessary components to perform the analysis, including an armored positive control (ARC+) and an armored internal control (IC). The study was done during the mission of specialized anti-epidemic team of the Russian Federation (SAET) in the Republic of Guinea in 2015-2018.

Based on sequencing data, LASV-specific assay was developed using synthetic MS2-phage-based armored RNA particles, RNA from Lassa virus strain Josiah, and further, evaluated in field conditions using samples from patients and Mastomys natalensis rodents.

2011, Tropical Infectious Diseases: Principles, Pathogens and Practice

2011, Tropical Infectious Diseases

2006, Journal of Clinical Virology

Anti-Lassa antibodies are detected by indirect immunofluorescence assay (IFA) or by enzyme-immunoassay (ELISA). Both methods have problems to detect low amounts of specific antibodies.

We report here highly sensitive and specific reverse ELISAs to detect Lassa virus IgG and IgM antibodies. Due to the reverse techniques, serum samples could be applied at dilutions of 1:10 without increasing non-specific background reactions.

For IgM antibody detection microtiter plates were coated with anti-IgM antibodies and for IgG antibody detection with rheumatoid factor (RF) (Sachers M, Emmerich P, Mohr H, Schmitz H. Simple detection of antibodies to different viruses using rheumatoid factor and enzyme-labelled antigen (ELA). J Virol Methods 1985;10:99-110). In both assays a tissue culture antigen was used in combination with a labeled anti-Lassa monoclonal antibody (Hufert FT, Ludke W, Schmitz H. Epitope mapping of the Lassa virus nucleoprotein using monoclonal anti-nucleocapsid antibodies. Arch Virol 1989;106(3-4):201-12).

The reverse ELISA turned out to detect virus-specific IgG and IgM antibody in all 20 samples of West African patients collected 2-8 weeks after onset of Lassa fever. Moreover, both IFA and reverse ELISA found IgG antibodies in 53 out of 643 samples of healthy West Africans (sensitivity of 100%). Six of the 643 samples were positive by reverse IgG ELISA only. Thus, the specificity compared to IIF was 99.0%, but it may be even higher, because compared to IFA the IgG ELISA was clearly more sensitive in detecting low antibody titers.

In Ghana 3% seropositives were found by IFA, but 4% by the reverse ELISA. The reverse ELISAs can be performed with high sensitivity and specificity under field conditions in Africa.

View all citing articles on Scopus

# data[].attributes.attachment_link.title
EARLY DIAGNOSIS OF HUMAN LASSA FEVER BY ELISA DETECTION OF ANTIGEN AND ANTIBODY

# data[].attributes.attachment_link.description
Sequential serum samples, beginning on the day of hospital admission, from three patients with Lassa fever were tested for the presence of Lassa-virus...

# data[].attributes.headline
the effectivity and cost measures of LF countermeasures

# data[].attributes.subject
the effectivity and cost measures of LF countermeasures

# data[].attributes.attachment_link.title
Mathematical analysis of a Lassa fever model in Nigeria: optimal control and cost-efficacy - International Journal of Dynamics and Control

# data[].attributes.attachment_link.description
In this study, we extend an existing Lassa fever model by incorporating four time-dependent control functions which are identified as a preventive strategy, treatment care, and rodent reduction. The six compartmental nonlinear optimal control problem is developed and analyzed. We employed Pontryagin…

# data[].attributes.attachment_link.readable_text
Abstract

Lassa fever (LF), a disease endemic to West Africa caused by infection with Lassa virus (family Arenaviridae), causes an estimated 100000-300000 human cases annually [ 1]. Most cases are caused by infection from ingestion, inhalation, or direct contact with the environmental reservoir, Mastomys natalensis rodents; human-to-human spread is uncommon [ 2, 3]. Mortality among hospitalized patients ranges from 15% to 20% [ 1]. Treatment with ribavirin during the first 6 days of illness decreases mortality rates to 5% [ 4]. Favipiravir, a novel antiviral agent with broad-spectrum activity against RNA viruses, decreases levels of Lassa viremia and increases survival in animal models [ 5, 6]. We describe 2 cases of LF acquired via secondary spread from the same index case who survived after treatment with ribavirin and favipiravir.

The index patient was a medical worker employed at a missionary institution in Oti District, Togo. The index patient was medically evacuated to Cologne, Germany, after 2 weeks of fevers, but died approximately 10 hours after admission despite intensive care. LF was diagnosed postmortem by reverse-transcription polymerase chain reaction (RT-PCR) from a liver biopsy (Gerd Fätkenheuer, submitted).

CLINICAL COURSE AND TREATMENT

Patient E

Patient E is a previously healthy 33-year-old man who provided 7 days of nursing care to the index patient in Togo and developed fevers 9 days after his last contact with the index patient. A thick blood smear was positive for malaria and he received 3 days of artemether/lumefantrine. LF was suspected when his fevers persisted and he developed sore throat, retro-orbital headache, intermittent diminished hearing, diarrhea, malaise, and generalized weakness. Laboratory testing in Togo revealed leukopenia with a nadir of 1 × 10 ⁹ cells/L, thrombocytopenia of 50 × 10 ⁹ cells/L, mild transaminitis (aspartate aminotransferase 131 U/L, alanine aminotransferase 77 U/L), and creatinine of 2.3 mg/dL. Patient E received ribavirin 2 g orally once followed by 1 g every 8 hours as intravenous ribavirin was not immediately available. He was medically evacuated to the Serious Communicable Diseases Unit at Emory University Hospital in Atlanta, Georgia, on day 8. An admission blood specimen was positive for Lassa virus infection by conventional RT-PCR and retrospective quantitative (q) RT-PCR (using laboratory-developed primers LV-Tog2 F (TCACAACTCATCGCCTCATAC) and LV-Tog2 R (AATCTGTATGACCACGCACTC) and of probe LV-Tog2 P (56-FAM/TCCATCTGT/ZEN/CCATCCCAAACTTCAACC/3IABkFQ) with a cycle threshold value (Ct) of 23.4 ( Figure 1A). On admission, intravenous ribavirin 16 mg/kg every 6 hours and oral favipiravir 2000 mg once followed by 1000 mg every 12 hours were administered after emergency investigational new drug approval was obtained from the US Food and Drug Administration. On day 12, favipiravir was discontinued and ribavirin was decreased to 8 mg/kg every 8 hours as per protocol from the manufacturer. He completed his ribavirin regimen on day 15.

Initial vital signs included temperature 37.1°C, heart rate 50 beats/minute, respiratory rate 24 breaths/minute, and blood pressure 140/91 mm Hg. Examination revealed a thin, ill-appearing male with mild conjunctival pallor, oral thrush, a soft precordial systolic murmur, and mild bladder distension with suprapubic tenderness. Tests for Ebola virus, human immunodeficiency virus (HIV), and malaria were negative. The patient had fevers to a maximum of 40.4°C, which resolved on day 12, and intermittent headaches. He developed nausea, which he attributed to favipiravir. His appetite improved and his sore throat resolved on day 14. He developed painful diffuse lymphadenopathy on day 14, epididymitis between days 19 and 21 (which later correlated to semen culture positivity for Lassa), and night sweats on day 22. He was discharged on day 25. His hearing slowly improved, although subjective right-sided decreased hearing persisted for 4 months despite normal audiological testing.

Patient F

Patient F is a mortician who prepared the index patient's corpse for repatriation 11 days after the patient's death. He was ill with mild upper respiratory tract symptoms prior to contact with the deceased patient, making the first day of symptoms due to LF difficult to clarify. Qualitative PCR for LF was ordered by the health authorities 2 days after contact with the corpse due to temperatures between 37.5°C and 38.0°C (illness day 1) with an inconclusive result (1 faint band in 1 of 3 parallel assays). His condition deteriorated over 3 days with fevers to 38.2°C, pronounced generalized weakness, myalgia, arthralgias, headache, and nausea. Repeat qRT-PCR testing 4 days later was positive (Ct 31.0; Figure 1B), and he was transferred to the Frankfurt University High-Level Isolation unit.

Initial vital signs were temperature 38.9°C, blood pressure 145/75 mm Hg, and heart rate 77 beats/minute. Examination revealed dry mucous membranes, pharyngeal erythema with patchy white exudates, conjunctival injection, a faint macular rash on the epigastrium, and slowed rapport and speech. Diagnostic testing was negative for HIV, hepatitis B and C, influenza, and group A Streptococcus pharyngitis. His fevers resolved on day 7 and his myalgias and arthralgias gradually improved. He developed nonproductive coughing without chest radiographic abnormalities. His pharyngitis worsened during the first week and resolved during week 3. He was discharged on day 39 after his symptoms resolved.

He received 1000 mg of intravenous ribavirin followed by 1000 mg every 6 hours for 4 days followed by 500 mg intravenously every 8 hours for another 6 days per the World Health Organization protocol [ 7]. Oral favipiravir 2000-mg loading dose was administered followed by 1200 mg twice daily for 4 days. He developed atrial fibrillation 2.5 hours after initiation of favipiravir and 10.5 hours after ribavirin. Therapeutic heparin treatment was administered. Four hours after discontinuation of favipiravir and 14 hours after ribavirin dose reduction, he converted to sinus rhythm.

LABORATORY STUDIES

For both patients, clinical laboratory support was provided as described in Supplementary Data 1 and initial molecular testing was performed as previously described [ 8, 9]. Patient-specific qRT-PCR assays were developed, designated LV-Tog2 for patient E and LV-Tog1 for patient F (Supplementary Data 1). Pertinent laboratory results for both patients are shown in Figures 2A and 2B, respectively. Lassa immunoglobulin M (IgM) antibodies were detected on day 13 in patient E and immunoglobulin G (IgG) antibodies were detected on day 46 in patient E and day 38 in patient F. IgM was not tested in patient F.

Cell culture by Vero cell culture for Lassa virus was positive from whole blood specimens from patient E on days 8, 9, and 11 and patient F on day 5. Lassa virus nucleic acid was detected in blood samples by qRT-PCR in patient E through day 59 ( Figure 1A) with 1 exception and in patient F through day 28 ( Figure 1B). Patient E had 6 positive RT-PCR assays from urine samples but virus was not isolated from urine by cell culture. Urine testing on patient F yielded 1 positive RT-PCR on day 22. Saliva RT-PCRs were intermittently positive from patient E through day 46 and pharyngeal swab RT-PCRs from patient F were positive through day 28. Three semen samples from patient E (days 15, 20, and 48) and 4 samples from patient F (days 35, 46, 82, and 103) all had detectable Lassa virus RNA by qPCR. Culture yielded Lassa virus in 1 semen sample from patient E on day 20.

DISCUSSION

We report 2 epidemiologically linked patients with LF secondarily acquired from the same index case treated with favipiravir and ribavirin and who made full recovery. Ribavirin, the only previously described anti-LF therapy associated with reduced mortality in humans, was initiated on day 6 (patient E) and day 5 (patient F). Favipiravir is licensed for influenza treatment in Japan and may reduce mortality in other RNA virus infections, including Ebola virus disease [ 10]. Favipiravir was started on day 8 (patient E) and day 5 (patient F) but was discontinued after 5 days of treatment in both patients due to nausea and worsening transaminitis. Transaminitis has been described in LF in both untreated and ribavirin-treated patients [ 4, 11], so it was unclear if the patients' transaminitis was due to their underlying disease vs a drug effect. In both patients, aminotransferase levels declined shortly after discontinuation of favipiravir and decreases in the ribavirin dose. Both patients also showed a decline in hemoglobin ( Figure 2A and 2B) with an elevated lactate dehydrogenase and bilirubin in patient F, which may partially be explained by a toxic effect of ribavirin and metamizole. Viral copy number values declined on therapy, although given the lack of comparator patients or sufficient historical data using PCR methods, it remains unclear whether this was due to an effect of combined antiviral therapy or the normal course of Lassa viremia. The utility of combined ribavirin and favipiravir treatment for LF should be evaluated in further clinical studies.

Viral nucleic acid was detected for a prolonged period in both patients in blood (days 27 and 62) and semen (days 48 and 103). The long-term persistence of viable virus in convalescent patients in immune-privileged compartments has been described for Ebola virus in ocular and seminal fluid [ 12-14]. Arenavirus escape attributed to evasion of immune system antigen presentation has been shown to contribute to long-term virus excretion in natural and dead-end hosts [ 15]. Well-documented longitudinal molecular detection of Lassa virus in semen has not been previously published, raising the possibility that Lassa virus could be sexually transmitted.

Supplementary Data

Supplementary materials are available at Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Emory Serious Communicable Diseases Unit

SCD Unit Coordinator: Sonia Bell. Administration: Catherine Maloney, Pam Cosper, Nancye Feistritzer, John Lewin, Bryce Gartland, Ira Horowitz, David Pugh, Chad Ritenour, Jerry Lewis, David Hatcher, Linda Scott-Harris, Lynne Ometer, Kip Hardy, Jeff Broughton, Robert Jackson, Samantha Thomas. Nursing: Toni Ash, Christopher Barnes, Jamie Breedlove, Bob Bridgman, Lane Darragh, Tracey Daye, Dustin Hillis, Crystal Johnson, Julie-Ann Johnson, Danni LaFond, Courtney Lyons, Josia Mamora, Anna McCord, Samantha McDaniel, Haley Morgan, Jill Morgan, Alexander Sanchez, Marissa Simon, Jason Slabach, Kevin Tirado, Sally Watkins, Terrica Wilson, Ken Logan. Emory Medical Labs: Juli Buchanan, Eileen Burd, John Cardella, Brenda Eaves, Crystal Evans, Charles Hill, Doris Igwe, Karen Jenkins, Maureen Lindsey, Jordan Magee, Stacy McCarthy, Randall Powers, James Ritchie

Pharmacy: Jan Pack, Susan Rogers. Emory Health and Safety: Patricia Olinger, Kalpana Rengarajan, Scott Thomaston, Esmeralda Meyer. Occupational Health: Emily Beck, Paula Desroches, Cynthia Hall, Celeste Walker Infection Prevention: Esther Baker, Betsy Hackman. Environmental Services: Jeff Broughton, Robert Jackson, Jerry Lewis

Pastoral Care: Robin Brown-Haithco, Faith Richardson. Emory Faculty and Staff Assistance: Art Krasilovsky, Clevevoya Jordan, Sue Matthews, Marilyn Hazzard Lineberger, Paula G. Gomes. Supplies/Logistics: Gentrice McGee, Porcia Jones. EUH Security: Linda Scott-Harris, James Cain, Roderick Davis, Tyrone Johnson, Tyrone Pickett, Anthony Shaw, Tenina Truesdale. Emergency Medicine: Alex Isakov, Sam Shartar, Wade Miles, Aaron Jamison, John Arevalo, Gail Stallings. Communications/Media Relations: Janet Christenbury, Vince Dollard, Melanie De Gennaro, Holly Korschun. Nutrition: Tom Ziegler, Daniel P. Griffith, Nisha Dave

Notes

Author contributions. T. W., G. K., and C. S. K.: Treated the patient, collected data, wrote the manuscript. S. B. and M. E.: Performed virological analysis, assisted in writing the manuscript, provided discussion of results. T. S.: Performed virological analysis, analyzed data. P. L.: Treated the patient, collected data. G. S.: Treated the patient. C. S.: Treated the patient, provided discussion of the manuscript. H. R.: Assisted in sample and data collection, provided discussion of the manuscript. V. N. R.: Collected data, wrote the manuscript. A. M.: Collected data, edited the manuscript. B. S. R., J. B. V., A. K. M., G. M. L., S. V., and K. F.: Treated the patient, collected data, edited the manuscript. S. B., J. D. K., P. E. R., U. S., S. N.: Performed virological analysis, analyzed data, edited the manuscript. K. P.: Performed virological analysis, analyzed data. A. M.: Performed virological analysis, assisted in writing the manuscript, provided discussion of results.

Acknowledgments. We thank Sarah Katharina Fehling and Svenja Wolff for virus isolation work and Michael Schmidt and Gotthard Ludwig for technical support in the biosafety level 4 facility at the Philipps University of Marburg. We express our gratitude to the nursing staff of the intensive care pool and the Infectious Diseases Department, University Hospital Frankfurt. We thank Debi Cannon, Cheng-Feng Chiang, William Davis, Aridth Gibbons, James Graziano, Mary Jenks, Maria Morales-Betouille and Nishi Patel for their assistance in performance of the laboratory testing at the Centers for Disease Control and Prevention (CDC). We are appreciative of the support of the US Food and Drug Administration, Valeant, and Medivector for the provision of the therapeutics for these patients. We thank the Investigational Drug Service at Emory University and the Emory University Institutional Review Board.

Disclaimer. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the CDC.

Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

References

Author notes

# data[].attributes.attachment_link.title
Favipiravir and Ribavirin Treatment of Epidemiologically Linked Cases of Lassa Fever

# data[].attributes.attachment_link.description
Abstract. Two patients with Lassa fever are described who are the first human cases treated with a combination of ribavirin and favipiravir. Both patients survi

# data[].attributes.headline
According to this report the replication of LF viruses was suppressed

# data[].attributes.subject
According to this report the replication of LF viruses was suppressed

# data[].attributes.attachment_link.readable_text
Abstract

Lassa fever (LF) is a viral hemorrhagic fever caused by Lassa virus (LASV), an arenavirus endemic in West Africa. Standard of care for LF is the nucleoside analogue ribavirin [ 1]. However, because of the small therapeutic window [ 1] and the fact that still one third of patients with LF die despite ribavirin treatment [ 2], there is a need for more-effective drug therapies against LF. A promising candidate is the broad-spectrum antiviral drug favipiravir (Toyama Chemical) [ 3], which is approved for emerging influenza virus in Japan. Besides influenza virus, favipiravir shows potent antiviral activity in vitro and in vivo against a wide range of negative-strand RNA viruses, including arenaviruses [ 3, 4]. We have tested the efficacy of favipiravir alone and in combination with ribavirin in a novel, chimeric mouse model, which is based on lethally irradiated type I interferon receptor knockout () mice transplanted with wild-type bone marrow progenitor cells (termed " mice"). mice feature a fully functional immune system and are susceptible to wild-type LASV infection (Oestereich et al, unpublished data).

METHODS

Antiviral and Toxicity Testing In Vitro

Vero E6 cells (4 × 10 ⁴ cells per well in a 24-well plate) were inoculated with LASV Ba366 at a multiplicity of infection of 0.01, and compound was added 1 hour after infection. Concentration in cell culture supernatant of infectious virus particles was measured 3 days after infection, using an immunofocus assay. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) method. The concentrations that reduced the virus titer by 50% (IC ₅₀), 90% (IC ₉₀), and 99% were calculated from dose-response curves, using Prism GraphPad 6.0 (GraphPad Software). Drug interaction was tested in a 8 × 8 matrix; both favipiravir and ribavirin were tested at concentration of 0, IC ₉₀/4, IC ₉₀/2, IC ₉₀/1.5, IC ₉₀, IC ₉₀ × 1.5, IC ₉₀ × 2, and IC ₉₀ × 4 in all possible combinations. The data were analyzed according to the Bliss independence model [ 5]. Effects of compounds on the LASV replication complex were tested in the context of the cellular RNA polymerase I-based LASV replicon system.

Antiviral Testing In Vivo

Chimeric C57BL/6 mice and A129 mice were infected intraperitoneally with 1000 focus-forming units of LASV Ba366 and monitored daily for signs of disease, body weight, and body temperature. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in blood were analyzed using a colorimetric assay (Reflotron, Roche Diagnostics). Organs were collected from randomly chosen mice 7 or 8 days after infection and from mice that died from the infection. Infectious virus particles in blood and organs were determined by immunofocus assay. Organs were fixed in 4% formaldehyde-phosphate-buffered saline (PBS), embedded in paraffin, and stained with hematoxylin-eosin. Ribavirin in 0.9% NaCl was administered intraperitoneally; 0.9% NaCl served as placebo. Favipiravir was suspended in methylcellulose-PBS and was administered twice daily per os, using a stomach probe; 0.5% methylcellulose-PBS served as placebo.

See Supplementary Methods for an extended description of methods.

RESULTS

Favipiravir and Ribavirin Show Comparable Antiviral Activity Against LASV In Vitro

Favipiravir suppressed LASV replication by ≥5 log ₁₀ units with IC ₅₀ of 29 µM and IC ₉₀ of 43 µM ( Supplementary Figure 1). Ribavirin displayed comparable inhibitory activity with IC ₅₀ of 26 µM and IC ₉₀ of 33 µM. Neither drug significantly affected cell viability. The antiviral activity of favipiravir was confirmed in the LASV replicon system; the IC ₅₀ was approximately 15 µM.

Favipiravir Increases Survival Rate Among LASV-Infected Mice

All LASV-infected and placebo-treated chimeras died within 10 days after infection (Figure 1 and Supplementary Figure 2A). They showed high levels of virus in blood and organs, rapidly lost weight, and displayed features of severe human LF, such as elevated AST and ALT levels, with AST levels increasing by a greater percentage than ALT levels, and hypothermia [ 2, 6]. To test the therapeutic efficacy of favipiravir, treatment was commenced at day 4 after infection, when viremia had reached a level of about 10 ⁴ infectious virus particles/mL. Oral doses of 75 mg/kg per day and 150 mg/kg per day did not prevent or significantly delay death (Figure 1). However, treatment suppressed the viremia level by 2 log units between days 4 and 8, and it reduced virus levels in organs around the time of death. In addition, AST and ALT levels were significantly lower and terminal hypothermia was absent. Increasing the favipiravir dose to 300 mg/kg per day from days 4 to 11 after infection rescued 100% of LASV-infected mice (Figure 1). Viremia decreased within the first 3 days of treatment to levels below the limit of detection of the assay, and after 4 days of treatment the virus levels in organs were up to 6 log units lower than those in organs from placebo-treated animals. Decrease in body weight stopped after 4 days of treatment, hyperthermia was absent, and transaminase levels were hardly elevated or returned to normal during treatment. However, histopathological analysis revealed that widespread liver tissue damage remained even after 4 days of treatment with the high dose of favipiravir ( Supplementary Figure 3A).

To confirm the antiviral activity of favipiravir in a nontransplant model, we performed the same set of experiments in mice on the A129 genetic background ( Supplementary Figure 4). These mice represent a nonlethal model for LASV infection [ 7]. As in the chimeric mice, favipiravir showed a dose-dependent beneficial effect on body weight, transaminase levels, viremia, and virus load in organs.

Moderate Effect of Ribavirin In Vivo

Ribavirin was tested at a dose of 80 mg/kg per day, with commencement of treatment at day 0 ( Supplementary Figure 5A) and day 4 (Figure 2 and Supplementary Figure 2B) after infection. None of the treatment regimens increased the survival rate, compared with the rates in the respective placebo groups, and only treatment starting at day 0 after infection prolonged the survival duration. No significant effect was seen on viremia, although the virus levels in organs were somewhat decreased. However, both treatment regimens prevented increases in AST and ALT levels. Treatment from days 4 to 11 with a high dose of 160 mg/kg per day significantly prolonged the survival duration, and 1 of 5 animals (20%) survived (Figure 2). No reduction in levels of viremia or virus in organs was seen, while transaminase levels were suppressed as in the low-dose treatment groups. At day 8 after infection, widespread liver tissue damage was present in mice treated with ribavirin, although this finding seemed less pronounced than that for animals treated with placebo or favipiravir, suggesting a cell-protective effect of ribavirin ( Supplementary Figure 3B).

In analogy to the favipiravir experiments, we tested ribavirin in A129 mice (80 mg/kg per day during days 0-7 after infection; Supplementary Figure 5B). Consistent with the findings in the chimeric mice, the drug reduced signs of LASV-associated disease such as weight loss and elevated aminotransferase levels, while it failed to influence the levels of viremia and virus in organs.

Beneficial Effect of Combination Treatment of Favipiravir With Ribavirin In Vitro and In Vivo

As ribavirin is the standard of care for LF, clinical trails may consider a combination of both drugs. Therefore, we first tested the antiviral activity of 64 combinations of ribavirin and favipiravir in cell culture ( Supplementary Figure 6). The 8 × 8 concentration matrix was designed around the IC ₉₀ values of both drugs. The dose-response surface demonstrated that all combinations of ribavirin and favipiravir exhibited strong antiviral effects, with suppression of virus replication by ≥5 log units. Possible antagonistic or synergistic effects were evaluated according to the Bliss independence model [ 5]. This analysis revealed clear synergistic effects when the drugs were combined in concentrations of around IC ₉₀/2 to IC ₉₀. In this area of the matrix, the experimental virus titer was 1-2 log units lower than the titer predicted by the Bliss independence model for additive effect. The MTT test did not reveal drug toxicity over the whole matrix.

To explore whether combination of 2 subeffective doses may result in effective treatment in the lethal mouse model, we administered a ribavirin dose of 80 mg/kg per day plus a favipiravir dose of 150 mg/kg per day from days 4 to 11 after infection (Figure 2 and Supplementary Figure 2B). While none of the single-dose treatments improved the survival rate or prolonged the survival duration (Figure 1 and Figure 2), the combination treatment prolonged the survival duration by 6 days, compared with both single-drug treatments ( P < .01; see Supplementary Table 1 for all statistical comparisons of combination vs single-drug treatments). The combination treatment led to a decline in viremia level until end of the treatment at day 11. However, a rebound in viremia level occurred following cessation of therapy, which may have caused the death of the animals around day 15 after infection Therefore, the combination treatment was extended until day 15 after infection (Figure 2), which resulted in survival of 3 of 5 mice (60%), with surviving mice effectively clearing virus from blood and regaining weight. Alternatively, the ribavirin dose was doubled to 160 mg/kg per day (for the single-drug experiment, see Figure 2) and administered in combination with a favipiravir dose of 150 mg/kg per day from days 4 to 11 after infection (Figure 2). This treatment led to survival of 4 of 5 mice (80%) during the observation period. However, a rebound in viremia level was not prevented. Surviving animals did not completely clear the virus from blood and still showed weight loss with biochemical evidence of disease at day 21 after infection. Increased AST and ALT levels, as seen in placebo controls, were effectively suppressed by all combination treatments. Histological analysis at day 8 after infection supported again a cell-protective effect of ribavirin ( Supplementary Figure 3C). Liver tissue damage was still abundant but was slightly ameliorated in mice that received combined treatment, compared with those treated with favipiravir alone.

DISCUSSION

Favipiravir strongly suppressed LASV replication in vitro and in vivo, consistent with its potent antiviral effect against other negative-strand RNA viruses [ 3]. Even if treatment was commenced 4 days after infection, the drug led to a rapid decline in viremia level, ameliorated disease, and increased survival rate. The replicon data indicate that the drug is targeting the LASV polymerase complex, consistent with previous findings for influenza virus and lymphocytic choriomeningitis virus [ 4, 8]. We found a synergistic interaction between favipiravir and ribavirin in vitro and increased protection from mortality and extended survival durations when combining suboptimal doses as compared to treatment with each drug alone.

Despite the marginal antiviral effect of ribavirin in the mouse model, which may not be explained by insufficient metabolization of the drug in mouse cells [ 9], we observed a clear cell-protective effect. This resembles observations in influenza virus-infected mice [ 10]. Here, ribavirin improved survival rate and lung pathology, while it hardly reduced the virus titer in lungs. These findings led the authors to conclude that "it seems unlikely that the activity of ribavirin (in vivo) is based primarily upon its antiviral properties" [ 10, p. 1070]. Several hypotheses have been put forward to explain in vivo effects of ribavirin, including inhibition of induction of proinflammatory cytokines in virus-infected macrophages [ 11] and induction of interferon-stimulated genes [ 12].

An important finding of our study is the synergistic interaction of favipiravir and ribavirin in vitro and in vivo. Various scenarios may explain the synergism. First, depletion of the intracellular GTP pool by ribavirin [ 9] may facilitate the incorporation of favipiravir into viral RNA, as the favipiravir-nucleotide complex competes against GTP and ATP during RNA synthesis and exogenous elevation of the GTP and ATP levels reverses the inhibitory effect of favipiravir [ 4, 8, 13]. Second, it may be that both ribavirin and favipiravir are incorporated into the viral RNA and hamper progeny RNA synthesis and/or cause lethal mutagenesis in a synergistic manner [ 14, 15]. Third, the synergistic effect in vivo may also stem from combination of direct suppression of virus replication by favipiravir with modulation of pathophysiological or immunological host pathways by ribavirin [ 11, 12].

In conclusion, our data hold promise that favipiravir is of value in the treatment of LF and provide a rationale for clinical trials evaluating favipiravir in combination with ribavirin.

Notes

Acknowledgments. We thank the animal caretakers at Heinrich Pette Institute and Bernhard Nocht Institute for their excellent technical assistance.

L. O., C. M.-F., and S. G. conceived and designed the experiments. L. O., T. R., A. L., P. R., S. W., E. P., S. B., S. K., and C. M.-F. performed experiments. L. O., C. M.-F., and S. G. analyzed the results and wrote the manuscript. All authors reviewed the manuscript.

Financial support. This work was supported by the Leibniz Center of Infection (grant LCI/HPI/2012/2) and the European Commission (European Union Seventh Framework Programme grant agreement 228292 "European Virus Archive").

Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

References

Author notes

# data[].attributes.attachment_link.title
Efficacy of Favipiravir Alone and in Combination With Ribavirin in a Lethal, Immunocompetent Mouse Model of Lassa Fever

# data[].attributes.attachment_link.description
Abstract. We studied the therapeutic potential of favipiravir (T-705) for Lassa fever, both alone and in combination with ribavirin. Favipiravir suppressed Lass

# data[].attributes.attachment_link.title
Enhanced Treatment of Lassa Fever by Immune Plasma Combined with Ribavirin in Cynomolgus Monkeys

# data[].attributes.attachment_link.description
Abstract. Lassa virus-infected cynomolgus monkeys were treated with Lassa virus-immune monkey serum containing a high concentration of neutralizing antibody, th

# data[].attributes.headline
efficacy of LF therapeutics on infected monkeys

# data[].attributes.subject
efficacy of LF therapeutics on infected monkeys

# data[].attributes.attachment_link.readable_text
Europe PMC requires Javascript to function effectively.

Either your web browser doesn't support Javascript or it is currently turned off. In the latter case, please turn on Javascript support in your web browser and reload this page.

This website requires cookies, and the limited processing of your personal data in order to function. By using the site you are agreeing to this as outlined in our privacy notice and cookie policy.

Abstract

The therapeutic efficacy of ribamydil and virasol was evaluated in experimental Lassa fever in monkeys which received these drugs at various intervals after the onset of the clinical illness. Ribamydil or virasol administered in the first day of fever protected from death 60% to 66% of the infected animals, but when the drugs were given 2 days after the onset of fever the number of survivors declined to 0.20%. When the treatment was started 4 days after the onset of fever none of the drugs prevented deaths of Lassa virus-infected baboons.

Citations & impact

Impact metrics

Citations of article over time

Article citations

Similar Articles

To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation.

# data[].attributes.attachment_link.title
[The therapeutic efficacy of ribamidil and virazole in experimental Lassa fever in monkeys].

# data[].attributes.attachment_link.description
The therapeutic efficacy of ribamidil and virazole in experimental Lassa fever in monkeys].

# data[].attributes.attachment_link.readable_text
Abstract

Lassa fever (LF), a disease endemic to West Africa caused by infection with Lassa virus (family Arenaviridae), causes an estimated 100000-300000 human cases annually [ 1]. Most cases are caused by infection from ingestion, inhalation, or direct contact with the environmental reservoir, Mastomys natalensis rodents; human-to-human spread is uncommon [ 2, 3]. Mortality among hospitalized patients ranges from 15% to 20% [ 1]. Treatment with ribavirin during the first 6 days of illness decreases mortality rates to 5% [ 4]. Favipiravir, a novel antiviral agent with broad-spectrum activity against RNA viruses, decreases levels of Lassa viremia and increases survival in animal models [ 5, 6]. We describe 2 cases of LF acquired via secondary spread from the same index case who survived after treatment with ribavirin and favipiravir.

The index patient was a medical worker employed at a missionary institution in Oti District, Togo. The index patient was medically evacuated to Cologne, Germany, after 2 weeks of fevers, but died approximately 10 hours after admission despite intensive care. LF was diagnosed postmortem by reverse-transcription polymerase chain reaction (RT-PCR) from a liver biopsy (Gerd Fätkenheuer, submitted).

CLINICAL COURSE AND TREATMENT

Patient E

Patient E is a previously healthy 33-year-old man who provided 7 days of nursing care to the index patient in Togo and developed fevers 9 days after his last contact with the index patient. A thick blood smear was positive for malaria and he received 3 days of artemether/lumefantrine. LF was suspected when his fevers persisted and he developed sore throat, retro-orbital headache, intermittent diminished hearing, diarrhea, malaise, and generalized weakness. Laboratory testing in Togo revealed leukopenia with a nadir of 1 × 10 ⁹ cells/L, thrombocytopenia of 50 × 10 ⁹ cells/L, mild transaminitis (aspartate aminotransferase 131 U/L, alanine aminotransferase 77 U/L), and creatinine of 2.3 mg/dL. Patient E received ribavirin 2 g orally once followed by 1 g every 8 hours as intravenous ribavirin was not immediately available. He was medically evacuated to the Serious Communicable Diseases Unit at Emory University Hospital in Atlanta, Georgia, on day 8. An admission blood specimen was positive for Lassa virus infection by conventional RT-PCR and retrospective quantitative (q) RT-PCR (using laboratory-developed primers LV-Tog2 F (TCACAACTCATCGCCTCATAC) and LV-Tog2 R (AATCTGTATGACCACGCACTC) and of probe LV-Tog2 P (56-FAM/TCCATCTGT/ZEN/CCATCCCAAACTTCAACC/3IABkFQ) with a cycle threshold value (Ct) of 23.4 ( Figure 1A). On admission, intravenous ribavirin 16 mg/kg every 6 hours and oral favipiravir 2000 mg once followed by 1000 mg every 12 hours were administered after emergency investigational new drug approval was obtained from the US Food and Drug Administration. On day 12, favipiravir was discontinued and ribavirin was decreased to 8 mg/kg every 8 hours as per protocol from the manufacturer. He completed his ribavirin regimen on day 15.

Initial vital signs included temperature 37.1°C, heart rate 50 beats/minute, respiratory rate 24 breaths/minute, and blood pressure 140/91 mm Hg. Examination revealed a thin, ill-appearing male with mild conjunctival pallor, oral thrush, a soft precordial systolic murmur, and mild bladder distension with suprapubic tenderness. Tests for Ebola virus, human immunodeficiency virus (HIV), and malaria were negative. The patient had fevers to a maximum of 40.4°C, which resolved on day 12, and intermittent headaches. He developed nausea, which he attributed to favipiravir. His appetite improved and his sore throat resolved on day 14. He developed painful diffuse lymphadenopathy on day 14, epididymitis between days 19 and 21 (which later correlated to semen culture positivity for Lassa), and night sweats on day 22. He was discharged on day 25. His hearing slowly improved, although subjective right-sided decreased hearing persisted for 4 months despite normal audiological testing.

Patient F

Patient F is a mortician who prepared the index patient's corpse for repatriation 11 days after the patient's death. He was ill with mild upper respiratory tract symptoms prior to contact with the deceased patient, making the first day of symptoms due to LF difficult to clarify. Qualitative PCR for LF was ordered by the health authorities 2 days after contact with the corpse due to temperatures between 37.5°C and 38.0°C (illness day 1) with an inconclusive result (1 faint band in 1 of 3 parallel assays). His condition deteriorated over 3 days with fevers to 38.2°C, pronounced generalized weakness, myalgia, arthralgias, headache, and nausea. Repeat qRT-PCR testing 4 days later was positive (Ct 31.0; Figure 1B), and he was transferred to the Frankfurt University High-Level Isolation unit.

Initial vital signs were temperature 38.9°C, blood pressure 145/75 mm Hg, and heart rate 77 beats/minute. Examination revealed dry mucous membranes, pharyngeal erythema with patchy white exudates, conjunctival injection, a faint macular rash on the epigastrium, and slowed rapport and speech. Diagnostic testing was negative for HIV, hepatitis B and C, influenza, and group A Streptococcus pharyngitis. His fevers resolved on day 7 and his myalgias and arthralgias gradually improved. He developed nonproductive coughing without chest radiographic abnormalities. His pharyngitis worsened during the first week and resolved during week 3. He was discharged on day 39 after his symptoms resolved.

He received 1000 mg of intravenous ribavirin followed by 1000 mg every 6 hours for 4 days followed by 500 mg intravenously every 8 hours for another 6 days per the World Health Organization protocol [ 7]. Oral favipiravir 2000-mg loading dose was administered followed by 1200 mg twice daily for 4 days. He developed atrial fibrillation 2.5 hours after initiation of favipiravir and 10.5 hours after ribavirin. Therapeutic heparin treatment was administered. Four hours after discontinuation of favipiravir and 14 hours after ribavirin dose reduction, he converted to sinus rhythm.

LABORATORY STUDIES

For both patients, clinical laboratory support was provided as described in Supplementary Data 1 and initial molecular testing was performed as previously described [ 8, 9]. Patient-specific qRT-PCR assays were developed, designated LV-Tog2 for patient E and LV-Tog1 for patient F (Supplementary Data 1). Pertinent laboratory results for both patients are shown in Figures 2A and 2B, respectively. Lassa immunoglobulin M (IgM) antibodies were detected on day 13 in patient E and immunoglobulin G (IgG) antibodies were detected on day 46 in patient E and day 38 in patient F. IgM was not tested in patient F.

Cell culture by Vero cell culture for Lassa virus was positive from whole blood specimens from patient E on days 8, 9, and 11 and patient F on day 5. Lassa virus nucleic acid was detected in blood samples by qRT-PCR in patient E through day 59 ( Figure 1A) with 1 exception and in patient F through day 28 ( Figure 1B). Patient E had 6 positive RT-PCR assays from urine samples but virus was not isolated from urine by cell culture. Urine testing on patient F yielded 1 positive RT-PCR on day 22. Saliva RT-PCRs were intermittently positive from patient E through day 46 and pharyngeal swab RT-PCRs from patient F were positive through day 28. Three semen samples from patient E (days 15, 20, and 48) and 4 samples from patient F (days 35, 46, 82, and 103) all had detectable Lassa virus RNA by qPCR. Culture yielded Lassa virus in 1 semen sample from patient E on day 20.

DISCUSSION

We report 2 epidemiologically linked patients with LF secondarily acquired from the same index case treated with favipiravir and ribavirin and who made full recovery. Ribavirin, the only previously described anti-LF therapy associated with reduced mortality in humans, was initiated on day 6 (patient E) and day 5 (patient F). Favipiravir is licensed for influenza treatment in Japan and may reduce mortality in other RNA virus infections, including Ebola virus disease [ 10]. Favipiravir was started on day 8 (patient E) and day 5 (patient F) but was discontinued after 5 days of treatment in both patients due to nausea and worsening transaminitis. Transaminitis has been described in LF in both untreated and ribavirin-treated patients [ 4, 11], so it was unclear if the patients' transaminitis was due to their underlying disease vs a drug effect. In both patients, aminotransferase levels declined shortly after discontinuation of favipiravir and decreases in the ribavirin dose. Both patients also showed a decline in hemoglobin ( Figure 2A and 2B) with an elevated lactate dehydrogenase and bilirubin in patient F, which may partially be explained by a toxic effect of ribavirin and metamizole. Viral copy number values declined on therapy, although given the lack of comparator patients or sufficient historical data using PCR methods, it remains unclear whether this was due to an effect of combined antiviral therapy or the normal course of Lassa viremia. The utility of combined ribavirin and favipiravir treatment for LF should be evaluated in further clinical studies.

Viral nucleic acid was detected for a prolonged period in both patients in blood (days 27 and 62) and semen (days 48 and 103). The long-term persistence of viable virus in convalescent patients in immune-privileged compartments has been described for Ebola virus in ocular and seminal fluid [ 12-14]. Arenavirus escape attributed to evasion of immune system antigen presentation has been shown to contribute to long-term virus excretion in natural and dead-end hosts [ 15]. Well-documented longitudinal molecular detection of Lassa virus in semen has not been previously published, raising the possibility that Lassa virus could be sexually transmitted.

Supplementary Data

Supplementary materials are available at Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Emory Serious Communicable Diseases Unit

SCD Unit Coordinator: Sonia Bell. Administration: Catherine Maloney, Pam Cosper, Nancye Feistritzer, John Lewin, Bryce Gartland, Ira Horowitz, David Pugh, Chad Ritenour, Jerry Lewis, David Hatcher, Linda Scott-Harris, Lynne Ometer, Kip Hardy, Jeff Broughton, Robert Jackson, Samantha Thomas. Nursing: Toni Ash, Christopher Barnes, Jamie Breedlove, Bob Bridgman, Lane Darragh, Tracey Daye, Dustin Hillis, Crystal Johnson, Julie-Ann Johnson, Danni LaFond, Courtney Lyons, Josia Mamora, Anna McCord, Samantha McDaniel, Haley Morgan, Jill Morgan, Alexander Sanchez, Marissa Simon, Jason Slabach, Kevin Tirado, Sally Watkins, Terrica Wilson, Ken Logan. Emory Medical Labs: Juli Buchanan, Eileen Burd, John Cardella, Brenda Eaves, Crystal Evans, Charles Hill, Doris Igwe, Karen Jenkins, Maureen Lindsey, Jordan Magee, Stacy McCarthy, Randall Powers, James Ritchie

Pharmacy: Jan Pack, Susan Rogers. Emory Health and Safety: Patricia Olinger, Kalpana Rengarajan, Scott Thomaston, Esmeralda Meyer. Occupational Health: Emily Beck, Paula Desroches, Cynthia Hall, Celeste Walker Infection Prevention: Esther Baker, Betsy Hackman. Environmental Services: Jeff Broughton, Robert Jackson, Jerry Lewis

Pastoral Care: Robin Brown-Haithco, Faith Richardson. Emory Faculty and Staff Assistance: Art Krasilovsky, Clevevoya Jordan, Sue Matthews, Marilyn Hazzard Lineberger, Paula G. Gomes. Supplies/Logistics: Gentrice McGee, Porcia Jones. EUH Security: Linda Scott-Harris, James Cain, Roderick Davis, Tyrone Johnson, Tyrone Pickett, Anthony Shaw, Tenina Truesdale. Emergency Medicine: Alex Isakov, Sam Shartar, Wade Miles, Aaron Jamison, John Arevalo, Gail Stallings. Communications/Media Relations: Janet Christenbury, Vince Dollard, Melanie De Gennaro, Holly Korschun. Nutrition: Tom Ziegler, Daniel P. Griffith, Nisha Dave

Notes

Author contributions. T. W., G. K., and C. S. K.: Treated the patient, collected data, wrote the manuscript. S. B. and M. E.: Performed virological analysis, assisted in writing the manuscript, provided discussion of results. T. S.: Performed virological analysis, analyzed data. P. L.: Treated the patient, collected data. G. S.: Treated the patient. C. S.: Treated the patient, provided discussion of the manuscript. H. R.: Assisted in sample and data collection, provided discussion of the manuscript. V. N. R.: Collected data, wrote the manuscript. A. M.: Collected data, edited the manuscript. B. S. R., J. B. V., A. K. M., G. M. L., S. V., and K. F.: Treated the patient, collected data, edited the manuscript. S. B., J. D. K., P. E. R., U. S., S. N.: Performed virological analysis, analyzed data, edited the manuscript. K. P.: Performed virological analysis, analyzed data. A. M.: Performed virological analysis, assisted in writing the manuscript, provided discussion of results.

Acknowledgments. We thank Sarah Katharina Fehling and Svenja Wolff for virus isolation work and Michael Schmidt and Gotthard Ludwig for technical support in the biosafety level 4 facility at the Philipps University of Marburg. We express our gratitude to the nursing staff of the intensive care pool and the Infectious Diseases Department, University Hospital Frankfurt. We thank Debi Cannon, Cheng-Feng Chiang, William Davis, Aridth Gibbons, James Graziano, Mary Jenks, Maria Morales-Betouille and Nishi Patel for their assistance in performance of the laboratory testing at the Centers for Disease Control and Prevention (CDC). We are appreciative of the support of the US Food and Drug Administration, Valeant, and Medivector for the provision of the therapeutics for these patients. We thank the Investigational Drug Service at Emory University and the Emory University Institutional Review Board.

Disclaimer. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the CDC.

Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

References

Author notes

# data[].attributes.attachment_link.title
Favipiravir and Ribavirin Treatment of Epidemiologically Linked Cases of Lassa Fever

# data[].attributes.attachment_link.description
Abstract. Two patients with Lassa fever are described who are the first human cases treated with a combination of ribavirin and favipiravir. Both patients survi

# data[].attributes.attachment_link.readable_text
Antibodies are a crucial ingredient in preventing infection for a wide variety of viruses. Scientists are frequently looking for new antibodies that could help fight viral infections. One deadly virus, the Lassa virus, infects hundreds of thousands of people annually, predominantly in Africa, causing its namesake: Lassa fever. There is as yet no approved vaccine or therapeutic against Lassa fever.

Now, however, scientists have identified three major monoclonal antibodies - artificial proteins that act like antibodies in the immune system - that can be used to potentially form the basis of a new vaccine that could save tens of thousands of lives a year.

The research team was led by Erica Ollmann Saphire, the president and CEO of the La Jolla Institute for Immunology (LJI), an organization dedicated to understanding and combating infectious diseases. Researchers from LJI used the bright X-ray beams produced by the Advanced Photon Source (APS), a U.S. Department of Energy (DOE) Office of Science user facility at DOE's Argonne National Laboratory, to characterize three antibodies that target the Lassa virus's protein structure. The antibodies were isolated from survivors of the disease.

"The work done at the APS allowed us to see what was happening on a molecular level." - Haoyang Li, La Jolla Institute for Immunology

The result is a culmination of a 17-year project to look at the action of the Lassa virus and try to create a suite of biological molecules that could inhibit the virus's ability to infect human cells.

"These antibodies are protective, but people weren't sure why they were protective," said Haoyang Li, a researcher at LJI and the first author on a paper based on the study. ​"The work done at the APS allowed us to see what was happening on a molecular level."

"People may be familiar with monoclonal antibodies as a treatment for viruses such as the virus that causes COVID-19, but these kinds of antibodies can have positive effects on other viruses as well, like Lassa," added Argonne physicist Naga Venugopalan.

According to Li, viruses such as Lassa are enveloped by a substance called glycoprotein. This glycoprotein is in turn equipped with a ​"glycan shield" made of carbohydrate molecules that typically both protects the virus from antibody attack and enables it to infect cells.

"Normally, antibodies cannot find a way to get through the glycan shield to get access to the protein to destroy the virus, but in our research we found that some of the antibodies could actually exploit the shield itself to reach the glycoprotein and neutralize the virus," Li said.

The researchers strove to identify the virus's weak spots, and they found that the most effective approach involved the use of a ​"cocktail" of the three antibodies to neutralize the virus. The antibody cocktail has been tested in nonhuman primates but is still awaiting federal approval for further study in humans. In the meantime, the researchers were interested in more fully understanding how the antibodies worked.

To do so, they used a combination of cryo-electron microscopy (cryo-EM) technology provided by LJI, and X-ray crystallography with X-rays provided by the APS. Partial structures of the three different antibodies and interaction of the glycoprotein complex with one of the antibodies were determined by X-ray crystallography. This information was used as an initial model when helping to fill out the atomic maps produced by the cryo-EM.

"X-ray crystallography provides us with a way to see the finer details of interactions between two proteins, namely, the side chain interactions," Venugopalan said. ​"Information obtained from X-ray crystallography provides better clarity when fitting cryo-electron density maps."

The ultimate goal would be to develop a vaccine that could drive the body into producing these antibodies naturally. ​"Because each of these antibodies in the cocktail can simultaneously bind to the glycoprotein, it creates the potential for a more effective treatment against Lassa virus than we have had to date." Venugopalan said.

A paper based on the study was published in Science Translational Medicine on October 26, 2022.

# data[].attributes.attachment_link.description
Antibodies are a crucial ingredient in preventing infection for a wide variety of viruses. Scientists are frequently looking for new antibodies that could help fight viral infections. One deadly virus, the Lassa virus, infects hundreds of thousands of people annually, predominantly in Africa, causing its namesake: Lassa fever.

# data[].attributes.headline
potential treatment for LF by the UTMB (University of Texas Medical Branch)

# data[].attributes.subject
potential treatment for LF by the UTMB (University of Texas Medical Branch)

# data[].attributes.attachment_link.readable_text
New research out of the University of Texas Medical Branch potentially points to an effective treatment for Lassa fever, a dangerous, often fatal disease common to much of West Africa but considered a major threat to global health.

In a paper published in the journal Proceedings of the National Academy of Sciences, UTMB's scientists document how a new drug from Zalgen Labs successfully cured cynomolgus monkeys infected with Lassa virus.

There currently is no approved treatment for the disease, which is estimated to infect 300,000 to 500,000 people a year across the region and cause at least 5,000 deaths annually.

Women and children are at the highest risk from Lassa fever, which is characterized by bleeding and coagulation abnormalities, with overall mortality rates exceeding 25 percent and reaching 50 percent during epidemics.

Exposure to infected animals is the most common cause of infection in humans. A recent study that identified the top 50 agents with pandemic potential, Lassa fever was ranked first among known viruses regarded as a threat for zoonotic spillover.

Though less common, human-to-human transmission is caused through exposure to blood or secretions and through sexual contact.

Those in the early stages of the disease often show mild or no symptoms and therefore tend not to seek help until their condition is advanced. About 20 percent of patients progress to viral hemorrhagic fever.

Lassa virus has a high level of genetic diversity across much of West Africa. This creates a real challenge for therapeutic developers as it raises the question of whether a single drug could be used to treat all Lassa virus infections."

Dr. Robert Cross, associate professor in the Department of Microbiology & Immunology and one of the authors of the study

The UTMB research shows the potential of Zalgen Labs' Arevirumab-3 therapeutic, a monoclonal antibody cocktail, as a treatment against all major strains of the disease in advanced stages.

The study concluded that the Arevirumab-3 therapeutic successfully treated cynomolgus monkeys when administered beginning at advanced stages of disease caused by two different major strains of Lassa virus that were involved in the research.

The results indicated Arevirumab-3 potentially could be used to treat humans infected with Lassa virus.

Arevirumab-3 is on a path toward Investigational New Drug status with the U.S. Food and Drug Administration and toward projected Phase 1 clinical studies to assess its safety and efficacy in healthy human subjects.

"This study represents yet another proof that broad protective treatments, here against the highly complex and variable hemorrhagic fever Lassa virus, can be developed with cutting-edge research within secure high-containment laboratory environments," said Dr. Gary Kobinger, director of the Galveston National Laboratory.

The finding is the result of an ongoing research collaboration between UTMB, Zalgen and Tulane University. UTMB, along with Tulane, is conducting additional studies to further explore the use of Arevirumab-3 against divergent and emerging strains.

"This work is yet another example of our dedication of UTMB and its many collaborative partners, including Zalgen Labs and Tulane University, to refine monoclonal antibody therapies against Lassa virus." Cross said. 'It no doubt will serve as further support of the value of continued investment in immunotherapeutics research for the treatment of Lassa fever in humans."

# data[].attributes.attachment_link.title
UTMB research potentially points to an effective treatment for Lassa fever

# data[].attributes.attachment_link.description
New research out of the University of Texas Medical Branch potentially points to an effective treatment for Lassa fever, a dangerous, often fatal disease common to much of West Africa but considered a major threat to global health.

# data[].attributes.body
according to the report (Above) on the epidemiology trend of Lassa fever in Nigeria there has been a decline in Lassa fevers epidemiology (that can be found in the Results paragraph) in Nigeria (due to the use of PPE) plus a decline in fatality's from 2018 to 2021. (this can be found at the end of the "quantitative analysis and state distribution of LF in Nigeria").
more links>https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0279467