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Energy helping LD survive in harsh environments

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Energy helping LD survive in harsh environments

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Poly-3-Hydroxybutyrate in Legionella pneumophila, an Energy Source for Survival in Low-Nutrient Environments | Applied and Environmental Microbiology

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ABSTRACT
Chloroform-soluble material was extracted from two strains ofL. pneumophila serogroup 1 following growth in continuous culture. The purified material was identified
as poly-3-hydroxybutyrate (PHB) by nuclear magnetic resonance spectroscopy and by…

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the yellow fever vaccine and its  safety for HIV patients 

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the yellow fever vaccine and its  safety for HIV patients 

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Immunogenicity and Safety of Yellow Fever Vaccination for 102 HIV-Infected Patients

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Abstract. Background. Yellow fever vaccine (17DV) has been investigated incompletely in human immunodeficiency virus (HIV)-infected patients, and adequate immun

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in pages 6 and 7 it mentions the areas where Aedes aegypti breeds 

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this mentions a decline in population immunity due to a variety of factors including deforestation and urbanization,

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this mentions a decline in population immunity due to a variety of factors including deforestation and urbanization,

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the possible need for a booster vaccine for the YF vaccine

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the possible need for a booster vaccine for the YF vaccine

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Are booster doses of yellow fever vaccine needed?

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Yellow fever is a major public health problem. A live attenuated vaccine, developed
by Max Theiler and colleagues in the 1930s,1 has helped to control yellow fever, but
the duration of protective immunity has been questioned.

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Bacteria Stimulate Hatching of Yellow Fever Mosquito Eggs

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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…

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the current levels of yellow fever vaccination regimes in people might make the immunity aginst yellow fever ineffective 

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the current levels of yellow fever vaccination regimes in people might make the immunity aginst yellow fever ineffective 

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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

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# 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 ...

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re-emergence of YF because of a disruption in immunization (in the 60s and 50s people began to only be immunized with Smallpox as the WHOs plan to eredicate smallpox)

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re-emergence of YF because of a disruption in immunization (in the 60s and 50s people began to only be immunized with Smallpox as the WHOs plan to eredicate smallpox)

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[Reemergence of yellow fever in West Africa: lessons from the past, advocacy for a control program]

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Reemergence of yellow fever in West Africa: lessons from the past, advocacy for a control program]

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Since the 1980s, yellow fever has reemerged across Africa and in South America. The total of 18 735 yellow fever cases and 4522 deaths reported from 1987 to 1991 represents the greatest amount of yellow fever activity reported to the World Health Organization (WHO) for any 5-year period since 1948. There is an excellent vaccine against yellow fever. At present, a high proportion of travelers to at-risk areas are reported to be immunized, reflecting widespread knowledge about the International Health Regulations. In South America, yellow fever remains an occupational hazard for forest workers, who should be immunized. However, Aedes aegypti mosquitoes are now present in urban areas in the Americas (including southern parts of the United States), and there is concern that yellow fever could erupt in explosive outbreaks. In Africa, a large proportion of cases have occurred in children. The WHO, the United Nations Children's Fund (UNICEF), and the World Bank have recommended that 33 African countries at risk for yellow fever add the vaccine to the routine Expanded Programme on Immunization; studies show that this would be highly cost-effective. To date, financing yellow fever vaccine has been a major problem for these countries, which are among the poorest in the world. For this reason, WHO has launched an appeal to raise $70 million for yellow fever control in Africa.

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yellow fever and the endangerment of species 

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yellow fever and the endangerment of species 

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Abstract

Understanding the impact of zoonotic diseases on wild primate populations is important for assessing local extinction risks and for evaluating potential mitigating factors. Comparative data on demographic changes in two isolated populations of the northern muriqui ( Brachyteles hypoxanthus) during a severe yellow fever outbreak in southeastern Brazil provide unique insights into the potential effects of this disease in this Critically Endangered species. From October 2016 to April 2017, the muriqui population at the Reserva Particular do Patrimônio Natural-Feliciano Miguel Abdala (Caratinga) lost 31 of its 324 members, or nearly 10%, whereas the population at the Reserva Particular do Patrimônio Natural-Mata do Sossego (Sossego) declined from 34 to 25 individuals, or 26%. Greater per-capita risks to muriquis in the Sossego population could be related to ecological and anthropogenic differences, including a wetter climate and an absence of sympatric howler monkeys ( Alouatta guariba), which may have directly or indirectly buffered the Caratinga muriquis. Although we lack definitive confirmation that the muriqui population declines were caused by yellow fever, the timing and magnitude of the losses strongly implicate the disease. We highlight the risks of catastrophic population declines in small populations and emphasize the value of long-term demographic monitoring studies.

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Acknowledgements

We thank the Brazilian government, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and the Sociedade para a Preservação do Muriqui for permission to conduct research at the RPPN Feliciano Miguel Abdala. The research here was supported with funds from the Vilas Research Professorship and the Graduate School of the University of Wisconsin-Madison to KBS, with logistical and administrative support provided by Preserve Muriqui and Conservation Internacional-Brasil. Andreia Maria da Silva, Luana Vinhas, Leticia Almeida, José Gustavo Adler, Renan César, Joice Lima, and Valeria Ribeiro helped with the demographic monitoring of the Caratinga muriquis during the present study. Other funding sources and contributors to the long-term database can be found at the Strier lab website, https://strierlab.anthropology.wisc.edu/muriqui-project-of-caratinga-acknowledgements/. We also thank the Primate Action Fund/Conservation International and the Fundação Grupo Boticário de Proteção à Natureza for supporting the activities of the Projeto Muriquis do Sossego, and Fundação Biodiversitas, Muriqui Instituto de Biodiversidade (MIB), Mineração Curimbaba, the Associação dos Amigos do Meio Ambiente (AMA), Sr. Franscisco Portes, and Eduardo Bazém for logistical support. Sr. Jairo Joaquim de Andrade, Adriana Milagres, and Mateus Melo Dias helped with the demographic monitoring of the Sossego muriquis during the present study. We are grateful to Júlio César Bicca-Marques, Marco Antonio Barreto de Almeida, and an anonymous reviewer for their constructive comments, which have greatly improved our manuscript, and especially to Marco Antonio Barreto de Almeida for guiding us to key literature on yellow fever.

Author information

Authors and Affiliations

1. Department of Anthropology, University of Wisconsin-Madison, Madison, WI, 53706, USA

Karen B. Strier

2. Muriqui Instituto de Biodiversidade, Caratinga, MG, Brazil

Fernanda P. Tabacow, Carla B. de Possamai, Anderson I. G. Ferreira & Marcello S. Nery

3. Departamento de Biologia Animal, Universidade Federal de Viçosa, Viçosa, MG, Brazil

Marcello S. Nery

4. Departamento de Engenharia Florestal, Universidade Federal de Viçosa, Viçosa, MG, Brazil

Fabiano R. de Melo

5. Unidade Acadêmica Especial Ciências Biológicas, Universidade Federal de Goiás, Regional Jataí, Jataí, GO, Brazil

Fabiano R. de Melo

6. Departamento de Ciências Biológicas, Universidade Federal de Espírito Santo, Vitoria, ES, Brazil

Sérgio L. Mendes

7. Instituto Nacional da Mata Atlântica (INMA), Santa Teresa, ES, Brazil

Sérgio L. Mendes

Corresponding author

Correspondence to Karen B. Strier.

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Strier, K.B., Tabacow, F.P., de Possamai, C.B. et al. Status of the northern muriqui ( Brachyteles hypoxanthus) in the time of yellow fever. Primates 60, 21-28 (2019). https://doi.org/10.1007/s10329-018-0701-8
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DOI: https://doi.org/10.1007/s10329-018-0701-8

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Keywords

Disease

Epizootic event

Demographic changes

Primate population

Atlantic forest

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Status of the northern muriqui (Brachyteles hypoxanthus) in the time of yellow fever - Primates

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Understanding the impact of zoonotic diseases on wild primate populations is important for assessing local extinction risks and for evaluating potential mitigating factors. Comparative data on demographic changes in two isolated populations of the northern muriqui (Brachyteles hypoxanthus) during a severe yellow fever outbreak in southeastern Brazil provide unique insights into the potential effects of this disease in this Critically Endangered species.

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climate change and lass fever : a ever increasing risk of a pandemic

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climate change and lass fever : a ever increasing risk of a pandemic

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In the city and at the hospital where Dr Nnennaya Ajayi works, in Abakaliki in southeastern Nigeria, the ever more evident effects of climate change are prompting ever more frequent influxes of two kinds of animals: rats, and people.

And because the rats come with a viral disease, the people come into the hospital with it too.

"When they come into the clinics and the hospital, they have fever, nausea, body aches, muscle, joint and abdominal pain, and sometimes they also have red, bloodshot eyes," the doctor explains.

While it used to be that there was just one small cluster or local outbreak every two or three years, she says, those sporadic events have recently become a steady trickle. "And now this time, since August 2022, we've been seeing cases very regularly. We've had an ongoing outbreak."

Dr Nnennaya Ajayi outside Lassa fever treatment center.

The disease Dr Ajayi is describing is Lassa fever, a viral haemorrhagic infection named after the Nigerian town where it was first identified in 1969. The virus already causes tens of thousands of cases of infection and hundreds of untimely deaths each year across West Africa. With case fatality rates of as high as 15 percent in hospitalised patients with severe disease, and with no currently approved effective vaccines available, Lassa is rapidly climbing up epidemic worriers' watchlists.

CEPI is the leading funder of research for Lassa vaccines and has candidate shots in development with four partners across the world.

The vaccine candidates being developed by Emergent Biosolutions and by IAVI have begun Phase I clinical trials in Ghana and Liberia, respectively, to assess safety and immunogenicity.

To support the development of these vaccines, CEPI has also created and funded the largest ever Lassa fever study (the 'Enable' study) to provide a more accurate assessment of the incidence of Lassa fever infections in West Africa, and inform the design of late-stage Lassa vaccine trials. More than 20,000 participants are being recruited to take part in the study in Benin, Ghana, Guinea, Liberia, Mali, Nigeria, Sierra Leone, and Togo.

Dr Nnennaya Ajayi standing by hospital van.

DEADLY REACH

Rightly so, says Dr Ajayi: "Because it is considered a disease of the poor and the ignorant, it is one that has been undervalued. But we've already seen that people have transported Lassa across borders, so now it should be a disease of international importance."

Indeed, scientists at the University of Brussels in Belgium and at Scripp Research in the United States warned in a study published in September 2022 that climate change is causing Lassa fever to extend its deadly reach far beyond its Nigerian and West African origins.

By 2070, the study found, the number of countries across the whole of Africa that will develop the ecological conditions suitable for Lassa virus spread could drastically increase, potentially exposing hundreds of millions more people to the disease.

In Nigeria, Dr Ajayi explains, a warming planet has meant that in recent years, the dry seasons have become hotter, and the rainy seasons wetter. These "changes in the ecosystem" as she describes them, bring with them first fire, and then floods. In turn, both of these prompt rats and other rodents to get on the move and run for their lives. Fleeing flames and rising floodwaters, they invade towns and villages, seeking refuge and food in the homes of people living there.

MORE SPILLOVER

It's a scenario that is familiar to Professor Aaron Bernstein, Head of the Center for Climate, Health, and the Environment at Harvard T.H Chan School of Public Health and an expert in the risks of infectious disease 'spillover'-a phenomenon where pathogens make a jump from animals or birds into humans and cause outbreaks of infectious disease.

Mastomys natalensis, the natural host for Lassa virus.

Bernstein spoke to CEPI late last year as global leaders were gathering in Egypt for the COP27 Climate Change Conference. When it comes to novel emerging pathogens that spill over from animal species and become major pandemic threats, he said, climate-prompted migration-of all kinds of species including insects, mammals, and people-is at the heart of the problem.

"Climate change...is forcing everything that can to get out of the heat. And that means running to the poles or up mountainsides," Bernstein said.

This means that species that were previously geographically isolated and didn't run into each other's paths, are now running into each other with increasing regularity, raising the risk they'll spread viruses and other pathogens from one to another. Just like the rats that invade and spread diseases in human homes when they flee the flames and floodwaters of Nigeria's drier and rainier seasons.

DOMINO EFFECT

Dr Tim Endy agrees. CEPI's Disease X Project Leader and, for most of his career, a professional virus hunter, Endy says that in an ever warmer, wetter and more populated world, we need to be ever more alert to the threat of infectious diseases with pandemic potential.

"Above all, climate change affects the distribution, the abundance and the spread of vectors," he said. "And there's a multi-layer domino effect. It's not just about warming, but about changing environments. It's multifactorial and-importantly-it affects everything in CEPI's portfolio." As well as every country and region of the world.

In Egypt, for example, the host nation for COP 27, there was an outbreak more than four decades ago of a little-known disease called Rift Valley Fever. The viral disease, which affects both animals and humans, was first identified in 1931 during an outbreak of sudden deaths and abortions among sheep along the shores of Lake Naivasha in Kenya's Rift Valley, and had caused sporadic outbreaks in other parts of Africa since then.

But the 1977 Egyptian outbreak, which began in a man who came down with an acute febrile illness in the city of Belbies in the northeastern province of El-Sharquia, was the first big one in North Africa and by far the most explosive. Between 20,000 people and 40,000 people were estimated to have been infected in an outbreak that persisted well into the following year-and more than 600 of them died.

RIFT VALLEY FEVER RISK

Exactly what brought Rift Valley Fever to Egypt in this deadly outbreak is still not known for sure, but two factors are regularly cited as the most likely causes. The first is that the virus hitched a ride from sub-Saharan Africa with Egyptian troops who were returning home from a peacekeeping mission in Congo; and the second is that the virus came into Egypt with imported animals-most probably sheep and camels from Sudan.

What is certain, however, is that since that outbreak 45 years ago, Egypt has seen several other major Rift Valley Fever outbreaks, and the disease has steadily but surely expanded its presence across the whole of the African continent, as well as into Yemen and Saudi Arabia on the Arabian peninsular.

CEPI's vaccine development portfolio includes two projects working on Rift Valley Fever: one at Colorado State University (USA) and the other at Wageningen University (Netherlands).

The Wageningen Rift Valley Fever vaccine candidate, known as RVFV-4s, went into Phase 1 human trials in August last year.

CEPI's Director of Epidemiology and Data Science, Gabrielle Breugelmans, is in no doubt that with Rift Valley Fever, just as with Lassa and several other pathogens with pandemic potential, the rapidly changing climate is a driving force behind the rising numbers of cases and outbreaks, and also behind the increasing pandemic risk.

While with Lassa it is the rats and other rodents who are forced to seek new habitats due to climate change making it more difficult for them to find food and forcing them into new places to forage, with Rift Valley Fever it is disease-carrying insects-specifically mosquitos-that are expanding their reach as the climate becomes more mosquito-friendly in more and more regions of the world.

"If we don't act with urgency right now, the risk of spillover and the threat of pandemics is only going to increase as the climate change gathers pace and prompts all sorts of species-animal, human and insect-to migrate to areas where temperatures, rainfall and food availability suit them better," she says.

Numerous studies have warned about climate change exacerbating the frequency and range of infectious disease outbreaks. This one, published in 2019, found that a warming planet will lead to the expansion by thousands of miles of regions in which two of the most important mosquito vectors of disease- Aedes Aegypti and Aedes albopictus -can thrive and multiply.

"Within the next century, nearly a billion people could face their first exposure to viral transmission from either mosquito in the worst-case scenario, mainly in Europe and high-elevation tropical and subtropical regions," the study's authors warned.

And as well as for Rift Valley Fever, which is spread by many types of mosquitoes and as well as by flies and other biting insects, these two types of mosquitoes are key transmitters of other epidemic infectious diseases such as Yellow Fever, Zika, Chikungunya, Dengue Virus, Japanese Encephalitis and malaria.

ANIMAL-HUMAN CONTACT

But it's not just rodents and mosquitoes that are finding new places to survive and thrive as previously inhospitable environments become warmer, wetter, and unfortunately, more disease-friendly. Other species, such as bats and birds, are also being forced out of forests and wetlands and are, in the process, more frequently coming into closer contact with the people who have encroached on their natural habitats.

A large study published in last year pointed to bats as a particularly acute problem. "Owing to their unique dispersal ability, bats account for the majority of novel viral sharing and are likely to share viruses along evolutionary pathways that will facilitate future emergence in humans," it said.

Among the most worrying viral threats found in bats are the henipaviruses, of which the most famous is Nipah-a pernicious virus first discovered in Malaysia more than 20 years ago. Nipah causes a fatal inflammation of the brain, or encephalitis, and has so far killed seven out of every 10 people it has infected in a series of frightening outbreaks that have popped up thousands of miles apart-first in Malaysia and Singapore, and then in Bangladesh and India.

FLYING FOXES

Nipah comes from a group of pathogens known as the paramyxoviruses: the same family of viruses as measles, mumps, and the equally worrying Hendra virus. It is high on the World Health Organization's priority list of pathogens with deadly epidemic potential, and is a focus area for CEPI's research and development. It's also a virus that is finding increasingly frequent opportunities to get into people as the animals that carry it-fruit bats or 'flying foxes' as they are also known-see their once remote forest habitats being gradually eroded and encroached upon by humans.

Nipah virus transmission

In the case of the first known Nipah outbreak in Malaysia in 1999, it was pig farmers that had newly developed land that was once uncultivated forest.

A recent study on bat ecology and spillover risk by researchers at Montana State University in the United States, published in Nature in November 2022, focussed on Hendra, a henipavirus closely related to Nipah, and found that "interactions between land-use change and climate now lead to persistent bat residency in agricultural areas, where periodic food shortages drive clusters of spillovers".

As Ari Bernstein warns: "With huge livestock operations happening around the world in more places, there's more opportunity for pathogens to spill into people. We've seen that certainly with influenza pandemics, and we saw it with the outbreak of Nipah virus in Malaysia, where bats infected domesticated pigs who spread it to people."

Such bat-human contact was also the spark for an outbreak in January 2023 of Nipah virus in Bangladesh, in which a number of children and adults have so far been infected-at least 14-and ten cases have already been fatal. Local public health officials warned people not to drink raw date juice and avoid picking up fruits that may already have been nibbled on by bats, or been contaminated by bat urine or faeces.

CEPI is supporting the development of four early-stage Nipah vaccine candidates.

Two of these-one being developed by Auro in the United States, and another being developed by Public Health Vaccines, also in the United States-are currently being tested in Phase I clinical trials.

These two Phase I vaccines are currently among the most advanced Nipah vaccine candidates in the world.

So far at least outbreaks of Nipah have been relatively small and have also been confined to two main regions: South and Southeast Asia. But this virus is highly deadly, and its outbreak potential is increasing all the time, largely due to the fact that its animal vectors, the flying fox Pteropus fruit bats, are already common, and becoming more so, throughout tropical and sub-tropical regions all over the world. Those tropical areas are also now home to more than two billion people.

RAPID CHANGE

So what about Disease X? This as-yet-unknown virus is already a certain future threat, and one that rides high on the World Health Organization's as well as CEPI's pandemic risk list. More alarmingly, a changing climate is only making the risk yet higher that a new Disease X will spill over from animals and spark a deadly novel outbreak.

Dr Anne Spurkland, a molecular immunologist and professor of anatomy at the University of Oslo, says it's also the rapidity of change that heightens the risk.

"Humans are accustomed to the viruses and bacteria that have been around for many decades, but every time there is a change in living conditions for humans and other life forms, there are opportunities for shifting the balance," she said in an interview with CEPI.

"Environmental change is happening at a much higher speed now than before, so that increases the likelihood that there will be (spillover of) pathogens and microbes which none of us are adapted to. And where there is lack of immunity, things can spread quickly to the whole (human) population."

That's not to say we are helpless in the face of these increasingly imminent risks. Whether it be a re-emerging known viral threat-like Lassa-that's sparking more frequent and larger human outbreaks, or the next Disease X threat that will one day spill over and begin to spread as a new human virus, humanity has the scientific knowledge and the technical capability to contain such outbreaks before they run out of control. Investing now in boosting global disease surveillance and early warning systems, as outlined in the 1 00 Days Mission, and in scientific research to prepare for known and as-yet-unknown epidemic diseases, will mean that-just like with climate change-we can begin to cap the rising risks, turn the tide of threats, and eventually neutralise their pandemic potential.
Kate Kelland This article was originally published by CEPI

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How climate change increases pandemic risk

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Kate Kelland explores the interconnection between two of the biggest existential risks that threaten our way of life: climate change and pandemics.

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Lassa Fever (LF), an acute zoonotic disease caused by the Lassa fever virus (LFV), has approximately 2 million annual occurrences with 5000 to 10000 deaths. The disease has been identified to be endemic in Africa, particularly West Africa, where a larger percentage is recorded in Guinea, Liberia, Nigeria and Sierra Leone, especially as a result of its transmission pathways which is through rodents and warm‐blooded species via contact with their pathogenic infected body wastes around the house., ,

Studies have shown that one of the major challenges facing the fight against this disease is the similarities between its symptoms, that include, sore throat, pains in the joints, back and chest, and weakness, with many other popular diseases. Since the disease can progresses in serious cases to pharynx inflammation, conjunctivitis, abdominal pains, diarrhoea, vomiting and clinical manifestations that range from facial drops to respiratory symptoms, loss of hearing, acute hemorrhagic fever, multiple‐organ failure, foetal loss and death,, that under and misdiagnosis is a problem as early diagnosis is key to effective treatment with Ribavirin, which is recommended to be used in the first 6 days of infection. In addition, the presence of vaccines to help combat the virus and its spread, still remains unpopular to many.

The pre‐existing situation has been aggravated by the coronavirus disease 2019 (COVID‐19) pandemic. As expected, the pandemic has different annihilating public health and socioeconomic impacts across the world, resulting in a reduction in the epidemiological control of several infectious diseases, such as zoonotic diseases., , Africa, like every other continent has been affected, and the pre‐existing deficits in its health system add as a major disadvantage, and Nigeria, besides other West African nations, is also faced with a repetitive Lassa fever epidemic. As of May 3, 2021, the entire number of COVID‐19 cases in Nigeria amounted to 165.2 thousand. Lagos was the foremost affected state, tallying up to 58.5 thousand cases. Government Capital Domain (FCT) and Kaduna had the second and third most elevated number of aggregate cases, separately.

COVID‐19 in Nigeria is at 56,256 affirmed cases with 1082 deaths across the country, though LF comes to 1078 affirmed cases with 225 deaths. The yearly hike of human cases is more often than not watched amid the dry season (December-April) taking after the generation cycle of the vector within the monsoon season (May-June). This repetitive transient pattern related to LF implies that the pandemic started shortly after LF peaked in Nigeria, resulting in a double crisis of COVID‐19 and LF. Amid epidemiological Week 9 of 2020, when the first affirmed case of COVID‐19 was recorded in Nigeria, there was an increase in the number of affirmed LF cases. The expanded number of cases from the 2019 LF plague brought about the early rise of LF in 2020. In 2007, almost 6% of febrile adults and 3.5% of intensely febrile sick children in endemic ranges have affirmed infections. Indeed, with steady care and treatment with ribavirin, case fatalities were still as high as 24%-33% in tertiary clinic settings in recent times whereas almost 13.5% of survivors have sensorineural hearing loss. Also, LF accounts for almost 22% of hospital maternal mortality in endemic ranges. Unfortunately, LF control was quite ignored in Nigeria. This disappointment permitted the episodes to gain in recurrence and seriousness with numerous deaths, which also included healthcare workers.

The extent of cases shifted from 37% to 82% within the first quarter, 8%-30% within the second quarter, 6%-22% within the third quarter, and 11%-33% within the fourth quarter. The extent of cases within the to begin with quarter was less than 50% in 6 of the 10 years, 2009-2012, 2014, and 2017.

From January 1 to February 9, 2020, 472 research facility affirmed cases have been detailed in 26 out of 36 Nigerian states and the Government Capital Domain. Of the 472 affirmed cases, 75% have been detailed from three states: Edo (167 cases), Ondo (156 cases), and Ebonyi (30 cases). The other states that have recorded cases include Taraba (25), Bauchi (14), Level (13), Kogi (13), Delta (12), Nasarawa (4), Kano (4), Waterways (4), Enugu (4), Borno (3), Kaduna (3), Katsina (3), Benue (2), Adamawa (2), Sokoto (2), Osun (2), Abia (2), Kebbi (2), Gombe (1), Oyo (1), Anambra (1), FCT (1), and Ogun (1).

The situation is complicated due to the similarity within the clinical picture of COVID‐19 and LF, making the misdiagnosis at the onset of both illnesses with a critical chance with an expanded probability of coinfection. The underdiagnosis due to the overlap of symptoms between COVID‐19 and other infectious diseases, such as Dengue, Typhoid were also identified in other low‐income countries, such as Pakistan and India.,

The official declaration of COVID‐19 as a pandemic had further demonstrated the urgency of taking action globally. Yet in the case of Nigeria, resources directed for containing the COVID‐19 pandemic had to be compromised for fighting diseases with higher rate fatality ratio, such as LF. Therefore, healthcare professionals, policymakers and researchers assisted by international institutions should pay special attention to monitoring both diseases.

Given that the host of LFV is a rodent found in houses and surrounding fields and that there's no prophylaxis for LF; prevention can only be achieved through educating people living in endemic areas on ways to protect themselves from LF. Good "community hygiene" should be encouraged by providing of rodent‐proof containers and means for maintaining clean households and forming dump away from houses.

When it comes to the healthcare sector, early differential diagnosis should take place by testing febrile patients with COVID‐19 PCR testing to confirm the cases and proceed to the appropriate treatment. To ensure preparedness of health institutions in control of LF and COVID‐19, it is important to follow the written protocol of managing infectious diseases for achieving optimal safety precautions for the management and control of infectious diseases. Also, laboratory workers dealing with possibly infected samples should be trained and placed in equipped laboratories. Africa's health systems are undeniably fragile due to numerous continuous plagues that have debilitated health frameworks, including LF. Under the light of COVID‐19 widespread, healthcare systems may be overwhelmed in Africa. Skilled healthcare workers are in short supply, falling 60% underneath the UN's least limit, whereas sub‐Saharan Africa has only 1%-5% of the intensive care unit beds per capita, compared to European and East Asian countries. To date, over $160 billion has been made accessible for the COVID‐19 reaction over East, Central, West, and Southern Africa-80 percent of it from public and benefactor sources and 20 percent from private sources. The lessons of the past, where outbreak reactions have driven to declining wellbeing results in other regions, make a compelling case for donors and governments to consider utilizing the subsidizing for COVID‐19 to control and include financing for COVID‐19 to control and address the spread of the illness and construct long‐term strength in health frameworks.

Furthermore, to improve epidemiology and surveillance programs urgent investigation is required for both rat and human populations to enhance comprehension of LF and the reform of its management. This can be achieved through the formation of international research collaboration, mapping of the epidemiological and clinical history of the disease as well as geographical allocation.

National control and prevention strategies should be implemented by providing effective and affordable diagnostics and treatment for LF and COVID‐19, enhancing laboratory diagnostics, clinical management, environmental control and research. The involvement of the communities affected is highly recommended, and the development of an effective and affordable vaccine would be the key pillars for the absolute management of Lassa especially during the challenging times of the COVID‐19 pandemic. Hence, the need to create awareness on the vaccine availability and make it readily accessible, in addition to early diagnosis and treatment, is paramount in creating a LF free society.

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Lassa fever and COVID‐19 in Africa: A double crisis on the fragile health system

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Lassa Fever (LF), an acute zoonotic disease caused by the Lassa fever virus (LFV), has approximately 2 million annual occurrences with 5000 to 10000 deaths.

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example of disease in a post-conflict country: LF in post-civil war sierra leon

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example of disease in a post-conflict country: LF in post-civil war sierra leon

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Lassa Fever in Post-Conflict Sierra Leone

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Author Summary Lassa fever (LF) is a major public health threat in West Africa. After the violent civil conflict in Sierra Leone (1991 to 2002) ended, the LF research program at Kenema Government Hospital (KGH) was reestablished. Higher CFRs in LF patients were observed compared to studies conducted…

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case reports about LF and pregnant women

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case reports about LF and pregnant women

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Lassa fever (LF), an acute viral haemorrhagic fever, is an endemic zoonotic viral infection in West Africa countries with up to 15% case fatality rate. Though a rodent-borne infection, it can also be transmitted from person to person during the care of sick relations or more commonly in health care settings as a nosocomial infection. Vertical transmission from mother to child has been documented. We report 2 cases of LF among pregnant women which were managed at the University College Hospital, Ibadan, between September and October 2014. Both patients were in their early 20s with only one surviving the disease. Both had supportive therapy but none had antiviral therapy. This report emphasized the importance of early presentation, high index of suspicion, prompt diagnosis, and early commencement of supportive therapy in the management of patients suspected with LF especially in the era of other viral haemorrhagic infections.

1. Introduction

Lassa fever (LF) is an acute viral haemorrhagic illness caused by Lassa virus, a member of the virus family Arenaviridae. LF was first recognized in 1969 in Lassa, Northern Nigeria, and has since become endemic especially in West Africa countries like Sierra Leone, Guinea, Mali, Liberia, and Nigeria [ 1- 6]. It is of great public health concern such that a single case is referred to as an outbreak. There are estimated 300,000-500,000 cases of Lassa fever each year [ 3- 5] with a mortality rate of 15%-20% for hospitalized patients, which can become as high as 50% during epidemics and up to 90% in third-trimester pregnancies for both the expectant mother and fetus [ 4, 6]. Presently, there is no licensed vaccine or immunotherapy available for prevention or treatment of this disease and although the antiviral drug ribavirin is somewhat beneficial, it must be administered at an early stage of infection to successfully alter disease outcome, thereby limiting its utility. In addition, there is no commercially available Lassa fever diagnostic assay, which hampers early detection and rapid implementation of existing treatment regimens (e.g., ribavirin administration) [ 3- 6].

The documented prevalence of antibodies to the virus in Africa has varied between 7% in Guinea [ 2], 8-52% in Sierra Leone [ 6], and 21% in Nigeria [ 7]. The main mode of spread is from infected rodents to man although it can equally be transmitted from person to person commonly through ingestion and inhalation but also through exchange of body fluids in a Lassa virus infected individual, during the care of sick relations or in health care settings [ 1]. Secondary human spread can result from percutaneous or permucosal exposure to blood and other infected body fluids, especially if the fluids contain blood. Studies have shown that person-to-person transmission of this virus contributes less than rodent contact to human infection [ 8] and this type of transmission can be minimized by timely infection-control measures, careful management of infected patients, and, in some cases, administration of personal protective equipment (PPE) to health care workers [ 1, 9].

Transmission peaks in the acute phase of fever when the virus is present in the throat while in the early stage its symptoms and signs are indistinguishable from those of other common conditions in the tropics such as malaria, typhoid, and other viral haemorrhagic fevers [ 10]. The symptoms occur 1-3 weeks after the exposure though it could be asymptomatic thereby making the clinical diagnosis difficult [ 1, 3- 6]. About 80% of Lassa fever virus infections symptoms are mild and are undiagnosed but death may occur within two weeks after symptom onset due to multiorgan failure [ 1].

LF can be diagnosed by immunofluorescent antibody (IFA) or enzyme linked immunosorbent assay (ELISA) by detecting an increase in antibody titer or the presence of specific IgM and IgG as well as Lassa antigen [ 1, 9]. Reverse transcription-polymerase chain reaction (RT-PCR) can be used in the early stage of disease [ 1]. In facilities with high containment laboratories, the virus can be cultured in 7-10 days while immunohistochemistry performed on formalin-fixed specimens can be used to make a postmortem diagnosis [ 1, 9, 11]. There are currently commercially available rapid diagnostic kits that are effective in facilitating early diagnosis and prompt management [ 4].

In the absence of vaccine, LF can be prevented by keeping rats away from homes and rapidly investigating a suspected case which will help in facilitating prompt response in terms of treatment, implementation of isolation measures, using PPE, contact tracing, and the education of people in high-risk areas on ways of reducing rodent populations in their homes [ 11- 13]. Supportive treatment in form of fluid and electrolyte management is often necessary.

In pregnancy, Lassa virus causes high maternal and fetal mortality due to high viral concentrations in the maternal blood, placenta, and fetal tissue [ 4, 7, 14]. Good maternal outcomes have been previously documented [ 4] but spontaneous abortion complicates the infection with an estimated 95% mortality in fetuses of infected pregnant mothers, 92% in early pregnancy, 75% in the third trimester, and 100% in the neonatal period for term babies [ 1, 7, 14]. Maternal mortality is 7% in the first two trimesters, 30% in the last trimester, and 50% within one month of delivery compared to less than 15% in the general population of nonpregnant women being 13% [ 7, 11, 14].

The outbreak of LF in a community within Ibadan at a time between 2014 and 2015 when some West African countries were plagued with Ebola viral disease (another viral haemorrhagic disease with similar symptoms) made it pertinent to highlighting the challenges of care and management for these two pregnant women in our facility.

2. Case Presentation

Case  1. Mrs. AA, 23-year-old gravida 2, para 1 (1 alive) trader, presented on September 16, 2014, at the gestational age of 18 weeks and 5 days with 5-day history of fever and 12-hour history of altered level of consciousness. There was associated history of jaundice, vomiting, and mild abdominal pain. There was no history of ingestion of herbal concoction. She had no cough, skin reaction, haematuria, or diarrhea. History of travel or contact was negative. She had no known intercurrent illness. At examination, she was icteric and dehydrated but neither pale nor febrile. She also had nontender hepatomegaly (6 cm below the right costal margin) but other parameters were essentially normal.

An initial assessment was that of fulminant hepatitis with encephalopathy to consider acute fatty liver of pregnancy and exclude viral haemorrhagic fever. Laboratory investigations were essentially normal except for markedly deranged liver enzymes (alkaline phosphatase, ALP, 176 iu, aspartate aminotransferase, AST, 2877 iu, and alanine aminotransferase, ALT, 2229 iu, with elevated direct and indirect bilirubin). Hepatitis and human immunodeficiency virus (HIV) screening were negative while the obstetric ultrasound was essentially normal for the gestational age.

In the absence of rapid diagnostic kits, samples were obtained for serology and RT-PCR for Lassa and Ebola viruses while she was comanaged with the gastroenterologist. She had repeated seizures with associated dyspnea and worsening coma scale. Viral haemorrhagic screening test was positive for Lassa virus and negative for Ebola but unfortunately further management was hampered by nonavailability of space in the intensive care unit.

She remained on supportive therapy till the third day when she had cardiopulmonary arrest with failed attempt at resuscitation. Consent for autopsy was declined by the relatives on religious grounds but necessary control and surveillance measures were ensured on the first-line contacts.

Case  2. Mrs. OB, 24-year-old nulliparous legal practitioner, presented at the accident and emergency department of our facility on October 2, 2014, at the gestational age of 12 weeks with 2-day history of continuous high grade fever, body weakness, vomiting, and diarrhea though the review of the systems was essentially normal. About 10 days before the onset of symptoms, she had visited a part of Ibadan for one week which was later confirmed to have an outbreak of LF. She was conscious and alert with no associated constitutional symptoms.

Laboratory studies including the liver function parameters were within the normal limits in this particular patient. Viral screening was however positive for Lassa virus but negative for Ebola virus. She was nursed on the isolation ward and all her vital signs remained within normal limits except the temperature which was intermittently high but resolved after 2 days. The patient and her husband declined ribavirin based on the fear of its risks. They requested transferring their care to Lagos and have since been lost to follow-up.

3. Discussion

In 2012, there was an outbreak of Lassa fever in Nigeria in which 623 cases and 70 deaths were recorded [ 15]. In Ibadan, between September and November 2014, there were 80 suspected cases of viral haemorrhagic fever managed in our hospital, out of which 27 were positive for Lassa fever.

The first case presented in this study presented in an advanced stage of the disease which was evidenced by the development of complications such as loss of consciousness and seizure disorders. While waiting for the result of the viral screening, she was commenced on supportive therapy. Unfortunately, she died before the result was available and therefore could not be commenced on antiviral therapy. The antiviral drug of choice, ribavirin, is effective in the treatment of Lassa fever but only if administered early in the course of illness [ 1, 3- 6]. In a study of Lassa fever in Sierra Leone, it was observed that patients with a high risk of death who were treated for 10 days with intravenous ribavirin, begun within the first six days after the onset of fever, had a case fatality rate of 5% while patients whose treatment began seven or more days after the onset of fever had a case fatality rate of 26% [ 16]. In another study it was reported that Lassa fever carries a high risk to the fetus throughout pregnancy and to the mother, especially in the third trimester with a documented maternal death of 20% due to Lassa fever [ 14, 15].

The second case presented in the hospital at the early stage of the infection and with supportive therapy she improved clinically. Surviving LF without antiviral treatment has been documented but rare [ 17].

A case report written previously suggested that patients with laboratory confirmed Lassa fever who have episodes of falling temperature with low level of AST (usually <150 iu per liter) on admission and improving clinical state may not require treatment with ribavirin [ 17]. This further emphasized the importance of early presentation, high index of suspicion by the health workers, commencement of supportive therapy, and prompt diagnosis of Lassa fever. Elevated levels of interleukin 6 have been documented as biomarker for poor prognosis but its assay is yet to be commenced in our centre. However, ensuring an effective vaccine against LF will remain a desirable step [ 18].

4. Conclusion

These reports emphasized the importance of early presentation, high index of suspicion among health workers, and prompt diagnosis of patients with Lassa fever so as to reduce the mortality rate of this infection in our area. They also suggest that conservative management of symptoms of the disease and its complications may improve survival of individuals with this infection. Availability of rapid diagnostic kit will significantly contribute positively to good outcome for the patients if it can be made available in our hospitals.

Competing Interests

The authors declare that there are no competing interests regarding the publication of this paper.

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Lassa Fever in Pregnancy: Report of 2 Cases Seen at the University College Hospital, Ibadan

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Lassa fever (LF), an acute viral haemorrhagic fever, is an endemic zoonotic viral infection in West Africa countries with up to 15% case fatality rate. Though a rodent-borne infection, it can also be transmitted from person to person during the care of sick relations or more commonly in health care settings as a nosocomial infection.

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Institute of Lassa Fever Research and Control, Irrua Specialist Teaching Hospital, Irrua, Nigeria

Department of Paediatrics, Irrua Specialist Teaching Hospital, Irrua, Nigeria

Department of Community Medicine, Irrua Specialist Teaching Hospital, Irrua, Nigeria

Department of Obstetrics and Gynaecology, Irrua Specialist Teaching Hospital, Irrua, Nigeria

Department of Surgery, Irrua Specialist Teaching Hospital, Irrua, Nigeria

Department of Nursing Services, Irrua Specialist Teaching Hospital, Irrua, Nigeria

Department of Medical Records, Irrua Specialist Teaching Hospital, Irrua, Nigeria

Nigeria Centre for Disease Control, Abuja, Nigeria

Department of Tropical Medicine, Bernhard Nocht Institute for Tropical Medicine & I. Department of Medicine University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, United States

Department of Biological Sciences and African Center of Excellence for Genomics of Infectious Diseases, Redeemer's University, Ede, Nigeria

Department of Virology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany

Department of Microbiology, Ambrose Alli University, Ekpoma, Nigeria

Background: The general lack of comprehensive data on the trends of Lassa fever (LF) outbreaks contrasts with its widespread occurrence in West Africa and is an important constraint in the design of effective control measures. We reviewed the contribution of LF to admissions and mortality among hospitalized patients from 2001 to 2018 in the bid to address this gap.

Methods: Observational study of LF caseload and mortality from 2001 to 18 in terms of the contribution of confirmed LF to admissions and deaths, and case fatality (CF) among patients with confirmed LF at a specialist center in Nigeria. The diagnosis of LF was confirmed using reverse transcription polymerase chain reaction (RT-PCR) test, and medians and frequencies were compared using Kruskal-Wallis, Mann-Whitney and χ2 tests, with p-values <0.05 taken as significant.

Results: The contribution of confirmed LF to deaths (362/9057, 4.0%) was significantly higher than to admissions (1,298/185,707, 0.7%; OR [95% CI] = 5.9 [5.3, 6.7], p < 0.001). The average CF among patients with confirmed LF declined from 154/355 (43%) in 2001-09 to 183/867 (21.1%) (OR [95% CI] = 2.9 [2.2, 3.7], p < 0.001) in 2011-18. The annual CF declined from 94% in 2001 to 15% in 2018 whereas the caseload increased from 0.3 to 3.4%. The outbreaks were characterized by irregular cycles of high caseload in 2005-2007, 2012-2014, and 2016-2018, and progressive blurring of the seasonality.

Conclusion: LF outbreaks in Nigeria have upgraded spatially and temporally, with the potential for cycles of increasing severity. The strategic establishment of LF surveillance and clinical case management centers could be a pragmatic and cost-effective approach to mitigating the outbreaks, particularly in reducing the associated CF. Urgent efforts are needed in reinvigorating extant control measures while the search for sustainable solutions continues.

Introduction

Lassa fever (LF), one of the severe viral hemorrhagic fevers (VHF), was first reported from Nigeria in 1969 ( 1). Since then, there have been repeated outbreaks in Nigeria ( 2) and other West African countries ( 3, 4), particularly Sierra Leone ( 5) and Liberia ( 6) but also Togo ( 4), and the Republic of Benin ( 4). In addition, LF is the most exported VHF, particularly to Europe ( 4, 7, 8) and America ( 4, 8- 10).

The control of LF has been neglected ( 2), even within the context of neglected tropical diseases. Nonetheless, one of the foremost priorities should be how to reduce the high hospital case fatality ( 2, 6, 11), which has remained relatively high despite the availability of an arguably effective therapeutic agent, ribavirin, for several decades ( 12, 13). While many factors could account for the high case fatality, including late presentation ( 12) and other delays in diagnosis ( 14), the dearth of human and material resources for diagnosis and treatment may be important but somewhat overlooked factors. The place of adequate case management in the outcome of such severe VHFs as Ebola and Marburg is attested to by North-South ( 15) and even intra-country South-South ( 16) variations in outcome.

ISTH was designated a COE for the control of LF by the Federal Government of Nigeria in 2002. However, the subsisting situation before 2008 was far from that of a COE: Blood samples had to be sent to Europe and South Africa among others for confirmatory tests, and the results were usually not available for weeks to months; Patients with suspected LF were admitted in improvised isolation rooms or bays within the general wards, with attendant high risk of nosocomial transmission and workplace infection of staff; And, the supply of ribavirin, the agent primarily used for the treatment of LF cases, was erratic. This scenario was not good either for case management or the prevention of nosocomial transmission, nor the institution of public health measures. The ILFR&C was therefore established by the Board of Management of ISTH in 2007 as "an aggregate indigenous response to comprehensively address the LF epidemic" in seeking to address these and other challenges related to LF control in the pursuit of the hospital's mandate as a COE.

Among others, the activities of the Institute include surveillance, clinical virology, clinical case management, community engagement, outreach and enlightenment campaigns, and research. To carry out these activities, the Institute has a robust staff compliment inclusive of public health physicians, clinical virologists, nurses, biomedical scientists, medical laboratory scientists, community health extension workers, and clinicians. It also has research and training collaborations with Bernhard-Nocht Institute for Tropical Medicine (BNITM), Hamburg, Germany, Harvard and Tulane Universities, USA, and Public Health, England since 2007, 2007, and 2012, respectively. In addition, the Institute has established a close working relationship with the Nigeria Centre for Disease Control, NCDC.

A biosafety level-2+ Diagnostic and Research Laboratory (D&RL) that started operation in September 2008 is available at the Institute. There is also a 35-bedded purpose-built Lassa Fever Ward (LFW), with a dedicated Lassa fever Intensive Care Unit (LFICU), which was opened in September 2010. The LFW has sections for adult males and females including pregnant women, and children while the LFICU has a hemodialysis unit that has been in operation since 2011. A composite panoramic view of the ILFR&C including diagnostic activities in the D&RL, the LFW, hemodialysis unit, and potshots from one of the visits of the WHO during 2018 outbreak and a training session are shown in Figures 1A,B.

Figure 1. Panoramic view of the Institute of Lassa Fever Research and Control (ILFR&C), Irrua Specialist Teaching Hospital, Nigeria and its activities. (A) Panoramic view of the Institute, diagnostic activities, Training Center and training activities, and hand washing stand. (B) Panoramic view of clinical case management and diagnostic activities, and awareness campaign match on a national Lassa fever day.

The first onsite laboratory confirmation of the diagnosis of LF in ISTH was made in the D&RL on September 27th, 2008 while about 1,300 patients with confirmed LF were treated in the hospital between January 2001 and September 2018. The cost of confirmatory tests for LF has been free as part of on-going research programs as well as support from NCDC, while the Federal Government of Nigeria provides Ribavirin and other care commodities such as personal protective equipment without charge for the treatment of patients.

After almost two decades since the designation of ISTH as a COE, over a decade of establishment of the Institute, and about 8 years of operation of the LFW, we thought that it might be helpful to broadly review our experiences in surveillance and clinical case management. Specifically with the view to sharing our experience in the diagnosis and treatment of LF, we sought first to review the temporal and spatial variations of the caseload of LF as indices of the trend of LF outbreaks in Nigeria. Second, we sought to describe the trends in case fatality pre- and post-commissioning of the LFW as an index of the potential impact of dedicated treatment facilities. A third perspective was to describe the determinants of hospital outcome of LF cases as seen in our center. Nigeria stands out among LF endemic countries due to her large population and landmass, widespread nature of the outbreaks, and diversity of the Lassa virus (LASV) lineages in circulation ( 23, 24). In addition, the outcome of LF had usually been worse in Nigeria compared to other West African countries ( 6). It could therefore be expected that the experiences gained in the mitigation of LF in Nigeria may be of significance in the mitigation of LF in West Africa in general.

Subjects and Methods

The study was carried out at the ILFR&C, ISTH, Irrua, Edo State, Nigeria.

Ethical Considerations

The study was granted exemption from ethical review by the Research and Ethics Committee of ISTH being one of service evaluation, which "is exempt from ethical review according to the National Code of Health Research Ethics, National Health Research Ethics Committee, Federal Ministry of Health, Nigeria" ( 22). Neither the diagnosis of LF nor treatment with ribavirin or any of the other drugs or treatment modalities was experimental in nature but were part of regular clinical practice and the data in the manuscript were drawn from available records so generated. This paper is based on de-identified data on patients evaluated and/or treated for LF in ISTH since the inception of the Lassa fever control program of the hospital.

Methodology

Data on suspected cases and the results of confirmatory tests were retrieved from the electronic worksheet maintained at the ILFR&C. This contains information on the socio-demographic characteristics, clinical features, and results of laboratory tests carried out on patients. Data on admissions and deaths for the period 2011-2018 were retrieved from the ward register maintained in the LFW. The corresponding data on admissions and discharges of confirmed cases for the period 2001-2010 were obtained from the Medical Records Department of our hospital.

Clinical Case Management of Patients Admitted to the LF Ward

Team leads, Medical, Pediatric, Obstetric, Nursing care, Laboratory services/IPC, and LF Diagnostic Services, supervise the management of patients in the LFW. Currently, the usual bundle of treatment includes intravenous fluids at maintenance rate, intravenous ribavirin given as a single dose daily ( 28, 29) or as divided doses daily ( 12, 13, 30), broad spectrum antibiotics usually intravenous ceftriaxone, intravenous antimalarial drugs usually artesunate, and supportive care. The latter include monitoring of vital signs, correction for fluid and electrolyte imbalance, cardiovascular support with bolus doses of intravenous fluids and treatment with inotropic drugs in patients with septic shock, maintenance of fluid balance and hemodialysis for patients with acute kidney injury (AKI), intravenous anticonvulsants for the control of convulsions, and monitoring of fluid and electrolyte balance. Patients with suspected acute abdomen are co-managed with the surgical team ( 29, 30). Pregnant women are managed based on viability of the fetus as assessed clinically and with Ultrasound Scan (USS) examination; those with non-viable fetus undergo evacuation of the uterus while those with a viable fetus are placed on intravenous ribavirin ( 31). Details of the individual subspecialty management are described in separate communications ( 25, 29- 32).

However, the treatment regimen has been evolving, and the standard of care was not always the same throughout the period of the study. For example, hemodialysis was not accessible to patients with LF at ISTH until 2011. Before then, those with AKI were usually referred for dialysis at St. Nicholas Hospital, Lagos, a private hospital which as at then was the only one willing to accept infected patients for hemodialysis. As another example, recommendations for the treatment of septic shock have also been evolving.

Data Analysis

Categorical variables were summarized as frequencies and proportions or percentages while the quarterly distributions of caseload of LF and admissions to the LFW for the years 2009-2018, being skewed, were expressed as median and interquartile range (IQR) instead of mean ± standard deviation ( 33). Groups were compared using n × n Chi-square (χ ²) tests or non-parametric tests (Kruskal-Wallis and Mann-Whitney tests) as appropriate ( 33, 34), using Open Epi software ( http://www.openepi.com); p < 0.05 were taken as significant.

The results are presented as overall and disaggregated based on the origin of the patients (Edo State vs. other States) and the odds ratio (95% confidence interval) (OR [95% CI]) of observing a confirmed case given the origin of the patient (Edo State vs. other States) were calculated. The OR (95% CI) for death as an adverse outcome was also calculated for the period 2001-2009 vs. 2011-2018, the study periods pre- and post-commissioning of the LFW.

Results

Annual Variations in Numbers of Suspected and Confirmed Cases of LF, 2008-2018

Data for 2001-08 was not available because no systematic testing of suspected cases using the criteria described under "Subjects and Methods" was undertaken over that period as we did not have facilities for testing locally then. From September 2008 to August 2018, LF was confirmed in 1,637 (11.6%) of 14,168 patients with suspected LF using LASV-RT-PCR test. The variations in annual numbers of suspected and confirmed cases are shown in Table 1 and the trend in annual numbers of confirmed cases illustrated in Figure 2 while the variations in test output from suspected cases (number of confirmed cases as a percentage of the number of suspected cases) are shown in Table 2 and illustrated in Figure 3. The variations are shown as overall and disaggregated, Edo State vs. other states.

There were mild increases in the number of confirmed cases in 2009 and 2012/13 followed by a decreases in 2010-11 and 2014/15, after which there was a steady increase from 2016 through 2017 to 2018 overall, 2017-2018 in Edo State and 2016-2018 in the other States ( Figure 2). The trend of overall steady decline in the proportion of suspected ( p < 0.001) and confirmed ( p < 0.001) cases from Edo State was highly significant ( Table 1). Edo State accounted for >85% of both suspected and confirmed cases from 2008 to 2011, 70-83% of suspected cases vs. 44-63% of confirmed cases from 2012 to 2015 and 55-66% of suspected vs. 36-52% of confirmed cases from 2016 to 2018. There were significant differences ( p = 0.003- p < 0.001) between the proportions of suspected vs. confirmed cases from Edo State as a percentage of the total numbers of suspected and confirmed cases from 2012 to 2018 ( Table 1).

The involvement of geopolitical zones is also shown in Table 1. Suspected cases were drawn from an average of 3.5 (58.3%) of the six geopolitical zones in 2008-11 and 5.9 (98.3%) in 20012-18 while confirmed cases were from 2 (33.3) in 2008-11 and 5.3 (88.3%) in 2012-18. There were suspected cases from Abuja and the Federal Capital Territory (FCT) every year from 2009 to 18 while confirmed cases were recorded therefrom only in 2009, 2012-13, and 2018.

The annual variations in test output are shown in Table 2 and illustrated in Figure 3.The overall test output dropped from about 30% in 2008 through 16% in 2009 to 7-11% between 2010 and 2016, and then increased to about 13% in 2017 and 18% in 2018. The output from among suspected cases from Edo State followed a similar pattern but that among patients from other states increased again much earlier from 2012 after the initial decreases from 2008 through 2009 ( Figure 3). The trend in test output from 2008 to 2018 was highly significant overall ( p < 0.001), and among cases from Edo State ( p < 0.001) and the other States ( p < 0.001) ( Table 2). The test output among suspected cases from the other States was also significantly higher than that among cases from Edo State from 2012 to 2018 ( p < 0.001 yearly for the difference in test output between Edo State and the other States from 2012 to 18) ( Table 2).

Annual Variations in the Contribution of Lassa Fever to Admissions and Deaths, 2001-18

Between January 2001 and September 30th, 2018, a total of 185,707 patients were admitted at ISTH, Irrua and among them 9,057 (4.9%) died. Patients with confirmed LF constituted 1,298 (0.7%) of the admissions, and 362 (4%) of the deaths (OR [95% CI] of the contribution of LF to deaths vs. admissions = 5.9 [5.3, 6.7], p < 0.001). The overall case fatality among patients with LF was 362/1,298 (27.9%) while that among the other patients was 8,695/184,409 (4.7%). The difference was highly significant (OR [91% CI] = 7.8 [6.9, 8.8], p < 0.001).

The annual variation in contribution of LF to admissions and deaths are shown in Table 3 and illustrated in Figure 4. The contribution of LF to admissions was low (range = 0.3-0.8%) from 2001 to 2015, then rose sharply from 2016 (1.1%) through 2017 (1.4%) to 2018 (3.4%); the mean ± standard deviation of the proportion of admissions from 2001 to 15 (0.5% ± 0.2%) was significantly ( p < 0.001) lower than that from 2016 to 18 (1.9% ± 1.2%). The overall trend was that of an increasing caseload (both in terms of the number of cases and the proportion of admissions) ( p < 0.001), with a major surge in number and proportion of admissions with LF in 2018 ( n = 248, 3.4% of admissions) but which began from 2016 and lesser surges in 2005 (0.7%) and 2012 (0.8%) ( Figure 4). The trend of the contribution to deaths was that of irregular increases and decreases, with peaks in 2006 (5.4%), 2010 (4.1%), 2016 (6.6%), and 2018 (8.8%), and troughs in 2004 (2.1%), 2008 (1.6%), and 2015 (1.5%) ( Table 3 and Figure 4). The variation in contribution of LF to death among in-patients from 2001 to 18 was highly significant ( p < 0.001, Table 3).

Seasonality of Cases and Admissions

The proportion of cases varied from 37 to 82% in the 1st quarter, 8-30% in the 2nd quarter, 6-22% in the 3rd quarter, and 11-33% in the 4th quarter ( Table 4). The median and IQR of the proportions of confirmed cases in each quarter is shown in Table 4. The median proportion of the first quarter was significantly higher than that of the second ( p < 0.001), third ( p < 0.001), and fourth quarters ( p < 0.001). The difference between the third and fourth quarter medians approached significance ( p = 0.053) whereas the difference between the second and third quarter medians ( p = 0.579), and that between the second and fourth quarter medians ( p = 0.182), were not significant. Used as a "crude" index of the measure of seasonality of LF, the proportion of cases in the first quarter was <50% in 6 of the 10 years, 2009-12, 2014, and 2017 ( Table 4).

The quarterly variation in the proportion of cases of LF admitted to the LFW from 2011 to 18 is shown in Table 5 and illustrated in Figure 5. The proportion was highest in the 1st quarter from 2011 to 18 (35-76%) except in 2014 when it was highest in the 2nd quarter (42%) ( Table 5 and Figure 5). The median and IQR of the proportion of admissions in each quarter is shown in Table 5. The median of the first quarter was significantly higher than that of the second ( p = 0.001), third ( p < 0.001), and fourth quarters ( p < 0.001) whereas the differences between the others were not significant ( p = 0.105 for 2nd vs. 3rd quarters; p = 0.189 for 2nd vs. 4th quarters; and p = 1.000 for 3rd vs. 4th quarters). Used again as a "crude" index of the measure of seasonality of LF, the proportion of cases in the 1st quarter was ≤50% in 4 of the 8 years, 2011-12, 2014, and 2017 ( Table 5 and Figure 5).

Hospital Case Fatality Among Patients With LF, 2001-18 and 2001-9 vs. 2011-18

Table 6 shows the variations in case fatality (CF) among patients admitted with LF from 2001-18 while Figure 6 illustrates the trend; both Table 6 and Figure 6 also show the contribution of LF to admissions during the period for ease of comparison. The trend in CF among patients with LF showed 2 phases: the first was that of a sharp decline from 2001 to 05, while the second was that of irregular increases followed by decreases over a period of generally steady gradual decline from 2006 to 18 ( Table 6 and Figure 6). The trend in contribution to admissions also showed 2 phases, but these were the opposite of the trend in CF: an initial phase of gradual increase with irregular periods of alternating increases and decreases from 2001 to 15 followed by a period of sharp increases from 2016 to 18 ( Table 6 and Figure 6).

The averaged CF in 2001-2009 was 154/355 or 43.4% (range = 33.9-93.7%) while that in 2011-2018 was 183/867 or 21.1% (range = 15.3-38.7%). The averaged CF was significantly higher in 2001-09 (OR [95% CI] = 2.9 [2.2, 3.7], p < 0.001). The mean ± standard deviation of CF in 2001-2009 (50.3 ± 22.8%) was also significantly higher ( p = 0.007) than that of 2011-2018 (23.6 ± 8.1%). We excluded the admissions in 2010 from this analysis because some were in the "general" wards (CF = 24/70, 34.3%) and some in the LFW (CF = 1/6, 16.7%; p = 0.072 for the difference).

Review of Factors Associated With Case Fatality Among Hospitalized Patients With LF

We reviewed the factors associated with outcome in four observational studies from ISTH, two published, one in press, and one unpublished but under review for publication; two involved adults, one involved children, and one pregnant women. The presence of shock, bleeding, encephalopathy (as evidenced by seizures and/or coma), and acute kidney injury (AKI) were the major determinants of outcome. Presentation with abnormal bleeding, and ≥1 feature of AKI was associated with increased case fatality in all four studies while encephalopathy was associated with increased case fatality in three of the four ( Table 7). Presentation with ≥2 of these complications or danger signs would be equivalent to a SOFA score of ≥2 ( 35) as criteria for diagnosis of severe infections.

Origin/Sources of the Patients in the Study, 2008-18

As with the data on annual variations in numbers of suspected and confirmed cases of LF, data for 2001-08 was not available because we lacked facilities for laboratory diagnosis before 2008 and data availability is limited to Edo State only for the period. A total of 10,367 (73.2%) of the 14,168 suspected cases investigated between 2008 and 2018 were from Edo State while 3,801 (26.8%) were from other states/FCT and Abuja. The corresponding figures for confirmed cases ( n = 1637) were 957 (58.5%) and 680 (41.5%). The test output among suspected cases from Edo State (957/10,367; 9.2%) was significantly lower than that from other states (680/3,801; 17.9%) (OR [95% CI] of test output from Edo State vs. other states = 0.5 [0.4, 0.5], p < 0.001).

The geopolitical origins of suspected and confirmed cases are shown in Table 8 and illustrated in Figure 7. South-South Zone had the largest proportion of suspected cases, 76.4 vs. 23.6% from the other five zones. The zone also had the largest proportion of confirmed cases, 60.5 vs.39.5% from the other zones. Within the South-South Zone, Edo State had 95.7% of suspected and 99.7% of confirmed cases. Outside the South-South Zone, Northwest had the lowest proportion of both suspected and confirmed cases (0.3 and 0.2%, respectively) while Southwest had the highest (8.5 and 19%, respectively).

Discussion

We set out in this paper to review our experience of the contribution of LF to admissions and mortality among in-patients, and to determine the trend in outcome of hospitalized cases. The results show more fully the spread of LF in Nigeria, the variations in intensity of the outbreaks in affected areas, and the blurring of its seasonality. The results also show the continuing decline in case fatality in contrast to the increasing caseload. Furthermore, the results of mini meta-analysis of determinants of outcome in our center show the feasibility of classification of case severity using exclusively clinical indices. We discuss these and other observations in the study.

Caseload of LF

There was a major surge in caseload in 2018, which began earlier in 2016 and before which there were lesser irregular surges in 2005, and 2012-2014. The trend demonstrates the steadily increasing burden of LF in Nigeria and neighboring countries ( 4) in the absence of effective control measures ( 2). The results of recent studies, which show that increased rodent-to-human transmission may be a major factor in the recent surges ( 36- 38) suggests that anti-rodent measures and other measures designed to reduce rodent-to-human transmission should constitute a major thrust of the control measures in the country and in the sub region, if the trend is to be stemmed and/or reversed.

It was required to disaggregate the trend in Edo State from that observed with respect to the other states for two main reasons. First, much is already known about LF outbreaks in Edo State ( 22, 25- 27, 39), which remains the epicenter of outbreaks in the country ( 40, 41). Second, it seemed reasonable that the data on the contribution of LF to admissions and deaths should be from the established diagnosis and treatment center in the state, which was the only such center in the country until recently. We believe that the results, including the differences in test output and caseload between Edo and other states, justify the disaggregation.

The relative preponderance of both suspected and confirmed cases from Edo State decreased over the years, from 2008 to 2018, although the reduction in the proportion of confirmed cases was more from 2012. Thus, the proportion of suspected cases decreased from 89.4% in 2008 to 59.4% in 2018 and that of confirmed cases from 85.7 to 51.5%. The difference between Edo State and others might be because of greater public and health care workers' awareness owing to the longer standing and perhaps more intense efforts in enlightenment/awareness campaigns spearheaded by ISTH and its ILFR&C. The trend gives hope that investments in public awareness campaigns and capacity building of healthcare workers toward raising the index of clinical suspicion for the purpose of engendering early diagnosis and treatment can lead to reductions in caseload and improved outcome over time.

It is possible that the trend in test output is a reflection of the sampling efforts, which could be uneven between different years, and different geopolitical regions but this position is not supported by the available data. The trend is evident both with regard to the absolute numbers of confirmed cases and with regard to the test output from suspected cases, which should be capable of correcting for whatever bias there might be in the use of absolute numbers only. In addition, the trends both countrywide and in Edo State were similar; Edo State accounted for about 40% of the cases seen nationally during the 2018 and 2019 outbreaks ( 41), and might have accounted for much higher proportions in earlier outbreaks.

Seasonality of LF

We have in earlier reports ( 22, 39) drawn attention to the blurring of the seasonality of LF in Nigeria, and similar observations have been reported from Sierra Leone ( 11, 21). The results of this study, which covers a much longer period, confirms earlier observations ( 11, 21, 22, 39). The transition might be due to many factors including population displacements due to perennial flooding, perhaps due to the famed problem of global warming ( 42, 43), and the increasing frequency and severity of civil unrest and insurgency ( 44). There are also the growing problems of poverty ( 45), slum dwellings and poor management of solid wastes in the Nigeria. These coupled with the now regular burning of bushes, might be hitherto unacknowledged factors associated with increased opportunities for rodent-to-human transmission of infection. In addition, variations between geographic regions and the risk of LF in West Africa has been associated with variations in annual rainfall ( 18) and climatic changes due to global warming ( 42, 43) could therefore also by itself be a factor in the change in seasonality. Irrespective of the factors involved, however, the change in seasonality makes outbreak preparedness and response an all year-round necessity.

The need for all-year round preparedness is buttressed by the findings that although the proportion of cases of LF was highest overall (59.9% of the annual prevalence) in the 1st quarter from 2008 to 18, <50% of the annual prevalence was in the 1st quarter in six (2009-12, 2014, and 2017) of the 10 years. In addition, 27.6% of the combined annual prevalence, range = 14.5% in 2016-47.2% in 2017, occurred during the raining season period (2nd and 3rd quarters). Shaffer et al. ( 11) in Sierra has also observed the occurrence of a second peak of LASV antigenemia during the rainy season, the other peak being in the dry season.

Case Fatality of LF

The trend in case fatality of LF contrasts sharply with the trends of the contribution of LF to admissions and deaths among in-patients. These contrasts emphasize the place of deployment of effective medical response in the control of outbreaks, to give hope in place of despair. The occasional and irregular surges in case fatality in 2003, 2006, and 2016 were most probably due to acute shortages of ribavirin, which is unfortunately recurrent ( 2, 46). There might also have been occasions of an increase in case severity, particularly in 2016 ( 47), which are not readily explained. Increased virulence, perhaps due to the emergence of a new lineage or sub-lineage to which there might have been low herd immunity was one of the explanations advanced earlier for the apparent increase in case severity in 2016 ( 47) but this explanation is not supported by the results of genomic analysis of the 2018 surge in LF outbreak in Nigeria which showed that the increased caseload was more likely due to increased rodent-to-human transmission and not the appearance of new lineages ( 36- 38).

Other studies on CF during the 2016 outbreak in Nigeria have reported varied figures: Buba et al. ( 48) reported a CF of 59.6% among 47 confirmed cases, and noted that CF was up to 77.8% in some states; Shehu et al. ( 49) reported a CF of 36% among 11 confirmed cases and noted that this was lower than the 83% from an earlier study in 2012 in the same state; and the European Centre for Disease Prevention and Control (ECDC) reported a CF of 60% among 57 confirmed cases ( 4). The CFs reported by Buba et al. ( 48) and ECDC ( 4) are about double the CF of 31.4% in our series in 2016, a year during which there were shortages of ribavirin, while the CF of 36% reported by Shehu et al. ( 49) in 2016 is not markedly different from ours. Neither Buba et al. ( 48) or ECDC ( 4) attempted to explain the high CFs while Shehu et al. ( 49) thought that the much higher CF in 2012 was due to late presentation, low index of suspicion among clinicians and lack of access to ribavirin.

Early diagnosis and treatment, engendered by regular public awareness campaigns and capacity building of health care workers toward the development of a high index of clinical suspicion, as well as the build up of experience in clinical case management, might be factors in the steady reductions in case fatality at our center. The ready availability of diagnostic facilities locally since 2008 ( 22), which facilitates early diagnosis, would clearly also be an important factor. There are also the on-going improvements in treatment facilities at the center, particularly since the commissioning of the LFW. For example, dedicated facilities for hemodialysis of infected patients with AKI became available in 2011 and facilities for respiratory support toward the end of the surge in outbreak in 2018 while the facilities for component blood therapies are under development.

It is however, notable that there were already marked decreases in CF before the establishment of the LFW and that the rate of reduction after its commissioning was lower than expected. In relation to the first observation, it is possible that the more severely ill patients were assessed and treated for LF in the days before establishment of the Institute and availability of diagnostic facilities. This situation, which could be associated with a low index of suspicion, low levels of awareness and late presentation, acting in conjunction with the frequent lack of ribavirin and limited capacity for supportive care, would not surprisingly be associated with high CFs as in the first few years covered by this report. As awareness and index of suspicion increased, and there was earlier presentation and diagnosis/institution of treatment, decreases in CF could be expected. This might also partly explain the sharp drops in CF in the earlier part of the study period. Furthermore, the practically of onsite diagnosis, and potential for early diagnosis, following the establishment of the D&RL before the coming on board of the LFW may also have contributed to the further declines in CF in the initial part of the study.

The apparent lack of a marked impact of establishment of the LFW on the trend in CF could be due to several factors: First, marked improvements in clinical case management experience might have predated its establishment. Second, it could be that the impact of treatment with ribavirin has approached saturation. Third, the supportive clinical care facilities, other than those for hemodialysis, are still at an early stage of development, and it should be reasonable to expect that the availability of more comprehensive and specialized care could result in further improvements. Thus, we have had patients who might have died if the provision of hemodialysis for AKI or assisted ventilation for respiratory failure was unavailable. These factors taken together could mean that further significant reductions in case fatality might not be forthcoming without the establishment of full facilities for enhanced supportive or critical care and/or the development of pharmacologic therapies that are synergistic with or alternative to ribavirin ( 50- 52). However, although this underlies the need for acceleration of the development of additional treatment resources, it would also not be unreasonable to further explore public health measures directed at further addressing delays in presentation. Most of the cases originate at the community level and a rigorous exercise of community engagement coupled with active community-based surveillance could address the problems of late presentation and case detection. This requires deliberate efforts in building the capacity of volunteers at the community level.

The lack of results from this study that are strongly supportive of the impact of dedicated treatment facilities on CF notwithstanding, we strongly believe that the pooling of human and material resources for clinical case management of severely ill patients with LF and other severe VHFs could greatly impact on the hospital outcome. Although there was no dramatic decline in CF after establishment of the LFW, it is still nonetheless notable that the trend of a steady decline in CF was sustained. For emphasis perhaps, the expectation of further rapid reduction could have remained unmet because the process of provision of facilities for enhanced supportive/critical or intensive care for severely ill patients has been protracted. We can attest from our experience in the treatment of severe LF ( 25, 53, 54), and from the experience of others in the treatment of severe VHFs such as Ebola both overseas and in the sub region ( 16, 55- 57), to the benefits of such provisions as hemodialysis and respiratory support among others. We yet lack adequate facilities for the treatment of patients with encephalopathy, as well as facilities for provision of adequate cardiovascular support while those for hematologic support are under development and facilities for blood gas determination, et cetera, are awaited. Also, the provision of point-of-care facilities for radiologic, microbiologic, and even clinical chemistry assessment has remained a challenge. And, even though hemodialysis has been available for some time, it is expensive and many patients are not able to meet the cost. We anticipate that it should be possible to more fully assess the impact of the dedicated diagnosis and treatment facility in years to come when these provisions have been made.

Thus, we believe that the establishment of dedicated, strategically located treatment centers can help to address major aspects of the clinical challenge of LF, particularly the (currently) unacceptably high case fatality in endemic areas ( 6, 11, 22, 25, 48, 49, 58). Adequate treatment is crucial to reduction of case fatality in severe VHFs ( 15, 16) and the establishment of dedicated centers and triaging of patients for referral, working in tandem can enable the pooling of personnel and facilities for the optimization of treatment approaches in reducing the risk of death. Therefore, we propose that treatment centers, preferably coupled with surveillance centers, be established in strategic "zones" in endemic areas as "hubs" with "spokes" drawn from surrounding health facilities in a "hub and spoke" referral system. For example, Nigeria with her 6 geopolitical zones could have one center per zone. The centers should be capable of accommodating surges in caseload and illness severity and could also serve the clinical care needs of patients with other severe VHFs. We are gratified to note that drawing on our experience, the strategic establishment of dedicated treatment centers in Nigeria has commenced as part of the national medical response to LF. And, it could clearly be quite helpful to compare the experience of the new centers with ours in future.

To be effective, these centers should have capacity for enhanced supportive/critical care ( 59- 62) for the goal-directed treatment of patients with severe infections. This is because the pathophysiologic changes in severe VHFs ( 59, 63, 64) are akin to those of severe sepsis/septic shock ( 65) and should be addressed accordingly ( 59, 64). Septic shock, less commonly hypovolemic shock from fluid loss or severe bleeding, AKI, and respiratory failure are the common complications associated with death in severe sepsis ( 35, 66, 67), including severe VHFs such as Ebola ( 35, 65, 68) and LF ( 22, 25).

We should also emphasize the added medical and public health advantages in having dedicated surveillance and treatment centers. None of the staff in the ILFR&C and its LFW has taken ill with LF since establishment of the LFW in 2010. On the other hand there have been regular reports of infection of healthcare workers (HCW) from other places ( 29, 40, 69). For example, among 381 confirmed cases of LF from 21 states reported between January 1st and February 24th 2019, 15 (3.9%) were infected HCWs ( 70), and 45 (7.1%) out of 633 confirmed cases between January 1st and December 31st in 2018 were also HCWs ( 71). Mustapha ( 72) has detailed the incidence of infection of HCW with LF in recent years in an editorial aptly titled "Lassa fever: Unveiling the misery of the Nigerian health worker"; "as at January 28, 2018" for example, 10 (13%) of 77 confirmed cases of LF were among HCWs and 4 of the 10 died ( 72).

The trend in CF and the factors associated with outcome among our patients also merit discussion from another perspective, which could be a further pointer to some of the actions required in improving the outcome of severe LF/other severe VHFs in West African. The association between mortality and presentation with ≥2 of four clinical features including abnormal bleeding, shock, indices of AKI, and features of encephalopathy, was crosscutting among the various demographic groups of patients with LF ( 22, 25, 31, Akpede et al., personal communication). This could form the basis for the classification of patients and choice of treatment pathway on presentation. We suggest that the presence of ≥2 risk factors could be taken as indicative of severe infections and the need for referral to higher levels of care while patients with mild infections (absence of a major risk factor) are managed locally. Operational research is however, required to validate this recommendation.

Other Issues

There are at least three other issues arising from or during this study that should be discussed. First, at least two ribavirin dosage regimens in the treatment of LF are in use in Nigeria. The first entails the administration of single or once daily doses ( 22, 28, 29) while the second entails the administration of multiple daily doses ( 12, 13, 30, 32). Beside this difference, the total dose of ribavirin administered over the course of treatment in the first regimen is lower than that in the second. The first regimen might theoretically be attended with fewer side effects, lower risk of nosocomial transmission of infection, and lower cost of care. However, there have as yet been no formal comparative analysis of the associated course and outcomes of illness associated with the two regimens, but anecdotal observations suggest that they might be comparable. Further studies are required to guide recommendations on the optimal dose and dosage of ribavirin, and could also be useful in addressing the growing concern with treatment costs ( 73).

Second, the costs of diagnosis and treatment might become major issues in access to care, with implications for the sustainability of surveillance and treatment programs. The dependence on overseas partners is becoming more strenuous and exerting. It is clearly not sustainable, and calls for urgency in the development of more affordable reagents and diagnostic alternatives. It also calls for the development of indigenous methods of diagnosis that do not depend on the importation of all component reagents from overseas.

Reducing the cost of care is of a growing concern ( 73), and is an important issue with the dependence on out-of-pocket expenditure as a major source of healthcare financing in many developing countries ( 74, 75). The Federal Government of Nigeria has been subsidizing the clinical care of infected persons but this has sometimes been plagued unfortunately by recurrent and severe shortages of ribavirin ( 46), with serious implications. A sense of urgency in the development of less expensive and preferably sub regionally sourced pharmaceutical products is desirable.

Third, the prevalence of LF of about 12% among patients with suspected infections in 2008-2018 is higher than the 6% reported from among a small group ( n = 31) of febrile patients evaluated in 2003-2004 ( 26) but lower than the 42% reported from a larger group ( n = 60) of patients investigated while on admission with "suspected Lassa fever" in 2005-2008 ( 27). In the latter study ( 27), LF was suspected in the presence of negative laboratory investigations for malaria and bacterial infections or persistence of fever despite treatment with antimalarial and antibiotic drugs or presentation with known signs of LF ( 27). Although the criteria are like some of those used in the current study, there might have been an over-selection of severely ill patients who were more likely to be positive for LF. In contrast, the criteria used in the selection of patients in the current study were more robust. In addition, the patients involved in this study were recruited from all settings in the hospital, and not limited to those on admission as in the earlier study ( 27). The test output in our study might thus be more representative of the true prevalence of LF among febrile patients seen in endemic areas in Nigeria. The increase in prevalence over that reported from the earlier study ( 26) might be reflective of a true increase in the prevalence of LF rather than being simply a reflection of improvement in diagnostic capacity and increased clinical index of suspicion.

Ilori et al. ( 41) reported a decline in test output among suspected cases from 31% during the peak of the 2017 outbreak to 21.6% in 2018. In contrast, there was an increase from 12.8% in 2017 to 17.8% in 2018 among our patients. The decrease reported by Ilori et al. ( 41) is difficult to explain against the background that the 2018 outbreak was the largest reported so far from Nigeria. On the other hand, the lower test output in our study could be because Ilori et al.'s report ( 41) was in relation to the peak of the outbreaks in 2017 and 2018 whereas ours represented yearly averages.

The trends in test output and caseload further strengthens the general case for the screening of febrile patients in endemic areas for LF ( 2, 26, 32) and thus the imperative of accelerating the development of rapid diagnostic tests/point of care tests for the diagnosis of LF, and other VHFs in endemic areas. The trends also underlie the need for a sense of urgency in scaling up the institution of general control measures and the further development of medical counter measures as advocated by the WHO/Welcome Trust ( 80). LF has the potential to frustrate attainment of the sustainable development goals in West African countries.

Limitations of the Study

There at least two limitations inherent in this study, which should be discussed. First, the LF case fatality component of the study was restricted to patients with RT-PCR-confirmed infections whereas some patients might have died at the emergency units of the hospital (Accident and Emergency Unit, Children Emergency Ward, Labor Ward) with unconfirmed infections. In addition, none of the confirmatory tests used in the study has 100% sensitivity ( 81). Thus, the estimates of the contribution of confirmed cases to admissions and deaths as well as the estimates of caseload and case fatality of LF could be different from that reported in this paper. However, we have no reasons to think that the differences are substantial.

Second, a detailed analysis of the factors associated with hospital outcome of cases of LF was not done, partly because it was beyond the scope of the study and also because it was the subject of other studies ( 22, 25, 31, Akpede et al., personal communication). Furthermore, the lack of uniformity in the standard of care during the period covered by the report would have made it quite difficult controlling for the many potential confounding factors that might feature owing to the variation. Despite this, however, we believe that the results give at least a broad view of the trend in outcome over the years.

Conclusion and Recommendations

This is the first "comprehensive" report on LF in Nigeria that assessed the trends for at least a decade and inter-related the spatial and temporal aspects of the outbreaks. It shows that LF has strengthened both spatially and temporally, with the potential for cycles of outbreaks of increasing severity. The report should serve to reduce the level of uncertainty regarding the spatial and temporal epidemiology of LF in Nigeria and further calls for urgent efforts in reinvigorating control measures and reinvigorating the search for sustainable solutions ( 2).

In addition, the study demonstrates the gains of a sustained trend in the reduction of LF case fatality, which might be related to the establishment of a dedicated clinical case management center. Thus, replication of the establishment of "hubs" of LF surveillance and treatment centers could be a pragmatic and cost-effective approach in rising to the clinical challenge of LF in endemic countries.

Author Contributions

GA, DAA, SO, CH, PS, DEA, MR, SG, MM, and AD: conceptualization of study. NA, GO, EM, FO, OO, CO, and CIk: data retrieval. GA and AC data analysis. GA: initial draft of manuscript. GA, EO-E, DAA, SG, MR, CH, AC, CIk, and CIh: revision of draft manuscript. GA, SD, MO, RA, AA, FO, AD, MM, and SO: clinical care of the patients. IO, DIA, and EM: laboratory work. GA, ET, DAA, SO, SD, MM, AD, CIh, NA, and EO-E: administration. SG, CH, PS, DAA, SO, GA, and CIh: securing funding for the project.

Funding

This work was supported by grants GU 883/1-1 and GU 883/4-1 from the German Research Foundation (DFG), the Volkswagen Foundation, the Global Health Protection Program of the German Government, and the German Partnership Program for Excellence in Biological and Health Security, and by US Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Grant Numbers U19AI110818 and R01AI114855. The National Human Genome Research Institute, National Institutes of Health, Department of Health and Human Services, under Grant Numbers U01HG007480 and U54HG007480, also supported the work while the Federal Government of Nigeria through the Federal Ministry of Health supports Irrua Specialist Teaching Hospital. The World Bank Project ACE019, the Henry M. Jackson Foundation and the Bill and Melinda Gates Foundation in addition supported CH and PS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We acknowledge the support of the Federal Government of Nigeria over the years through the FMOH, the support of our partners (BNITM, Harvard University and Public Health England, among others), the support of the NCDC, the support of Hon. Patrick Ikhariale, Member House of Representatives, representing Esan West, Esan Central, and Igueben Federal Constituency, 2007-2014, the support of the successive Managements and Boards of Management of the ISTH, and the recent interest and support of Edo State Government. The history of the ILFR&C would be incomplete without emphasizing the role of the partners, particularly BNITM and Harvard, who assisted with the design, provision of equipment, and training of laboratory scientists for the takeoff of the Diagnostic and Research Laboratory (D&RL) and have continued to support our research activities in many ways. Recently also, at the height of the 2018 outbreak Edo State Government assisted with the provision of dialysis machines, ventilators, and a borehole. We also acknowledge the support of the Management of St. Nicholas Hospital, Lagos in the provision of dialysis for infected patients.

References

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Caseload and Case Fatality of Lassa Fever in Nigeria, 2001-2018: A Specialist Center's Experience and Its Implications

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Background: The general lack of comprehensive data on the trends of Lassa fever (LF) outbreaks contrasts with its widespread occurrence in West Africa and is an important constraint in the design of effective control measures.

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Lassa fever–induced sensorineural hearing loss: A neglected public health and social burden

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Although an association between Lassa fever (LF) and sudden-onset sensorineural hearing loss (SNHL) was confirmed clinically in 1990, the prevalence of LF-induced SNHL in endemic countries is still underestimated. LF, a viral hemorrhagic fever disease caused by Lassa virus (LASV), is endemic in West…

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hearing loss associated with lassa fever along with Ebola and zika

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hearing loss associated with lassa fever along with Ebola and zika

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Abstract.

The neglected tropical diseases Zika, Ebola, and Lassa fever (LF) have all been noted to cause some degree of hearing loss (HL). Hearing loss is a chronic disability that can lead to a variety of detrimental effects, including speech and language delays in children, decreased economic productivity in adults, and accelerated cognitive decline in older adults. The objective of this review is to summarize what is known regarding HL secondary to these viruses. Literature for this review was gathered using the PubMed database. Articles were excluded if there were no data of the respective viruses, postinfectious complications, or conditions related to survivorship. A total of 50 articles were included in this review. Fourteen articles discussing Zika virus and subsequent complications were included. Across these studies, 56 (21.2%) of 264 Zika-infected individuals were found to have HL. Twenty-one articles discussing Ebola virus and subsequent complications were included, with 190 (5.7%) of 3,350 Ebola survivors found to have HL. Fifteen additional articles discussing LF and subsequent complications were included. Of 926 individuals with LF, 79 (8.5%) were found to have HL. These results demonstrate a relationship between HL and infection. The true prevalence is likely underestimated, however, because of lack of standardization of reporting and measurement. Future studies of viral sequelae would benefit from including audiometric evaluation. This information is critical to understanding pathophysiology, preventing future cases of this disability, and improving quality of life after survival of infection.

INTRODUCTION

Tropical diseases have immense societal impact due in large part to their myriad long-term sequelae. Classically, these disabilities include physical impairments such as blindness, limb and physical deformities, an increased number of negative maternal and neonatal outcomes, and delayed physical or mental development. Furthermore, the association of these illnesses with poverty and the loss of productivity resulting from these disabilities lead to increased levels of stigma and social isolation, which contributes to the total burden of disease. The calculation and comparison of the number of disability-adjusted life years (DALYs) lost because of neglected tropical diseases (56.6 million DALYs) to other more common diseases such as HIV/AIDS (84.5 million DALYs) and malaria (46.5 DALYs) illustrates the large impact of these diseases on the populations they affect.1,4,5 Hearing loss (HL) is an often neglected and understudied sequelae of these infections, which contributes to the number of DALYs lost. Hearing loss affects more than 1.3 billion people worldwide and is now the 4th leading cause of years lived with disability.7 The effects of HL are lifelong and span from speech and language delays in childhood to restricted employment opportunities in adults and accelerated cognitive decline in older adults. The global burden of HL is unequally distributed, with more than 80% of affected individuals living in low- and middle-income countries, the very places where access to hearing care is limited.

Viruses were first established as an etiology of HL in the 1950s and are suspected to contribute to 12.8-25% of sudden-onset HL cases. Zika, Ebola, and Lassa fever (LF) are all tropical diseases which have received little worldwide attention until recent epidemics, and each of these viruses has been reported to be associated with HL. By comparing the prevalence reported for these and other viruses, Zika, Ebola, and LF may be associated with HL prevalence, that is, up to 300× greater than that of more common and better understood viral etiologies. The true burden of HL secondary to Zika, Ebola, and LF is unknown, however, and may be underreported because of lack of proper measurement of this chronic disability. Despite the paucity of data, the World Health Organization (WHO) recognizes the potential public health impact of these associations and has requested a review of the existing literature on Zika, Ebola, and LF for the upcoming World Report on Hearing, to be released in 2020. The objective of this review is, therefore, to describe what is known regarding HL secondary to these three tropical diseases, identify gaps in knowledge, and propose areas of research to increase our understanding of pathophysiology and potentially lead to new treatment modalities for viral-mediated HL.

METHODS

This literature search and analysis was conducted from August 2018 through April 2019. All study designs, publication dates, and languages were considered. Literature was gathered from PubMed using key terms and Boolean operators. Key terms used included the following: Zika, Ebola, Lassa, Survivors, Sequelae, HL, Hearing Impairment, Deafness, Complications, Congenital, and Post-Ebola Syndrome. Abstracts and titles of all retrieved studies were reviewed for mention of secondary complications, and the full texts of relevant articles were obtained. Articles were excluded if there were no data or discussion of the respective viruses, postinfectious complications, or conditions related to survival but not directly caused by the virus itself. Data regarding demographics and HL were gathered and aggregated according to the respective cause of infection. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed as applicable in creation of this review.

RESULTS

Two thousand nine hundred ninety-three total articles were identified by this methodology. Of these, 2,909 articles were excluded based on abstract and title review and 34 articles were excluded based on full article review. A total of 50 articles were included in the analysis ( Figure 1 ).

Hearing loss and Zika.

Fourteen articles discussing Zika virus and subsequent complications in 347 individuals were included in this review ( Table 1 ). Across studies, 56 (21.2%) of 264 individuals were found to have some degree of HL. Four of the fourteen articles described acquired HL in adults following Zika infection ( Table 1 ). The HL in these cases varied from moderate to severe and was reported as both unilateral and bilateral, with most patients experiencing recovery to normal or previous thresholds. Ten articles presented complication data of congenital Zika syndrome related to HL ( Table 1 ).18,22,25-29,33-35 The majority of these studies used standard HL screening methods for infants, including measurement of auditory brainstem response and otoacoustic emission, which assesses cochlear function.18,25-29,31,33,34 The proportion of infants with reported HL in these studies varied from 6% to 68%. One article presented in-depth testing of two individuals, one of whom had moderate unilateral HL and one with normal hearing thresholds. Importantly, the patient with HL was also found to have poor speech recognition scores in the same ear. These studies suggest the association of Zika virus not only with HL but also with auditory processing disorders such as auditory neuropathy. The wide range of HL prevalence found by this analysis indicates the need for further research on this disability.

Table 1

First author

Publication year

Study type

Sample size (n)

Age group

HL Screening Method

HL result (n, %)

Unilateral HL (n)

Bilateral HL (n)

Control group (n, % HL)

Does not look for sound

8 (17)

No response to word "No"

20 (43)

Calle-Giraldo ³⁴

2019

Prospective cohort

68

Neonatal

ABR

6 (9)

3

3

ND

Hearing loss and Ebola.

Twenty-one articles discussing Ebola virus and subsequent complications in 5,055 individuals were included in this review ( Table 2 ). Of the 3,385 individuals studied, 223 (6.6%) were found to have some degree of HL using audiometric evaluation and survey instruments. Only one of 21 articles used audiometry to objectively measure HL. This study, by Rowe et al.,19 recruited convalescent Ebola survivors and household contacts following the conclusion of the 1995 Ebola outbreak in Kikwit, DRC. The study defined HL as an inability to hear at least 1 frequency between 0.5 and 4 kHz at 25 dB. The authors reported that 18 (64.3%) of 27 individuals developed HL after surviving Ebola infection, and 11 of these patients had developed HL within the first 6 months following discharge from Ebola treatment centers. At the end of the 21-month follow-up period, seven individuals (26%) were found to have persistent HL. The remainder of the articles that described HL as sequelae of Ebola relied on questionnaires or self-report of symptoms.36-46,50,52-54 The proportion of individuals reporting HL in these articles varied widely, from 0% to 22%. In these studies relying on self-report, HL typically arose late in the course of the disease and persisted throughout recovery. Self-reported timing of HL onset was broad, spanning from the initial hospital admission to as many as 350 days post discharge. Despite continued complaints of HL, the lone study to measure HL by audiometry demonstrated resolution in several individuals.19 Thus, it is possible that HL related to Ebola may resolve spontaneously. Data regarding Ebola-related HL is scarce, and more studies are required to elucidate the persistence of this disability.

Table 2

First author

Publication year

Study type

Sample size (n)

Median age (years)

HL screening method

HL results (n, %)

Days to HL onset (median DPI)

Control group (n)

Shantha41

2017

Cross-sectional

96

38.6

Self-reported

10 (10.4)

NR

ND

Hearing loss and LF.

Fifteen articles discussing LF and subsequent complications in 1,207 individuals were included in this review ( Table 3 ). Of 15 articles, 11 presented HL data. Across studies, 53 (6.0%) of 898 individuals were found to have some degree of HL using audiometric evaluation and survey instruments. Thirty-eight (71.7%) affected individuals were found to have bilateral HL, and 15 (28.3%) individuals demonstrated unilateral HL. Audiometry was used to characterize HL in five of 11 studies. The mean pure-tone average (PTA) for all reported data was 66.5 dB, which is consistent with severe HL. This measurement was gathered from 139 (15.5%) of 898 individuals with an average age of 33.7 years. Several studies monitored progression of HL. Eleven of 22 individuals were found to have residual HL at 1 year, and one reported residual loss 4 years after the initial infection.20,57-59,62,63 At the end of the 1 year period, nine of these individuals were found to have severe HL, including three cases of bilateral HL and six cases of unilateral.20 Cummins and colleagues included three separate evaluations to characterize the HL secondary to LF infection.20 In the third evaluation, a case-control study of 32 individuals with HL in comparison with 32 individuals without, 26 (81.2%) of 32 individuals with HL were found to be seropositive for LF antibodies versus only six (18.7%) of those without HL. Interestingly, only 13 (50%) seropositive individuals with HL were aware that LF might be the cause of HL.20 These studies indicate that LF may be an underappreciated cause of HL in LF endemic areas. This analysis finds that the prevalence of LF-related HL ranges widely, from 0% to 81.25%. More robust studies are needed to determine the relationship between symptomatic disease, HL and seropositivity.

Table 3

First author

Publication year

Study type

Sample size

Mean age (years)

HL screening method

HL results (n, %)

Average severity of HL*

Unilateral HL

Bilateral HL

Days to HL onset (median DPI)

Control group (n, % HL)

DISCUSSION

The major lifelong sequelae of neglected tropical diseases are secondary disabilities following infection. HL is an understudied morbidity following these viral tropical disease pathogens. This review examines the association between HL and Zika, Ebola, and LF viruses, summarizing what is known regarding this complication. Results of this analysis demonstrate the range of HL prevalence associated with each of these viruses. The major limitation is the small amount of data that exist to fully characterize this disability and the lack of studies that follow any standard protocol of measurement or reporting, which likely underestimates the true prevalence and undermines data quality. Furthermore, few studies attempt repeat screening to detect progression or late-onset HL. Social stigma and psychiatric complaints are reported complications of both HL and these tropical diseases.8,9,70-74 However, no studies have examined the relationship between these complications.

Important similarities and differences were observed in HL associated with the three viruses. Zika virus, a well-described cause of birth defects, may also lead to various levels of HL in both acquired and congenital cases. Interestingly, in-depth audiology testing in the acquired cases of Zika-related HL suggested the presence of a sound processing disorder called auditory neuropathy. The association of Zika with this type of retrocochlear HL has important public health implications because auditory neuropathy is not identified with typical methods of hearing screening. Hearing loss in congenital Zika cases was commonly reported in children older than 1 year, suggesting that Zika may lead to delayed onset of HL and emphasizing the need for recurring screening beyond the newborn period.21 The most well-supported hypothesis of the mechanism for Zika-related HL is direct viral invasion of neurons in the auditory pathway. The virus has been shown to have preference for undifferentiated neurons, which may explain the devastating complications resulting from congenital cases in comparison with adults and may serve as a possible explanation for the observed auditory neuropathy findings.

In contrast to the auditory neuropathy observed in Zika, HL associated with Ebola and LF is similar to common viral-mediated etiologies of HL and can be easily detected with traditional hearing screening. Prevalence of Ebola- and LF-mediated HL is likely underestimated in this review because of the paucity of studies using objective audiometric measurements. The one study that used objective audiometric testing found a staggering 64.3% of Ebola survivors developed some form of postinfectious HL.19 However, because of the low quality of data presented by these studies, it must be emphasized that these results must be interpreted with caution, highlighting the urgent need for future studies of HL secondary to tropical diseases. Various mechanisms have been proposed for the etiology of post-Ebola syndrome symptoms, depicted by detection of the virus in areas such as the eye and semen, including the persistence of virus in immune-privileged sites leading to a direct cytopathic effect.37,40,81-84 It has been theorized that the penetration of the virus into cerebrospinal fluid may allow passage into the perilymph via the cochlear aqueduct, leading to the development of Ebola-associated HL. Although most cases of LF are thought to be asymptomatic or subclinical, sequelae, including HL, have been noted to occur across all severities of disease.20,62,85-87 The pathogenesis responsible for LF-related HL is poorly understood, and both immunologic and cytopathic mechanisms have been proposed.

Future directions.

Hearing loss is a growing public health concern with lifelong impact. As DALYs and the economic impact secondary to HL continue to increase, it is imperative that steps are taken to address this disability. These results demonstrate that the prevalence of HL associated with Zika, Ebola, and LF infection may be up to 300× greater than common viral etiologies of HL. It is critical that future studies of these tropical infections include objective audiological evaluation using a standard, WHO-supported definition of HL, coupled with longitudinal rescreening to accurately determine the prevalence and fully characterize the natural course of HL secondary to these viruses. Furthermore, future studies of sequelae can provide evidence of causal relationships between these tropical viruses and HL, identify risk factors for diagnosis and prognostication, and elucidate mechanisms leading to HL. Such knowledge is crucial to the development of public health interventions to prevent this understudied disability and improve the quality of life after survival from these devastating and neglected tropical diseases.

Acknowledgments

This project was supported by NIH Research Training Grant #D43 TW009340 funded by the NIH Fogarty International Center, NINDS, NIMH, and NHBLI and serves as background for the World Health Organization World Report on Hearing.

REFERENCES

19. Rowe AK, et al. 1999. Clinical, virologic, and immunologic follow-up of convalescent Ebola hemorrhagic fever patients and their household contacts, Kikwit, Democratic Republic of the Congo. Commission de Lutte contre les Epidémies à Kikwit. J Infect Dis 179 (Suppl 1): S28-S35. [[ PubMed] Google Scholar]

35. Wheeler AC, 2018. Development of infants with congenital Zika syndrome: what do we know and what can we expect? Pediatrics. 141 (Suppl 2): S154-S160. [[ PubMed] PMC free article] [ Google Scholar]

42. Bwaka MA, et al. 1999. Ebola hemorrhagic fever in Kikwit, Democratic Republic of the Congo: clinical observations in 103 patients. J Infect Dis 179 (Suppl 1): S1-S7. [[ PubMed] Google Scholar]

70. Arlinger S, 2003. Negative consequences of uncorrected hearing loss-a review. Int J Audiol 42 (Suppl 2): 17-20. [[ PubMed] Google Scholar]

75. Parra VM, Matas CG, Neves IF, 2003. Estudo de caso: neuropatia auditiva. Rev. Bras. Otorrinolaringol 69: 283-288. [ Google Scholar]

Articles from The American Journal of Tropical Medicine and Hygiene are provided here courtesy of The American Society of Tropical Medicine and Hygiene

# data[].attributes.attachment_link.title
A Review of Hearing Loss Associated with Zika, Ebola, and Lassa Fever

# data[].attributes.attachment_link.description
The neglected tropical diseases Zika, Ebola, and Lassa fever (LF) have all been noted to cause some degree of hearing loss (HL). Hearing loss is a chronic disability that can lead to a variety of detrimental effects, including speech and language delays ...

# data[].attributes.headline
decline in seeking healthcare in the aftermath of the 2013 - 2016 ebola and increasing cases of Lassa fever as a result

# data[].attributes.subject
decline in seeking healthcare in the aftermath of the 2013 - 2016 ebola and increasing cases of Lassa fever as a result

# data[].attributes.attachment_link.readable_text
Mikaela R. Koch,

Lansana Kanneh,

Paul H. Wise,

Lianne M. Kurina,

Foday Alhasan,

Robert F. Garry,

John S. Schieffelin,

Jeffrey G. Shaffer,

Donald S. Grant

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Abstract

Background

The West African Ebola epidemic of 2013-2016 killed nearly 4,000 Sierra Leoneans and devastated health infrastructure across West Africa. Changes in health seeking behavior (HSB) during the outbreak resulted in dramatic underreporting and substantial declines in hospital presentations to public health facilities, resulting in an estimated tens of thousands of additional maternal, infant, and adult deaths per year. Sierra Leone's Kenema District, a major Ebola hotspot, is also endemic for Lassa fever (LF), another often-fatal hemorrhagic disease. Here we assess the impact of the West African Ebola epidemic on health seeking behaviors with respect to presentations to the Kenema Government Hospital (KGH) Lassa Ward, which serves as the primary health care referral center for suspected Lassa fever cases in the Eastern Province of Sierra Leone.

Methodology/Principal findings

Presentation frequencies for suspected Lassa fever presenting to KGH or one of its referral centers from 2011-2019 were analyzed to consider the potential impact of the West African Ebola epidemic on presentation patterns. There was a significant decline in suspected LF cases presenting to KGH following the epidemic, and a lower percentage of subjects were admitted to the KGH Lassa Ward following the epidemic. To assess general HSB, a questionnaire was developed and administered to 200 residents from 8 villages in Kenema District. Among 194 completed interviews, 151 (78%) of respondents stated they felt hospitals were safer post-epidemic with no significant differences noted among subjects according to religious background, age, gender, or education. However, 37 (19%) subjects reported decreased attendance at hospitals since the epidemic, which suggests that trust in the healthcare system has not fully rebounded. Cost was identified as a major deterrent to seeking healthcare.

Conclusions/Significance

Analysis of patient demographic data suggests that fewer individuals sought care for Lassa fever and other febrile illnesses in Kenema District after the West African Ebola epidemic. Re-establishing trust in health care services will require efforts beyond rebuilding infrastructure and require concerted efforts to rebuild the trust of local residents who may be wary of seeking healthcare post epidemic.

Author summary

The West African Ebola epidemic of 2013-2016 killed nearly 4,000 Sierra Leoneans and devastated health infrastructure. There is limited information regarding the effects of the epidemic on health seeking behavior. Lassa fever is clinically similar to Ebola and is prevalent in Kenema District, located in the Eastern Province of Sierra Leone. There was a significant decline in the number of individuals seeking care for suspected Lassa fever following the epidemic. Responses to a questionnaire completed by 194 Sierra Leonean residents suggested that confidence in the healthcare system was not fully restored. Cost was identified as a major deterrent to seeking healthcare. Additional community sensitization is needed to convey the need, and importance, of seeking care for Lassa fever.

Citation: Koch MR, Kanneh L, Wise PH, Kurina LM, Alhasan F, Garry RF, et al. (2021) Health seeking behavior after the 2013-16 Ebola epidemic: Lassa fever as a metric of persistent changes in Kenema District, Sierra Leone. PLoS Negl Trop Dis 15(7): e0009576. https://doi.org/10.1371/journal.pntd.0009576

Editor: Christopher M. Barker, University of California, Davis, UNITED STATES

Received: June 26, 2020; Accepted: June 19, 2021; Published: July 14, 2021

Copyright: © 2021 Koch et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: Supported by National Institute of Allergy and Infectious Diseases grants/contracts AI104621, AI114855, AI115754, U19AI135995, and U01AI151812 (to LK, FA, JSS., RFG., JGS, and DSG) and by a Stanford University Undergraduate Research Grant (to MRK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: L.K., F.A., J.S.S., R.F.G., J.G.S., and D.S.G are members of the Viral Hemorrhagic Fever Consortium ( www.vhfc.org). The VHFC is a partnership of academic and industry scientists who are developing diagnostic tests, therapeutic agents, and vaccines for Lassa fever, Ebola, and other severe diseases. Tulane University and its various academic and industry partners have filed U.S. and foreign patent applications on behalf of the consortium for several of these technologies. Technical information may also be kept as trade secrets. If commercial products are developed, consortium members may receive royalties or profits. This does not alter our adherence to all policies of the NIH and PLOS Neglected Tropical Diseases on sharing data and materials. Financial and non-financial competing interests that the editors consider relevant to the content of the manuscript have been disclosed. R.F.G. is a co-founder of Zalgen Labs, LLC. All other authors declare no competing interests.

Introduction

The largest known Ebola epidemic in history began in 2013 ultimately spanning over 30 months and killing over 11,000 people in Guinea, Sierra Leone, Liberia and other countries [ 1, 2]. The epidemic was reportedly fueled and perpetuated by misinformation. Erroneous messaging, rumors, conspiracy theories-including that Ebola virus (EBOV) was a hoax and that consumption of bush meat was sustaining the outbreak-and predictions by government advisors that the outbreak would soon burn out, contributed to the public health disaster [ 3- 5]. Healthcare facilities, which were plagued by large numbers of heath care worker (HCW) deaths, became known by some as places where people went to die, and thus many individuals stopped seeking care altogether [ 6- 10]. The long-term effects that a traumatic event, such as the West African Ebola epidemic, pose to trust in the healthcare system are not well-documented in the scientific literature.

While Ebola was directly responsible for thousands of deaths in Sierra Leone (approx. 4000), changes in health seeking behavior (HSB) due to documented fear, misinformation, distrust, and a reduction in medical services, resulted in considerable non-Ebola mortality that continues to this day [ 11- 14]. Maternal and child health services were particularly affected [ 10]. At the height of the epidemic, there was a 31-37% drop in facility-based child deliveries and an 80% decrease in pediatric admissions for malaria in Sierra Leone [ 15]. Such reduction in healthcare provision was predicted to result in an additional 4,022 maternal deaths per year and 6,700 additional infant deaths per year in the region [ 10]. Additionally, the number of major surgeries conducted, in the region, during the West African Ebola epidemic decreased by 50% [ 16, 17]. Beyond the barriers present and inherent to the region (limited health service availability, poverty, lack of educational access), the epidemic may have left a lasting impact on the perceptions of, and attitudes towards, the healthcare system [ 18- 20]. Health seeking behavior, as assessed in this study, is defined as "a sequence of remedial actions individuals undertake to rectify perceived ill health" [ 21]. The underlying assumption is that health is influenced by behavior and behavior itself is subject to modification, as influenced by external and internal factors [ 22]. Proxies used for health seeking behavior in the literature include, healthcare consumption patterns, provider visits, and self-reported care seeking [ 23- 26].

In the wake of the Ebola epidemic, there has been significant investment and attention in health systems strengthening for the affected countries of West Africa [ 27, 28]. In April 2015, the Sierra Leone MoHS detailed a strategy to strengthen the health system which included, as its first key areas, establishing safe work settings and increasing the workforce [ 29]. NGO's in the area, such as Partners in Health, joined this objective working to improve the infrastructure of local health facilities [ 27]. In Kenema specifically, a new 48 bed Lassa fever ward was built following the epidemic [ 30]. While the desire to strengthen the health system is often the 'dominant narrative' after an epidemic, whether individuals are availing themselves of these services has not been adequately addressed in this setting [ 31]. This study sought to comprehend that knowledge gap by 1) assessing HSB of patients with suspected Lassa fever and 2) assessing general HSB of individuals through use of a questionnaire, in the Kenema district of Sierra Leone, a region that was severely impacted by Ebola. Based on the incredible shock to the system (societal, healthcare, governmental, economic etc.) and clear evidence of a dramatic change in health seeking behavior during the epidemic across the nation, the authors hypothesized that there would remain an impact on hospital attendance for Lassa fever observed in the population years after the epidemic concluded.

Lassa fever and Ebola are both hemorrhagic fevers-severe acute viral infections induced by Lassa virus (LASV), an arenavirus, and EBOV, a filovirus, respectively [ 1, 32]. Patients present with similar symptoms, including fever, malaise, vomiting, and bleeding. While many individuals infected with LASV do not present to a health care provider or facility, both prior to, and following the epidemic, those that do are often misdiagnosed as having influenza or malaria given the initial nonspecific symptoms and aggressive local treatment programs for malaria [ 32- 35]. LASV is transmitted through contact with Mastomys natalensis, the multimammate mouse, and other rodent reservoirs [ 36, 37]. Human to human transmission of LASV can also occur through contact with bodily fluids, similar to EBOV, although less frequently [ 38- 40]. The incubation period of Lassa fever is 6-21 days and symptoms may be mild and subclinical [ 35, 41, 42]. Severe cases progress to multi-organ failure and internal hemorrhaging. For those requiring hospitalization, the suspected case fatality rate is variable-between 20% in Nigeria to 70% in Sierra Leone [ 43, 44]. While there are no precise data on the incidence or prevalence of Lassa fever, it has been estimated that there are tens of thousands of deaths each year in West Africa [ 45]. The case fatality rate among hospitalized Lassa fever (LF) patients has been reported to be 69% [ 34].

The first goal of this study was to measure potential changes in HSB for suspected Lassa fever. Given the similarity in the diseases, HSB for Lassa fever may serve as a metric for the impact of the West African Ebola epidemic on HSB. The second goal of this study was to evaluate attitudes toward healthcare in a Lassa fever endemic region to identify barriers (e.g cost, time, information, trust etc.) and provide insights into whether there was a persistent effect of the epidemic on communities' relationship to, and perception of, their healthcare system.

Methods

Ethics statement

Compilation of surveillance data regarding patients presenting to Kenema Government Hospital (KGH) with suspected Lassa fever was approved by the Sierra Leone Ethics and Scientific Research Committee. Informed consent was obtained from suspected Lassa fever subjects presenting to KGH or one of its public health units or non-identifiable presentation frequencies were captured as part of routine hospital surveillance activities. Administration of health seeking behavior (HSB) questionnaires was approved by the Sierra Leone Ethics and Scientific Research Committee and the Institutional Review Board of Stanford University (#45598). All subjects who participated in the HSB questionnaire and/or their legal guardians provided written informed consent. Consent was obtained for each participant in the presence of a witness. Illiterate participants consented with a thumbprint rather than a signature. Given Mende, the most common local language, is written and read by very few, all consents and questionnaires were administered orally in the resident's preferred local dialect (Krio or Mende). As per the standard outreach team practice, upon entering the village, permission from the village leaders were obtained. All methods were carried out in accordance with relevant guidelines and regulations, including the Declaration of Helsinki.

Lassa fever surveillance database

Surveillance and demographic data on suspected Lassa fever cases have been captured using a suite of case report forms (CRFs) and log books at KGH since 2006. These data were captured from patient referral CRFs, pre-admission evaluation CRFs, patient medical charts (for admitted subjects), and a paper-bound log book maintained at the KGH Lassa Ward. Data for subjects presenting to KGH (regardless of admission status) were verified against pre-admission evaluation CRFs, and data for admitted subjects were verified against patient medical charts. Patient survival outcome was considered with the hierarchical order of validity: 1) Lassa Ward log book; 2) pre-admission evaluation CRF; 3) patient referral CRF; and 4) patient medical chart and nurses notes. Data were managed and linked across data sources using the SAS statistical package (version 9.4, SAS Institute, Cary, NC). The complete data capture and management process for suspected Lassa fever cases at KGH is described in further detail by Shaffer et al. [ 46].

Database analysis

For this study, the number of annual visits by age, gender, and survival outcome were abstracted from the Lassa fever surveillance database from 2011-2019. During the height of the West African Ebola epidemic (between May 24, 2014 and January 26, 2015), resources were devoted to containing the outbreak, and the data capture processes were adapted for incoming Ebola subjects. During this time, data for Lassa cases was sporadic and unreliable due to limited human resources available for Lassa fever surveillance; therefore, a detailed analysis on Lassa presentations during this time-period is not covered in this work.

HSB questionnaire

The questionnaire used in this study was adapted from a questionnaire on general health seeking behavior employed by Medair and Mercy Corp in Uganda, along with demographic data including age, gender, village of residence, and education [ 18]. Four specific study pertinent questions were added. The HSB questionnaire was provided in the two major written languages used in the villages, English ( S1 Table) or Krio ( S2 Table), but given low literacy rates, was delivered orally in Krio or Mende. Questionnaires were numbered and did not contain any personal identifiers. The villages were selected to represent a cross-section of the district based on distance from KGH (ranging from 22-51 km away) and population size (ranging from 600 to 2100 individuals). The outreach team had contacted each village in previous years either for case identification, educational programming directed at Lassa fever awareness, or for participation in Viral Hemorrhagic Fever Consortium (VHFC) research studies. The twenty questions took on average of ten minutes to complete. Overall, 200 participants were interviewed.

Statistical analysis

Surveillance data included both categorical and continuous responses and were presented as frequencies and percentages, or means and standard deviations, respectively. Data were classified in terms of pre-Ebola (2011-2013); Ebola (2014-2016); and post-Ebola (2017-2019) time periods according to data of clinical presentation to a hospital or public health unit in Sierra Leone. Nonparametric approaches (Fisher's Exact and Kruskal-Wallis tests) were used to account for departures in diagnostics for their analog parametric approaches (Pearson's chi square and ordinary one-way analysis of variance). The logistic regression approaches were applied due to the categorical nature of the data captured in the HSB survey. The Type 1 error threshold was set at 5%, and those p-values less than this threshold were considered as statistically significant. Statistical significance focused on differences observed between pre- and post-Ebola time periods (2011-13 and 2017-19, respectively) that were not due to chance alone. Significance tests were performed using the SAS statistical application (version 9.4, Cary, NC).

General HSB questionnaire responses (questions 1, 2, & 4-12) were analyzed to identify potential predictors of responses to the specific study pertinent questions (3 & 13-15). The analysis was performed by running logistic regression models in STATA v. 15.1 (Stata Corp, College Station, Texas). The logistic regression analyses were performed using full main effects (no interaction) models, where outcomes were dichotomized and modeled against subject age, gender, religion, education, and village of residence. For this analysis, age categories, 18-27, 28-36, 37-45, and 45+, were selected to accommodate the age inclusion criteria for the HSB questionnaire. All logistic regression analysis were run with Village 1 as the reference village. Village 1 was selected to be the reference village as it was one of two villages with the most respondents. One of these two villages was arbitrarily chosen as the reference village. When appropriate, responses were contracted to dichotomous measures. All chi-square tests and trends were analyzed using SAS/STAT software version 9.4 (Cary, NC). All statistical tests applied a type I error threshold of 5%.

Results

Persistent decline in suspected Lassa fever cases following the West African Ebola epidemic

In the three-year period (2017-19) following the West African Ebola epidemic, as compared to the three years prior to the epidemic, there was a decline in the numbers of subjects presenting to the Kenema Government Hospital (KGH) Lassa ward with suspected Lassa fever and the numbers of patients admitted to the KGH Lassa ward ( Fig 1, S3 Table). The average annual number of suspected cases of Lassa fever presenting to KGH was 698 from 2011 to 2013, the three years before the beginning of the epidemic ( Fig 1A). During the epidemic an average of 473 suspected cases presented, although, as noted, data collection was suboptimal during this period. In the three years following the epidemic, an average of 326 suspected cases presented to KGH ( Fig 1A). The proportion of cases post-epidemic was statistically significantly lower than the proportion pre-epidemic (p = .049, Table 1).

The decline in suspected cases was further reflected in a persistent decline in the numbers of admitted cases ( Fig 1B). The average annual number of cases admitted to the KGH Lassa ward was 148 from 2011 to 2013. During the West African Ebola epidemic an average of 128 cases were admitted. In the three years following the epidemic, an average of 38 cases were admitted ( Fig 1B).

Decline in suspected Lassa fever cases is not due to any major demographic shifts

The decline in suspected Lassa fever cases presenting to KGH did not appear to be due to a major reduction in presentation by any demographic group ( Fig 2). However, there were modest, but perceptible, changes in the demographic make-up of the cases presenting to KGH after the epidemic. From 2011 to 2018, 55-59% of patients evaluated for suspected Lassa fever were women. ( Fig 2A). However, in 2019 more males (179) than females (172) presented to KGH. There was a shift in the proportion of female suspected cases from pre-epidemic to post-epidemic of 59% to 55% respectively (p = .005; Table 1).

Age distributions were based on the 2015 Sierra Leone Census and suspected cases were analyzed in four groups: 0-4, 5-19, 20-34, and 35+. There were no major changes in the proportion of all annual suspected cases of Lassa fever associated with each age group, pre- versus post-epidemic ( Fig 2B, Table 1). The 20-34 year old age group was the age group with the largest proportion (34%) of suspected Lassa fever cases in the three year period before the West African Ebola epidemic (2011-13) and also during the three year period (2017-19) following the epidemic (37%, Fig 2B, Table 1). However, there was a reduction in the proportion of children and teenagers (5-19 years old) who presented as suspected Lassa fever cases. The decline in the total number of suspected Lassa fever cases pre-epidemic versus post-epidemic among each age group was significant (p < .001, Table 1).

Fatality rate of suspected cases pre-epidemic compared to post-epidemic

The case fatality rate of suspected cases (CFR; the proportion of deaths to deaths plus discharges) among persons admitted to the KGH Lassa Ward in the three-year period (2017-19) following the West African Ebola epidemic was similar to the CFR in the three-year period prior to the epidemic (2011-2013), 43% and 40% respectively, which was not significantly different ( Fig 3, Table 1). There was an increase in CFR among suspected Lassa fever cases during the epidemic (2014-16), which was likely due to patients presenting to the hospital at advanced stages of illness or with Ebola virus (EBOV). Over the time-period studied, the numbers of deaths and discharges amongst cases admitted to the Lassa Ward declined between the pre-Ebola and post-Ebola periods in parallel with the decline in number of admitted cases.

Assessment of health seeking behavior questionnaire

The questionnaire was successfully completed for 194 individuals (97%). For six respondents, the full questionnaire was not completed in a timely fashion, and they were excluded from analysis. Of the religions specified by respondents, 89% were Muslim, followed by 11% that were Christian. Of the genders specified by respondents, 66% were female and 34% were male. Of the education levels specified by respondents, 68% received no education, followed by 10% receiving primary education, 20% receiving a secondary education, and 2% receiving a tertiary education ( S4 Table). Of female respondents, 16% were currently pregnant, and 87% reported having at least two living children; culturally, given many female respondents had lost children, it was accepted to refer to children that were still alive, whereas medically, any full-term pregnancy may have been relevant Eighty-eight percent of women reported that they go to a government hospital to give birth, and 95% go to a government hospital for postnatal follow up and immunizations ( S4 Table).

Analysis of the responses to the questionnaire enabled several features of health seeking behavior (HSB) in Kenema District to be identified ( Fig 4, S5 Table). When seeking health care, 91% of respondents reported that they go to a government hospital ( Fig 4A). Only 2% reported they used a form of self-treatment or a traditional healer, and 68% of respondents confirmed receiving all their needed services at their health facility ( Fig 4B). Moreover, 60% of respondents reported they were likely to seek care within 24 hours of symptoms arising ( Fig 4C). According to 87% of respondents, cost was the most common barrier to seeking care at a health facility ( Fig 4D). Seventy-two percent of respondents said their attendance at a healthcare facility had increased since the epidemic, and 19% said it had decreased ( Fig 4E). Seventy-eight percent of respondents stated that they felt that the system was safer post-Ebola epidemic, 5% reported less safe, and 17% said safety of health facilities had remained the same ( Fig 4F).

Self-reported satisfaction for health services was higher among males than females ([aOR] = 2.1, [95% CI] = [0.87, 5.10]) ( S7 Table). Respondents were likely to say they received all the services they needed at their health facility ( S5 Table). There were no other statistically significant differences amongst age groups (with the exception of the 37-45 year age group, but its confidence interval included one); level of education and religious affiliation did not predict answers to either question. There were only three individuals who reported being a survivor of Lassa fever, insufficient for meaningful subgroup analysis.

Symptoms triggering heath seeking behavior

Responses to the questionnaire were used to identify how likely individuals are to initiate HSB for specific symptoms, both for themselves and their communities ( Fig 5). The frequency of symptoms was not determined through the questionnaire and could explain varying levels of HSB by symptom. Fever was the most common symptom prompting individuals to seek care for themselves ( Fig 5A). When asked 'which health issues prompt your community to seek care for you', fever was also the most common response ( Fig 5B). Pain was the next most frequent sign that caused individuals to initiate HSB, followed by headache/nausea, vomiting, and diarrhea. Headache/nausea was the second most frequent health issue noted to prompt community sought care, followed by vomiting, pain, and diarrhea. Bleeding was infrequently reported as a symptom triggering heath seeking behavior in either the individual or community.

Variation in health utilization and satisfaction among villages

The eight villages in which the questionnaire was administered varied in a variety of parameters ( S6 Table). The villages examined ranged in size from approximately 600 to 3000 individuals. The villages were 22-51 km away from KGH, with a mean distance from KGH of 38 km. More females answered the questionnaire than males except in villages 1 and 3. The median age of respondents was 35. The level of education varied between villages with a range of 52% to 85% of respondents indicating that they had no formal education. There were statistically significant differences in responses to health utilization and satisfaction among villages ( Fig 6). When comparing villages to index village 1, respondents in villages 4 and 6 were less likely to say they got all the services they needed at their health facility ( Fig 6A). In response to feelings of safety at hospitals post-epidemic, the majority of residents in all villages said they felt as safe or safer at hospitals ( Fig 6B); differences between villages were not statistically significant.

Discussion

Lassa fever (LF) and Ebola share common characteristics in etiology, clinical presentation, and need for strategic medical intervention such as isolation and intensive hospital care of patients with severe disease. The current study used LF as a metric to assess health seeking behavior (HSB) for febrile illnesses in Kenema District Sierra Leone-a region that was severely impacted by the West African Ebola epidemic. Previously, we reported that the annual numbers of suspected cases seen at Kenema Government Hospital (KGH) from 2008-2012 were similar reflecting the expected "steady-state" incidence for a region with endemic LF [ 43]. The number of patients evaluated in 2013 for potential LF was consistent with this trend.

Following the 2013-16 West African Ebola epidemic, there has been a notable decline in the number of suspected LF patients presenting to KGH. Potential explanations for the observed decline in Lassa evaluations, include: 1) A decrease in disease incidence, 2) A decrease in population, 3) A change in referral patterns to hospitals and 4) A change in HSB for febrile illnesses. The evidence indicates that the incidence of LF may actually be increasing in West Africa [ 47]. The largest outbreaks of LF in Nigeria have occurred in the past three years [ 48, 49]. From 2017-2018, alone, Nigeria experienced their largest ever recorded LF outbreak, which was longer, and included more severe cases than previously seen [ 50]. Additionally, LF has been detected with increasing frequency in neighboring countries Togo and Benin [ 51]. While we cannot rule out a localized decrease in Lassa virus (LASV) infection, the incidence of LF is unlikely to have declined by 50% over this brief time frame. Moreover, the population in Sierra Leone has been growing steadily for years. According to census data, since 2012, the average increase in population annually has been 2.3% in Sierra Leone [ 52]. Outside of the more than 4000 deaths from Ebola, census estimates have indicated ongoing population growth indicating that a population decline substantial enough to explain the dramatic decline in patients is unlikely.

There were changes in the demographics of the suspected LF cases presenting to KGH, including a decrease in the proportion of females and children, including teenagers. However, these demographic changes were modest and do not readily explain the large overall decrease in suspected LF cases. Since the epidemic, considerable resources have helped rebuild the health-care infrastructure at KGH [ 53]. A new, state of the art Lassa Ward has been built and the research and clinical teams have been fortified. Factors beyond infrastructure constraints are influencing residents seeking care for febrile illnesses. Such changes in HSB for LF are likely multifactorial with almost one in five respondents still reporting not feeling safer in hospital care and nearly 20% of respondents reporting a decreased attendance at hospitals since the epidemic. Given the documented decline in HSB during the epidemic, individuals who reported feeling similar safety levels at hospitals since the epidemic are still likely a reflection of a decline in HSB as compared to pre-epidemic levels. As such, for the purposes of this study, they are being considered within the category of individuals who are not reporting increased safety in hospital care.

There have been no programmatic or governmental interventions to drive a change of long existing referral patterns to KGH. A program, started by the government in 2010, to provide free medical care to children under 5 and to pregnant or lactating women has continued [ 54, 55], albeit with major challenges for implementation [ 56- 60]. KGH has served as the regional center of excellence for Lassa care for decades [ 61]. No other facility has comparable expertise or infrastructure to handle patients with suspected LF. However, other facilities that are capable of testing for LF have recently been established in Sierra Leone including a new Child/Maternal health hospital operated by Médecins Sans Frontières (MSF)/Doctors Without Borders located outside of Kenema town. The West African Ebola epidemic did impact rural health centers and staff [ 8, 56]. For example, a major health center in Gondama town Bo District, was closed during the epidemic. Prior to the epidemic, Gondama routinely referred suspected Lassa cases to KGH. We did not assess knowledge of LF among rural health care workers (HCW) after the epidemic. Similarly, how the epidemic impacted HCW behavior with regard to LF is unknown. However, a 2017 study assessing community HCW diagnosis in febrile patients in Kenema district found that rapid malaria testing (banned under no touch policies) had been replaced during the epidemic with empiric testing and had not yet returned to pre-epidemic levels [ 62]. After the epidemic, HCW's continued to presumptively treat significantly more individuals for malaria than pre-epidemic (p < . 001). This change in HCW behavior for febrile illnesses may account for underreporting or decreased recognition of LF patients. Whether the role and or effectiveness of the rural health centers has changed was beyond the scope of this study.

Given the public health threat Ebola and LF pose, case identification, patient isolation, and vector trapping, or avoidance, are all essential interventions to contain outbreaks. Outreach and educational efforts with local communities and regional health outposts are essential to aid in early case detection to identify at-risk individuals with a febrile illness. Educated providers in health outposts need to identify those with more severe cases to allow transport to better equipped facilities that can provide supportive services and isolation to prevent disease spread. LF poses an ongoing public health threat in West Africa which could be exacerbated if an absence of trust in local or regional medical providers impacts HSB.

Changes in HSB are likely contributing to the notable decline in presentations of suspected LF to KGH. Responses to the HSB questionnaire suggest that confidence in the health infrastructure has not fully rebounded after the epidemic. While the majority of respondents reported hospitals as "safer after the epidemic," and have "increased their attendance at health facilities since the epidemic," around 20% of respondents did not report increased attendance healthcare facilities. This group of respondents, if extrapolated to the regional population, could account for the identified decline in hospital presentation for suspected LF. A persistent wariness, and perhaps distrust, of existing healthcare facilities for care related to possible hemorrhagic fever may remain.

While questionnaire responses varied by village, the sample sizes were too small to identify which specific differences among villages might explain the overall observation. Based on the analysis completed, there were differences among villages in the proportion of respondents that felt they received all the services they needed at their health facility. However, this difference was not explained by gender, religious affiliation, or level of education. Distance from KGH, while considerable, did not explain the different responses. While gender and age have been reported to impact HSB in a variety of studies, we were unable to identify an association given our limited sample size [ 63- 65]. Financial resources of individuals or villages could be a factor, but was not assessed. For each village, neither the incidence of antecedent Lassa illnesses (or death), nor the incidence of Ebola were known. First-hand experience with Ebola or Lassa deaths could influence HSB for febrile illnesses-Ebola survivors are more likely to seek care at a hospital rather than other treatment options [ 66]. There may be the sentiment that, given the severity of Ebola, LF may be perceived as less severe, associated with a decreased sense of urgency to seek care for symptoms. In conjunction with a potential decrease in concern for the early signs of LF, one can imagine a scenario in which any cost, which is the primary barrier to seeking care reported by 87% of the respondents, would be sufficient to override any inclination to seek medical attention.

We present results from two data sets from two distinct groups of subjects in the Region: one of individuals in the general population and the other of symptomatic patients with suspected LF at KGH. While were unable to draw a direct linkage between the two data sets, we were able to identify some factors that may have facilitated and hindered HSB. Moreover, assessing HSB by an orally administered questionnaire had limitations primarily in sample size and scope. Furthermore, five different individuals delivered the questionnaire and variations in presentation and translation are likely. Bias in self-reporting could further limit the validity of this HSB questionnaire. For example, individuals may report more favorable attitudes towards their local health service for fear that doing otherwise may lead to a decline in service provision. Further understanding the nuances and limitations of the survey can provide additional context; the 78% of respondents reporting 'safer hospitals' and 72% reporting greater 'utilization' could be a reflection of a willingness to seek care for conditions other than suspected hemorrhagic fever. That is, individuals may show increasing HSB for immunizations, maternal/child health care or even HIV or malaria care and remain reticent to seek care for symptoms that may raise concern over a life-threatening illness that mimics Ebola. While it was not within the scope of this project to study, the demand for some health services may rebound more quickly than others. Additionally, the role of local and regional healthcare systems post- the West African Ebola epidemic were not measured in this study. The local and regional healthcare providers serve as an essential bridge to the referral center and may have suffered unrecognized losses from the epidemic that curtail the number of appropriate patients being referred [ 67]. The need for further research in these areas is essential to understand the factors contributing to HSB in the region and to target interventions to improve the health of these communities long-term.

The goal of this study, to identify and evaluate the persistent impact of the Ebola epidemic on health seeking behavior for LF through hospital level data, and by survey questionnaire of general health seeking behavior, was met given the limitations of the methodology. The study identified a statistically significant decline in the number of individuals presenting to the hospital, or public health unit, with suspected LF. Although limited by sample size, the questionnaire of 194 individuals identified persistent reluctance to seek care for febrile illness since the Ebola epidemic among one fifth of respondents that could account for the decline noted at KGH.

Identifying cause(s) beyond those self-reported for the decline in presentations at the hospital was beyond the scope of our study. What impact changes in community healthcare workers, transportation access, financial constraint or other potential drivers of HSB, had on this decline were not assessed in this study, but likely play an important role. Further research into these, and potentially other, factors influencing underutilization of healthcare for LF is warranted.

To better achieve the goals of this study, a larger sample size, more villages, and more targeted questions regarding the interaction with public health units, and behavior more clearly linked to suspected LF could have strengthened the conclusions. Additionally, targeting multiple aspects of the care pathway would have been valuable. For example, interviewing or analyzing community health workers, observing any changes in cost, distance, or time to care and other barriers identified by respondents in this paper.

After an epidemic, or similar shock to the healthcare system and fabric of society, it is critical to be aware of the impact such an event has or does not have on morbidity, mortality, and healthcare utilization in communities. This research, and its findings, are critical not only for management during an emergency, but in the years following.

This study confirms that health seeking behavior of individuals with suspected LF has not rebounded after the Ebola epidemic despite the maintenance of a state-of-the-art facility. These findings suggest that to optimize healthcare delivery post epidemic, investments into rebuilding the healthcare system and health of communities need to be multifaceted. Beyond healthcare infrastructure, consideration of other important drivers of health seeking behavior should be considered.

Indirect effects on health and mortality during the Ebola epidemic are well known [ 10- 15]. However, factors that may persistently impact outcomes post-epidemic may be more nuanced and less well studied or understood. Monitoring the impact on health seeking behavior (across health indicators) not only during an epidemic but in the years following is important.

Similar to the West African Ebola epidemic, the current coronavirus disease (COVID-19) pandemic is overwhelming healthcare systems, and spreading fear among patients and providers with documentation globally of decreased health seeking behavior for vital health issues such as heart attack, stroke, and deliveries [ 68]. If, in line with our findings in this paper, HSB decreased during the COVID-19 pandemic and remains below baseline following the pandemic, such information will be vital for appropriate mitigation efforts. Particularly in lower resourced health systems already confronting a sustained impact on health seeking behavior from the West African Ebola epidemic, monitoring how that may be further impacted by COVID-19 will be critical. Optimal health outcomes can only be achieved when individuals seek care appropriately, particularly for infectious diseases that are contagious and often lethal. Improved understanding of the factors that impede HSB is vital to effectively mitigate public health crises as well as help to implement corrective strategies and allow overall health to rebound.

Acknowledgments

We thank the members of the Viral Hemorrhagic Fever Consortium (The Broad Institute, Harvard University, La Jolla Institute of Immunology, the Scripps Research Institute, Tulane University, the University of Texas Medical Branch, Zalgen Labs, the ISTH Lassa Fever Program, and KGH) without which this work could not have been conducted; and the patients with serious febrile illnesses who presented to KGH, as well as their families, without whose cooperation this study would not have been possible. Simbirie C. Jalloh, Allyson M. Haislip, Christopher M. Bishop, Tynette D. Hills, and Douglass Simpson provided program management and logistical support.

References

1. 4. Sabeti P, Salahi L. Outbreak Culture: The Ebola Crisis and the Next Epidemic. Cambridge, Massachusetts: Harvard University Press; 2018 11/26/2018. 288 p.

2. 22. Conner M. International Encyclopedia of the Social & Behavioral Sciences (Second Edition). 2015:582-7.

3. 51. ECDC. Lassa fever in Nigeria, Benin, Togo, Germany and USA. European Centre for Diseases Prevention and Control.

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Health seeking behavior after the 2013-16 Ebola epidemic: Lassa fever as a metric of persistent changes in Kenema District, Sierra Leone

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Author summary The West African Ebola epidemic of 2013-2016 killed nearly 4,000 Sierra Leoneans and devastated health infrastructure. There is limited information regarding the effects of the epidemic on health seeking behavior. Lassa fever is clinically similar to Ebola and is prevalent in Kenema District, located in the Eastern Province of Sierra Leone.

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a increase of LF in Nigeria from January to march 2018

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a increase of LF in Nigeria from January to march 2018

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the few months spike of cases was caused by

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A massive wave of laboratory-confirmed cases of Lassa fever occurred in Nigeria in 2018. Whether this high case count was caused by a new virus variant, increased seasonal incidence, improved case recognition, availability of laboratory diagnostics and therapy, or a combination of these factors is unknown. We set out to determine the factors that contributed to this outbreak using data available through the Nigerian Disease Surveillance System.

Lassa fever is endemic in Nigeria and peaks during the first 12 weeks of the year (January-March; ) ( https://ncdc.gov.ng/themes/common/files/sitreps/b7cd08c8047e52ceabb09e5318a3b0a7.pdf). A total of 107 laboratory-confirmed cases were reported during the first 12 weeks of 2017 and 394 during the same period in 2018. Among confirmed and probable cases, 50 deaths were reported during the peak season in 2017 and 104 in 2018. In the neighboring states of Edo and Ondo, 45.0% of confirmed cases occurred in 2017 and 66.0% in 2018. These states have access to the long-established and largest Lassa fever treatment center that has Lassa virus diagnostic capabilities in Nigeria. Although Edo and Ondo comprise only 4.6% of Nigeria's population, the population in these states accounted for 74.6% of the increase in confirmed cases during the 2017 and 2018 peak seasons. Another area with a strong increase in case numbers in 2018 was the nonadjacent Ebonyi State, where another laboratory was outfitted to conduct Lassa virus diagnostics by early 2018.

Evidence from sequencing studies yielded no indication that viruses circulating in 2018 were different from those found in previous years (). Phylogenetic data suggest multiple zoonotic infections instead of extensive person-to-person transmission.

The increased case numbers in Edo, Ondo, and Ebonyi could not be accounted for by gross population changes; these states are distant from the refugee emergency in northeastern Nigeria ( http://reporting.unhcr.org/node/21275). Studies on climatic and other environmental circumstances (e.g., new agricultural practices or larger harvests) that could have potentially contributed to changes in the way and frequency humans come into contact with the Lassa virus reservoir, Mastomys natalensis mice, have not been established. Also, data on rodent abundance are not available.

However, in 2018, many changes occurred in Nigeria regarding Lassa virus surveillance and treatment. Instead of only 1 Lassa virus laboratory for Edo and Ondo and 1 in Lagos, 2 additional laboratories (in Ebonyi and Abuja) started providing diagnostic services in early 2018. Sample transport logistics had improved, and patients' samples were transported and tested without charge. Considering that laboratory confirmation is a prerequisite for counting cases, laboratory capacity building directly affects surveillance case numbers. By early 2018, Lassa fever surveillance had been strengthened by the formulation of standard operating procedures, and rapid response teams provided capacity building and support to affected states 4 weeks earlier than they did in 2017. Clinical cases of Lassa fever need to be treated in isolation facilities, and by 2018, additional treatment centers had been assessed and were being supported by the federal government. At this time, ribavirin was also available to patients without charge, and the media actively advertised this message, encouraging patients to seek medical treatment. In addition, Lassa fever received strong media attention in early January 2018 because of an incident in a healthcare facility where 3 of 4 infected healthcare workers died.

Surveillance data are incomplete for 2018, but higher surveillance sensitivity is visible in the available data from the peak 2017 and 2018 seasons. The percentage of suspected Lassa fever cases testing positive decreased from 31.0% in 2017 to 21.6% in 2018 (30.2% decrease). Nosebleed, an indicator of disease severity in nonfatal confirmed cases, was noted on 10.8% of case report forms for surviving patients in 2017, but this frequency declined to 2.6% in 2018 (75.9% decrease), and the case-fatality ratio among confirmed and probable cases declined from 43.9% in 2017 to 25.8% in 2018 (41.2% decrease). Disease severity and case-fatality ratios are also influenced by the timeliness of patients seeking treatment and treatment availability. The decrease in the 3 aforementioned factors (percentage of Lassa virus-positive cases, percentage of nonfatal confirmed cases with nosebleeds, and case-fatality ratio) reflects lowering of the surveillance threshold to detect cases. More patients with comparatively mild disease probably sought treatment because of the increased Lassa fever publicity and communication about available therapy; moreover, additional suspected cases were probably detected in the community through enhanced contact tracing and active case finding.

In conclusion, we cannot exclude that early 2018 represents a particularly active Lassa fever season in Nigeria, especially in Edo, Ondo, and Ebonyi. However, no available evidence indicates that higher case numbers could be attributed to new virus strains. The addition of new laboratories with growing surveillance capacities, an overall intensified response, and increasing surveillance sensitivity are likely major drivers of the high number of Lassa fever cases reported in early 2018. The weekly case numbers reported in early 2019 slightly surpass those from 2018 ( https://ncdc.gov.ng/themes/common/files/sitreps/b94e459c79a59ca9d667a55539cda5db.pdf). Improved identification of Lassa fever cases in Nigeria provides the basis for epidemiologic studies of disease and effective disease control. Also, each identified case treated in isolation centers reduces the likelihood of person-to-person transmission.

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Increase in Lassa Fever Cases in Nigeria, January-March 2018

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We reviewed data pertaining to the massive wave of Lassa fever cases that occurred in Nigeria in 2018. No new virus strains were detected, but in 2018, the outbreak response was intensified, additional diagnostic support was available, and surveillance ...

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Low levels of interferon induced protein 10 ,interleukin 8 can cause a fatal infection from Lassa fever

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Low levels of interferon induced protein 10 ,interleukin 8 can cause a fatal infection from Lassa fever

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Abstract

To investigate the role of inflammatory mediators in the pathogenesis of Lassa fever, the levels of a number of pro- and anti-inflammatory cytokines and chemokines in serum samples collected from hospitalized patients with fatal and nonfatal acute Lassa fever were compared with those from 2 control groups: patients with other febrile illnesses and uninfected individuals. Serum interleukin (IL)-8 and interferon (IFN)-inducible protein (IP)-10 levels were significantly higher in patients with acute nonfatal Lassa fever than in control subjects. In striking contrast, levels of these chemokines were low or undetectable in patients with fatal Lassa fever. IFN-γ, IL-12, IL-6, and RANTES levels were elevated in all the febrile study groups. Tumor necrosis factor-α levels were not elevated in patients with fatal or nonfatal Lassa fever. These data indicate that acute nonfatal Lassa fever is associated with high levels of circulating IL-8 and IP-10 and that low levels or absence of these mediators correlates with a poor outcome

Lassa fever is a severe viral infection of humans caused by Lassa virus (LV), a member of the family Arenaviridae. The disease is associated with significant mortality (∼20% in hospitalized patients) and is characterized by a rapidly progressive shocklike syndrome preceding death in patients with fatal illness [ 1]. Histopathologic studies in humans and nonhuman primates have found a paucity of lesions severe enough to explain the cause of death, although there is widespread viral replication and development of shock in the terminal stages of illness [ 2-5]. These pathologic features and the presence of profound shock in fatal cases of Lassa fever have led to the speculation that inflammatory mediators likely are involved in the pathogenesis of this disease [ 6]

The establishment of a center for the study of Lassa fever in Guinea and the occurrence of an epidemic of Lassa fever in Sierra Leone in 1997 provided us with an opportunity to study patients with acute Lassa fever. We determined the levels of inflammatory cytokines and chemokines in West African patients with acute Lassa fever, to establish whether proinflammatory mediators derived from macrophages, endothelial cells, and other cellular sources are present during illness and to determine whether their levels correlate with disease severity. Our data, which to our knowledge constitute the first report of in vivo responses in humans with Lassa fever, indicate that several macrophage- and endothelial cell-associated inflammatory mediators are present in the circulation during acute Lassa fever. Some responses are associated specifically with Lassa fever, whereas others are elevated in both Lassa fever and other febrile illnesses. These findings have important implications for the pathogenesis of Lassa fever in humans

Patients and Methods

Patients and serum collectionBlood specimens were obtained from patients who presented with suspected cases of Lassa fever to any 1 of 5 surveillance hospitals in West Africa (Kindia, Faranah, Kissidougou, and N'Zérékoré hospitals in Guinea and Kenema Government Hospital in Sierra Leone). Specimens were obtained immediately upon clinical suspicion of the disease (usually at the time of admission) and then at various intervals over the following weeks. Samples were collected in 10-mL syringes without anticoagulants, and serum and clot were separated at room temperature in a bench-top centrifuge (2500 g). Samples were stored in liquid nitrogen (in Guinea) or in a freezer at −75°C (in Sierra Leone) until they were transported on dry ice in International Air Transport Association-approved safety shippers to the Centers for Disease Control and Prevention (CDC; Atlanta)

Laboratory confirmation of Lassa feverAt CDC, all serum samples were tested for Lassa-specific antigen and IgM and IgG antibody by ELISA, as described elsewhere [ 10]. Patients were considered to have acute Lassa fever if Lassa-specific antigen and/or IgM antibody (above background levels) were found in any sample. Those who were IgG antibody positive in the initial sample and who did not show a ⩾4-fold increase in antibody titer over the course of their illness were considered to have had previous exposure to LV and to be suffering from a non-Lassa illness at the time of the study. Some patients with acute Lassa fever, as determined by the presence of antigen or IgM antibody, also developed Lassa-specific IgG antibody responses as their illness progressed. They were considered to have acute Lassa fever. Patients negative by ELISA for Lassa antigen and antibodies were considered to be suffering from other tropical febrile illnesses, such as malaria or typhoid fever. Limited laboratory facilities usually prevented more specific diagnosis of these patients

Assignment to study groupsPatients were assigned to 1 of 5 groups on the basis of ELISA results and the outcome of their illness ( table 1). Since laboratory results only became available weeks to months after patients were seen, group assignment was retrospective. The 5 group designations included (1) Lassa fever, nonfatal (LF-nf; n=29); (2) Lassa fever, fatal (LF-f; n=6); (3) non-Lassa febrile illness, previously exposed to LV (NLF-pe; n=7); (4) non-Lassa febrile illness, no previous exposure (NLF-ne; n=9); and (5) uninfected, afebrile control (n=10). The afebrile group consisted of uninfected, afebrile individuals who participated in a village serosurvey for Lassa fever in Sierra Leone. Initial analyses confirmed no significant differences in any study parameters between NLF-pe and NLF-ne, so the 2 groups were combined and designated NLFI in subsequent data analyses

Antigen and antibody assaysLassa fever viral antigen levels were determined by a capture ELISA, as reported elsewhere [ 10]. Lassa antigen titers >1:4 were considered to be positive. Anti-Lassa IgM and IgG were also determined by ELISA, as reported elsewhere [ 10]. Anti-Lassa IgM and IgG titers >1:400 were considered to be positive, for the purpose of classification

Cytokine assays Serum levels of the cytokines interferon (IFN)-γ, interleukin (IL)-1β, IL-6, IL-10, IL-12, and tumor necrosis factor (TNF)-α and the chemokines IL-8, IFN-inducible protein (IP)-10, and RANTES were measured in irradiated serum samples by multiplex capture ELISA, using monoclonal antibody (MAb)-coated microspheres (Luminex System; Luminex) and flow cytometry, according to the manufacturer's directions. In brief, MAbs specific for each cytokine or chemokine were coupled to microspheres (Luminex), to provide a solid-phase capture surface. Samples diluted 1:2 and 1:4 in blocking buffer (PBS, 1% Tris-borate, and 1% bovine serum albumin) were incubated in 1.5-mL test tubes with a mixture of cytokine-specific microspheres (2000/sample) for 30 min at room temperature with rapid agitation. Next, biotinylated cytokine- or chemokine-specific MAbs were added (final concentration, 2 μg/mL), and the tubes were incubated at room temperature for 30 min. Cytokines bound to microspheres were detected with Alexa-avidin (4 μg/mL; Molecular Probes), using flow cytometry (FACScan; Becton-Dickinson). Cytokine and chemokine levels in samples were calculated by use of proprietary software, from the mean fluorescence of 100-200 microspheres (for each cytokine) by gating for forward scatter (size) and fluorescence. Standard curves were generated with a pool of recombinant human (h) IL-1β, hIL-6, hIL-8, hIL-10, hIFN-γ, hRANTES, hIP-10, and hTNF-α (all from Pharmingen). For comparison among groups, data were expressed in picograms per milliliter

Soluble TNF-α (sTNF) receptors Levels of soluble hTNF-α receptors I (p55) and II (p75) were measured with an ELISA kit (Quantikine; R&D Systems), used according to the manufacturer's directions. Data were expressed in nanograms per milliliter, using recombinant standards for the purpose of comparison

Statistical analysisSerum cytokine and chemokine levels were compared by use of the Kruskall-Wallis test, a nonparametric analysis of variance. When the test revealed significant differences between the study groups, 2-group comparisons were done with the Mann-Whitney U test with exact P values. The Wilcoxon signed-rank test was used for comparison of paired samples from each individual. P<.05 was considered a statistically significance difference between groups. Statistical analysis was done using the Statview package (SAS) on a personal computer work station

Results

Study population and clinical dataSerum samples from 59 individuals were selected for analysis and were categorized into the following groups: LF-nf (27), LF-f (6), NLF (16), and uninfected (10) ( table 1). The median interval between onset of symptoms and the first blood sample varied between the groups, but the differences were not statistically significant ( table 1). The first serum sample, which was obtained at the time of admission, represents the acute or early phase of illness, a time when ongoing inflammatory responses were likely to be at a maximum. Since the second sample was obtained close to discharge or to the death of the patient, it likely represents a late or convalescent phase of illness, a time when inflammation would be expected to be resolving or be in a preterminal stage in those who succumbed to disease. However, the short interval between samples, particularly in the most severely ill patients (LF-f), warrants caution in the interpretation of changes during the course of illness

Circulating inflammatory mediators associated with acute Lassa fever and other febrile illnessesIn the serum samples obtained at admission, the pro- and anti-inflammatory cytokines and chemokines we measured fell into 2 groups. Serum levels of the first group of mediators, IL-1β, IL-8, and IP-10, were highest in LF-nf patients, ranging from ∼10 pg/mL (the sensitivity of the assay for these proteins) to 11,214 ng/mL, 19,494 pg/mL, and 397 pg/mL, respectively (P<.05, Kruskall-Wallis test; figure 1). In the LF-nf group, 22 (81%) of 27 patients had serum IL-8 levels >500 pg/mL, compared with 1 (17%) of 6, 2 (13%) of 16, and 0 (0%) of 10, respectively, among LF-f, NLF, and uninfected individuals. Similar but less marked differences were apparent in levels of IL-1β and IP-10 ( figure 1). This strong clustering of elevated IL-8 and IP-10 levels in serum of patients with acute nonfatal Lassa fever and lower or undetectable levels of these mediators in patients with fatal cases suggests that more severe disease is associated with a weak pro- and anti-inflammatory response, at least by the time patients are hospitalized. The data are in agreement with a recently published report indicating that Lassa-infected human macrophages produce IL-8 in vitro as early as 24 h after infection [ 7]

In contrast to the Lassa-associated elevation observed with the cytokines and chemokines mentioned above, IFN-γ, IL-12, TNF-α, IL-10, and IL-6 were elevated in subjects in the LF-nf and NLF groups ( figure 2). LF-f subjects also had the lowest levels of these cytokines and chemokines at the time of admission, which is in accord with the low levels of IL-8, IP-10, and IL-1β ( figure 1). Of note, 1 patient with fatal Lassa fever had the highest measured serum level of TNF-α (6888 pg/mL). Among the LF-f group, this patient also had the highest levels of IL-8 (4677 pg/mL) and IL-1β (921 pg/mL) in the first serum sample, which was obtained on day 5 after onset of symptoms, but had low or undetectable levels of the other cytokines and chemokines that were measured. In addition and in contrast to others in the LF-f group, this patient was not antigenemic

Overall, the levels of a number of proinflammatory mediators were elevated in the serum samples of patients with Lassa and non-Lassa febrile illnesses; however, cytokine and chemokine levels were low or undetectable in serum samples from 5 of 6 patients with fatal Lassa fever. An unexpected observation was that the uninfected group had detectable IL-10 and TNF-α in their serum samples ( figure 2). However, since these individuals live in a region with a high prevalence of parasitic and bacterial diseases, it is possible that the circulating cytokines reflected underlying infections by other (non-Lassa) pathogens. The levels of TNF-α and IL-10 did not differ significantly from those of the other groups

Changes during the course of illness in chemokines that are elevated in acute Lassa feverWe compared the levels of IL-8 and IP-10, the chemokines found to be significantly elevated in acute nonfatal Lassa fever, between samples collected at admission and those collected later in illness (5-26 days later), to determine whether levels of these mediators are altered with resolution or progression of disease ( figure 3). In the LF-nf group, the levels of both IL-8 and IP-10 declined by 2-1000-fold in most patients who initially had elevated levels (21 [84%] of 25 for IL-8 and 16 [64%] of 24 for IP-10). Only one LF-nf patient had an increase in IL-8 levels, and 2 had increases in IP-10 levels between the first and second time points ( figure 3). In the other 2 groups (LF-f and NLF), initial values for IL-8 and IP-10 were low and remained low or undetectable in the second sample or were initially high and remained high, except for values for one LF-f patient who had an elevated IL-8 level that declined before the patient's death. Similar significant declines were seen in IL-1β, IL-6, and IFN-γ levels in serum samples from LF-nf patients (data not shown). These data indicate that IL-8 and IP-10 are elevated in association with the acute phase of nonfatal Lassa fever and decline with resolution of the disease. In contrast, patients with severe, fatal disease (LF-f) have low levels of IL-8 and IP-10 throughout the course of their illness

In the 3 study groups, there were no consistent patterns of change in the levels of other measured cytokines (IFN-γ, TNF-α, IL-1β, IL-10, and IL-12) and chemokines (RANTES) between the early- and later-phase serum samples (data not shown). Furthermore, there was no correlation (positive or negative) between the days between onset of symptoms and initial serum levels of these cytokines (data not shown). However, 2 of 6 patients with fatal Lassa fever and with no detectable cytokines in the earlier serum samples were found to have higher levels of IL-6 (3620 and 5330 pg/mL), RANTES (5080 and 570 pg/mL) and IL-10 (210 and 230 pg/mL; data not shown) in the serum samples obtained later in illness, which suggests that some patients with severe Lassa fever retained the capacity to secrete pro-inflammatory mediators

Elevated levels of sTNF receptors in Lassa fever and other febrile illnessesThe absence of markedly elevated levels of TNF-α in the febrile patients was unexpected, so we tested the possibility that serum levels of TNF-α receptors I and II (RI and RII) were elevated, possibly resulting in binding of TNF-α [ 11]. We, therefore, measured the levels of TNF-α RI and RII in the first serum samples from the study populations. Levels of TNF-α RI were elevated in all febrile groups (LF-nf, LF-f, and NLF), compared with levels in uninfected control subjects ( figure 4), but the elevation was most marked in patients with fatal Lassa fever (P<.05, Kruskall-Wallis test). Similarly, the median serum TNF-α RII levels in the febrile groups were 3-10-fold higher than those of uninfected individuals. The highest levels of TNF-α RII were also observed in the LF-f group, but the differences did not achieve statistical significance ( figure 4A 4B)

In a small subset of LF-nf (n=5) and LF-f (n=2) patients, serial serum samples (3-5 for each patient) that were collected during the course of illness and convalescence were available for the determination of sTNF receptor levels. sTNF-α RI and RII values in LF-nf patients were high initially and declined slowly with recovery from illness, eventually reaching values similar to those seen in uninfected control subjects ( figure 4C 4D). In contrast, LF-f patients, for whom intervals between specimen collection were short (1 day in both), reflecting the rapid course of disease, had very high initial levels of TNF-α RI and RII or levels that increased rapidly to a peak that was higher than levels in LF-nf patients. There were rapid declines in levels of sTNF-α RI in one LF-f patient and of sTNF-α RII in 2 LF-f patients before their deaths. These data demonstrate that sTNF-α RI and RII are higher in patients with fatal Lassa fever than in those with less severe disease

Discussion

The development of profound shock, despite the paucity of histopathologic evidence of cellular damage, vascular thrombosis, or hemorrhage has been taken as evidence for subtle immunologic dysfunction attributable to inflammatory mediators, such as in septic shock. Investigation of physiologic and immunologic function in animal models of Lassa fever has revealed abnormalities of platelet aggregation [ 8] and elevated levels and activity of prostaglandins [ 3, 9, 12], often preceding the onset of shock. Studies using guinea pigs have implicated TNF-α in the pathogenesis of Lassa fever [ 12-14] and Pichinde virus infections [ 15]. However, to our knowledge, no previous human studies have directly examined mediators of inflammation in Lassa fever. In this study, we compared the levels of circulating proinflammatory cytokines and chemokines derived from or involved in the activation of macrophages and endothelial cells in acute Lassa fever with those seen in patients with other febrile illnesses and in uninfected individuals from regions endemic for LV

Serum chemokine and cytokine levels are not the best measure of inflammatory responses, because these mediators usually act locally in an autocrine fashion, and in vitro antigen-driven peripheral blood mononuclear cell responses provide a more direct measure of chemokine and cytokine secretion by macrophages and lymphocytes. However, field conditions in regions endemic for LV and accessible to us precluded such studies, so we used serum levels of inflammatory mediators as a surrogate

Our observation that serum levels of IL-8 were low or undetectable in LF-f patients but high in LF-nf cases is consistent with the paucity of histologic evidence of neutrophil infiltration into the liver, adrenals, and other tissues demonstrating significant pathology in postmortem studies of fatal Lassa fever [ 5]. Unfortunately, there are no data available regarding neutrophil infiltration in LF-nf, the group in which we observed high circulating levels of the chemokine. Lukashevich et al. [ 7] reported that LV infection of human macrophages in vitro results in a down-regulation of IL-8 mRNA [ 7]. If similar down-regulatory mechanisms operate in vivo, the IL-8 observed in LF-nf patients may be derived from non-monocyte/macrophage sources, such as endothelial cells and T cells

IP-10 is also a CXC chemokine that is secreted by macrophages, endothelial cells, and T cells in response to a number of activating stimuli, such as IFN-α, IFN-β, IFN-γ, TNF-α, and lipopolysaccharide [ 18, 19]. IP-10 is secreted by monocytes and macrophages in response to IFN-γ and TNF-α that act via the NF-κB pathway, a pathway common to a number of inflammatory responses [ 20, 21]. IP-10 is thought to participate in the pathogenesis of infections and shock by inhibition of endothelial cell function and chemoattraction of resting T cells and NK cells

In addition to the Lassa fever-associated elevation of IL-8 and IP-10, several of the other chemokines we measured were elevated in non-Lassa febrile illnesses. This is not surprising, since inflammation mediated by macrophages is a common feature of many infectious conditions that are prevalent in regions endemic for LV. These mediators therefore are likely to account for many of the nonspecific features of Lassa fever that make it so difficult to differentiate the disease from other infections in its early phases [ 22]

The almost uniformly attenuated cytokine and chemokine profile of fatal Lassa fever that we observed in this study population was remarkable and may provide some clues to the determinants of severity in this disease. Previous studies demonstrated that severity of disease correlates with the levels of circulating virus in humans and nonhuman primates [ 23-25]; however, in these older studies, it was not possible to determine the contribution of altered host defense to the overall severity of disease. Studies in humans with Argentine hemorrhagic fever show a different pattern of serum cytokines, with the high levels of IL-6, IL-8, and IL-10 being associated with severe or fatal disease [ 26]. However, in that study, the patient population, the etiologic virus, and the disease phase when blood samples were obtained differed considerably from those in our study, which may explain the differences in the patterns of cytokine responses

The low levels of serum TNF-α in all groups was unexpected, since elevations of circulating TNF-α have been reported in a number of arenavirus infections, such Junin virus infections in humans [ 35] and Pichinde virus infections in guinea pigs [ 15]. An explanation for this observation may be the long interval between the onset of symptoms and the time when the first blood sample was obtained in these patients ( table 1). Peak elevations of serum TNF-α may have occurred earlier in the course of LV infections. The low levels of TNF-α in our patients ( figure 2) were in agreement with recently published data indicating a failure of induction of TNF-α mRNA in macrophages infected in vitro with LV [ 7]. The low TNF-α levels are also in agreement with findings in a study using Pichinde virus-infected guinea pigs [ 12]: The study reported up-regulation and activation of "downstream" components of the TNF-α pathways, such as leukotrienes and platelet-activation factor, which suggests a regulatory role for TNF-α in this disease

This study has shown that acute Lassa fever is associated with high levels of proinflammatory chemokines (IL-8 and IP-10) and with a number of proinflammatory mediators that are also elevated in other febrile illnesses. Although we did not find high levels of TNF-α accompanying the inflammatory state in our patients, the presence of elevated levels of TNF-α receptors in serum suggests that TNF-α may also play a role in the disease. Furthermore, we have shown, paradoxically, that severe disease is associated with low levels or the absence of these mediators, which indicates a complex interplay between inflammatory responses and immune cells in the pathogenesis of this disease. A closer examination of the production of these mediators and the activation of macrophages in the early phases of Lassa fever is warranted to attain a better understanding of the disease

Acknowledgments

We thank the staff and health care providers of Kindia, Faranah, Kissidougou, and N'Zerekore hospitals, Guinea, and Kenema Hospital, Sierra Leone, for assistance in the care of patients and in the collection and transport of samples. We thank A. Demby for sample processing and serology and J. O'Connor for editorial comments and suggestions

References

Author notes

# data[].attributes.attachment_link.title
Low Levels of Interleukin-8 and Interferon-Inducible Protein-10 in Serum Are Associated with Fatal Infections in Acute Lassa Fever

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Abstract. To investigate the role of inflammatory mediators in the pathogenesis of Lassa fever, the levels of a number of pro- and anti-inflammatory cytokines a