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This video talk about Candida (more specific and educational)

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This video talk about Candida (more specific and educational)

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The Truth about Candida: what works, what doesn't

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I think this is a video and quite detail about Candidiasis (general knowledge) 

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I think this is a video and quite detail about Candidiasis (general knowledge) 

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Top 8 Home Remedies for Yeast Infection (Candidiasis)

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Molecular and conventional methods for detection of Candida species isolated from a sample of immunocompromised Iraqi patients with pulmonary symptoms

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(PDF) Molecular and conventional methods for detection of Candida species isolated from a sample of immunocompromised Iraqi patients with pulmonary symptoms

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PDF | Objectives Candida species has emerged as a potentially pathogenic fungus rather than benefit mucosal commensal in patients with pulmonary diseases. Therefore, our study was carried out to detect Candida species in sputum samples from patients with pulmonary diseases using...

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This is the journal about Synthetic glycopeptide vaccines combining β-mannan and peptide epitopes induce protection against candidiasis

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This is the journal about Synthetic glycopeptide vaccines combining β-mannan and peptide epitopes induce protection against candidiasis

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Abstract

The first fully synthetic glycopeptide vaccines against a fungal disease have been used to combat disseminated candidiasis in mice. Six T cell peptides found in Candida albicans cell wall proteins were selected by algorithm peptide epitope searches; each was synthesized and conjugated to the fungal cell wall β-mannan trisaccharide [β-(Man) ₃] by novel saccharide-peptide linker chemistry to create glycopeptide conjugates. The six proteins were selected because of expression during human candidiasis and cell wall association and included: fructose-bisphosphate aldolase (Fba); methyltetrahydropteroyltriglutamate (Met6); hyphal wall protein-1 (Hwp1); enolase (Enol); glyceraldehyde-3-phosphate dehydrogenase (Gap1); and phospho glycerate kinase (Pgk1). By immunization protocols favoring production of protective antibody, the β-(Man) ₃-Fba, β-(Man) ₃-Met6 and β-(Man) ₃-Hwp1 induced protection evidenced by survival and reduced kidney fungal burden, the β-(Man) ₃-Eno1 and β-(Man) ₃-Gap1 gave moderate protection, and the β-(Man) ₃-Pgk1 slightly enhanced disease. For the β-(Man) ₃-Fba conjugate, protection was uniquely acquired through immunity against the carbohydrate and the Fba peptide. This approach based on fully synthetic chemically defined immunogens should be generally useful in vaccine development.

The rising incidence of life-threatening fungal diseases and associated high treatment failure rate are providing impetus for development of antifungal vaccines as a preventive strategy ( 1). Whereas some vaccines are constructed to induce cell mediated immunity, others favor protective antibody responses.

The role of antibody immunity against fungal disease has been controversial ( 1, 2), which is likely because of a lack of appreciation for critical parameters such as antibody specificity, isotypes and titers and the relationship between humoral and cell mediated immunity ( 3). The less understood phenomenon of immunoglobulins that may cause tissue damage provides additional complexity. IgE may represent one example, but although a relationship between allergic hypersensitivity and recurrent vaginal candidiasis has been speculated ( 4), the role in systemic or disseminated mycotic diseases requires more investigation. We showed that certain IgM and IgG ₁ antibodies with anti-mannan specificity may not be protective against disseminated candidiasis ( 5, 6), but whether other antibody classes and specificity enhance disease progression is unknown. Nonetheless, in recent years, attention to these various details has resulted in solid evidence for antibody defense against candidiasis and cryptococcosis, and perhaps other mycoses ( 1). Active immunization promoting positive humoral immune responses has mostly involved fungal surface antigens, including carbohydrates and proteins ( 5, 7- 11). Here, we have focused on candidiasis, which is the fourth leading cause of nosocomial blood stream infections; it causes mucosal disease in AIDS patients and >70% of females will experience one or more bouts of vulvovaginal candidiasis during their life ( 12).

We previously demonstrated that complement-fixing antibodies that recognize β-1,2-linked mannotriose [β-(Man) ₃] and β-1,2-linked mannobiose of the cell wall phosphomannan complex of Candida albicans, and a few other Candida species, protect against hematogenously disseminated candidiasis and vaginal infection in mouse models ( 5, 13- 16). The β-(Man) ₃ was chosen as the vaccine carbohydrate epitope, but unconjugated oligosaccharides are poorly immunogenic ( 5). Fungal cell wall peptides were sought that would serve as carriers and to provide an additional protective epitope. The latter attribute would induce protection against fungal strains not producing the carbohydrate epitope, especially because that moiety is not essential for C. albicans growth ( 17) and only ≈80% of isolates of C. glabrata, another important agent of candidiasis, produce β-linked mannosides ( 18). To satisfy these features, a highly efficient conjugation chemistry was developed. This allowed the facile conjugation of unprotected β-(Man) ₃ with six candidate peptide carriers derived from the cell wall of C. albicans. An antigen-pulsed dendritic cell (DC)-based vaccine strategy elicited specific antibody against both epitopes and thereby induced protective immunity in mice against candidiasis.

Results

Oligosaccharide-Peptide Glycoconjugates.

Our conjugation strategy allowed the efficient coupling of the two completely deprotected synthetic components-antigenic β-(Man) ₃ and specific T H-peptide epitopes-as the last step of the synthesis ( 19). This strategy readily permits the screening of a range of peptide carriers and facilitates the identification of the most suitable peptides. The heterobifunctional coupling reagent ( 1) ( Fig. 1) is based on a water-soluble triethylene glycol core functionalized at one end with an N-hydroxysuccinimide ester and a sulfhydryl-reactive acrylate moiety at the other ( 19). This linker permits efficient coupling of biomolecules under aqueous conditions while affording, as far as possible, an immunologically silent tether.

Assembly of a panel of antifungal vaccine candidates used a convenient readily scaled synthesis of the β-1,2-mannopyranosyl trisaccharide as an allyl glycoside ( 20). The allyl glycoside was further reacted with cysteinamine to incorporate an amino-functionalized tether ( I) that allows for the conjugation with heterobifunctional linker 1. Subsequent conversion with 1 in an aqueous buffer system furnished the stable building block 2, which was obtained after purification by preparative HPLC. The tether bound acrylate moiety incorporated in this way reacted smoothly under mild, aqueous conditions with a set of thiopropionic acid N-terminated peptide epitopes ( II) prepared by solid-phase synthesis to form a nonimmunogenic thioether linkage ( 21). The pure, synthetic target glycoconjugates were obtained in yields between 64 and 81% after preparative HPLC ( Fig. 1). [For details of the chemical synthesis see supporting information (SI).]

C. albicans carrier proteins were selected from previously identified cell wall proteins that are expressed during pathogenesis of human disseminated candidiasis ( 22, 23). The selected six candidate carriers were fructose-bisphosphate aldolase (Fba); methyltetrahydropteroyltriglutamate (Met6); hyphal wall protein-1 (Hwp1); enolase (Enol); glyceraldehyde-3-phosphate dehydrogenase (Gap1); and phospho glycerate kinase (Pgk1). By application of an epitope-finding algorithm (GenScript, Antigen Design Tool, OptimumAntigen), putative epitopes were selected comprised of 14 aa each located near the N terminus ( Fig. 1) as we presumed that this location might be accessible to the host immune system. Each peptide shows 100% homology only with C. albicans and none has identity with mammalian proteins (data not shown). Peptides Fba, Met6 and Pgk1 contain obligatory P1 anchor residues and are predicted to bind HLA-DR ( http://bioinformatics.uams.edu/mirror/propred/index.html), and multiple MHC class I binding sites are predicted for all six peptides ( www.imtech.res.in/raghava/propred1) ( 24, 25).

Six Glycopeptide Conjugates Were Immunogenic in BALB/c Mice.

The antigen-pulsed dendritic cell (DC)-based vaccine strategy was used in all immunizations for development of antibodies in mice. To determine the immunogenic efficacy of each, immune sera were tested for antibodies specific for the whole conjugates, synthetic β-(Man) ₃, synthetic peptide carriers and fungal cell surface phosphomannoprotein (PMP) extract from C. albicans cell walls ( Fig. 2). Surprisingly, all six conjugates were immunogenic as shown by high titers of specific antibody to each of the test antigens ( Fig. 2). Also, each antiserum, but not negative control sera, reacted directly with yeast and hyphal forms of the fungus as evidenced by indirect immunofluorescence microscopy (data not shown). The antibody response to the glycoconjugates was several fold greater over that of sera from control groups consisting of mice injected with buffer (Dulbecco's PBS), DCs alone, adjuvant alone (CFA) or DCs+CFA without antigen. Our previous work showed the protective potential of antibodies specific for β-(Man) ₃, and the presence of these antibodies in the sera from vaccinated animals was confirmed by dose-response ELISA inhibition as described in ref. 16 (data not shown).

Vaccination with Glycopeptide Conjugates Showed Protection Against Experimental Disseminated Candidiaisis in Mice.

The immunological properties of the conjugates prompted us to assess their potential in a vaccination strategy for disseminated candidiasis. Fourteen days after the second booster, the mice were challenged intravenously (i.v.) with a lethal dose (5 × 10 ⁵) of viable yeast forms of C. albicans that eventually killed all control mice. Conjugate-immunized animals, except for those that received the β-(Man) ₃-Pgk1, survived longer than each of four control groups consisting of DPBS alone, DCs alone, CFA alone or DCs+CFA alone.

The immunized groups that received the β-(Man) ₃-Hwp1, β-(Man) ₃-Fba, or β-(Man) ₃-Met6 conjugates showed 80−100% survival throughout the 120 day postchallenge observation period ( Fig. 3). Mice that received either β-(Man) ₃-Enol or β-(Man) ₃-Gap1 showed 40−80% survival. However, control groups given DPBS, DCs alone, CFA alone or DCs+CFA without antigen, or mice given the β-(Man) ₃-Pgk1 conjugate, had median survival times of 5, 6, 14, 16, and 11 days, respectively. Importantly, the survivors had greatly reduced or even nondetectable viable fungal units (colony forming units, CFU) in kidneys, a target organ in disseminated candidiasis, compared with animals that succumbed ( P < 0.0001).

The exception to the above trend exemplified by β-(Man) ₃-Pgk1 is in itself interesting. These animals died by day 13 after challenge compared with day 40 for the DC+CFA control. In a repeat experiment the numbers were 16 and 26 days for β-(Man) ₃-Pgk1 immunized mice and controls, respectively ( Fig. 3).

To confirm that induced antibodies were protective, sera from mice immunized against the β-(Man) ₃-Fba conjugate were transferred to naïve mice as before ( 5, 26) and challenged with live C. albicans. These mice had significantly prolonged survival times and reduced kidney fungal colony counts compared with those that received serum from nonimmunized controls or antiserum preabsorbed with fungal cells before challenge ( Fig. 4).

Vaccination with the Fba Peptide Induced Antibody Production and Protection Against Disease.

The previous results led us to consider whether protection was restricted to responses against the mannotriose, or whether responses to the carrier peptide also contributed. The Fba peptide and the synthetic β-(Man) ₃ were used independently to pulse DCs for subsequent immunizations. The peptide induced relatively strong specific antibody responses ( Fig. 5) that were not cross-reactive with β-(Man) ₃ (data not shown), whereas mice immunized against the mannotriose without carrier produced, as expected, only minimal antibody titers ( Fig. 5). Both test groups survived longer than DPBS controls following an i.v. challenge with live fungal cells, but all mice that received peptide survived. Based on our earlier findings that antibodies specific for the β-(Man) ₃ can be protective against candidiasis ( 5, 14, 27), the relatively high trisaccharide-specific titers in protected mice following immunization with three of the conjugates ( Fig. 3), ability to passively transfer protection to naïve mice, and slight protection in mice that developed low antibody titers to the mannotriose alone, led us to conclude that the β-(Man) ₃-Fba conjugate induced protective antibody specific for the β-(Man) ₃ part of the vaccine conjugate. This conclusion was further strengthened by an extended observation in which neither the Gap1 nor the Eno1 peptide alone induced protection against disseminated candidiasis (data not shown), yet the β-(Man) ₃-peptide conjugate of each induced high titers of anti-β-(Man) ₃ antibodies that correlated with an intermediate to high level of protection against disease ( Fig. 3). These various results indicate that both epitopes that comprise the synthetic vaccine induce independent and favorable responses against candidiasis.

Discussion

Six chemically defined glycopeptide vaccine conjugates have been prepared by a convenient and efficient conjugation strategy that avoids aromatic or other cyclic structural elements ( 28) that can themselves elicit a strong humoral immune response, which may suppress the desired antibody response against the target epitopes ( 29- 31). Each synthetic conjugate comprised a β-linked mannotriose (glycan) epitope corresponding to the Candida albicans cell wall β-mannan, a unique, nonimmunogenic linker, and one of six selected peptide carriers derived from cell wall proteins of the fungus that are known to be expressed during pathogenesis of human candidiasis. When tested for their ability to protect experimental animals against hematogenously disseminated candidiasis following a dendritic cell (DC) immunization these putative vaccine candidates produced antibody against the glycan epitope and the carrier peptide. Presumably responses against both epitopes can be anticipated because DCs present peptide epitopes via the MHC class II pathway. In our case, expression of mannose receptor (MR) on the DC surface was also likely advantaged because of its recognition of N-linked mannosyl residues ( 32), of which the β-linked mannotriose is a part ( 33). In preliminary work, we determined that the synthetic β-linked mannotriose coupled to protein carriers, such as BSA, were poorly immunogenic in mice even when given with complete Freund adjuvant. The antigen-pulsed DC immunization approach, however, proved to be a powerful way of overcoming the relatively weak immune response of the mouse to the defined small carbohydrate and peptide antigens of C. albicans.

The work reported here is intriguing from a number of standpoints. First, our results are in sharp contrast to observations that suggest glycoconjugate vaccines be constructed from oligosaccharides consisting of multiple repeating units ranging in size from 8 up to 200 saccharide residues ( 13- 16). Here, we show the feasibility of generating a synthetic glycopeptide-based vaccine where peptide and saccharide components are small and chemically defined by standard techniques such as NMR and mass spectrometry. In this case, we demonstrated utility in development of a vaccine against a fungal disease, but the process should be applicable to glycopeptide vaccine development in general. Second, the immunization approach that induces robust antibody responses to otherwise poorly immunogenic vaccines should be generally applicable to vaccine development in mouse models of human disease. Third, the work establishes the usefulness of epitope-finding algorithms for development of synthetic vaccines. That is, identification of an appropriate peptide carrier epitope lends itself to rapid synthesis in the laboratory and potentially obviates the need for working with large recombinant antigenic/carrier proteins. In fact, use of this approach led both to the identification of potentially useful vaccine candidates against candidiasis and the surprising finding of one conjugate, β-(Man) ₃-Pgk1 that enhanced disease susceptibility. This latter intriguing observation merits more investigation as it may yield insights, in addition to those discovered earlier in our lab ( 5, 14) into mechanisms by which antibody or other immune responses against C. albicans may not be of benefit to the host. Although we do not know a cause for the enhanced disease, the mechanism does not appear to be related to IgE induction because this Ig isotype was not enhanced in sera of immunized mice (data not shown). Regardless, the approaches outlined here allow for rapid screening of multiple peptide carriers to determine which will lead to optimal host immune responses. Finally, while synthetic glycopeptide vaccines reported by others induce potentially protective antibody responses against only the carbohydrate epitope of the vaccine conjugate ( 34, 35), at least one conjugate reported here uniquely induces protective responses against both the carbohydrate and peptide carrier components. Developers of glycan-based vaccines commonly employ bacterial toxoids, such as that of tetanus or diphtheria, as the protein carrier molecule ( 34, 36, 37). The immunological universality of these carriers and acceptance for human use are distinct advantages. An advantage of our approach is that the peptide carrier is derived from the infectious agent of interest, providing the possibility of a dual protective immune response against both the glycan epitope and the peptide carrier. In conclusion, our work shows the potential for chemical synthesis of relatively small molecular weight vaccines that induce protection against more than one unrelated epitope expressed by the infectious disease agent of interest.

Materials and Methods

Organism and Culture Conditions.

C. albicans 3153A (ATCC 28367) was grown as stationary-phase yeast cells in glucose-yeast extract-peptone broth at 37°C, washed and suspended in cold Dulbecco's PBS (DPBS; Sigma), and used to infect mice as described ( 5, 15).

Mice.

BALB/c female mice (National Cancer Institute Animal Production Program or Harlan) 5 to 7 weeks old were used throughout. Mice were maintained and handled in accordance with a protocol approved by the Institutional Animal Care and Use committee (IACUC) regulations at Children's Hospital Research Institute in New Orleans.

Synthetic Glycopeptide Vaccines.

Six cell wall proteins were selected as possible carriers on the basis of their known expression during pathogenesis of human candidiasis a cell wall location ( 22, 23) and included: hyphal wall protein-1 (Hwp1); enolase (Enol); phosphoglycerate kinase (Pgk1); glyceraldehyde-3-phosphate dehydrogenase (Gap1); fructose-bisphosphate aldolase (Fba); and methyltetrahydropteroyltriglutamate (Met6). By application of an epitope-finding algorithm ( www.genscript.com/cgibin/tools/antigenic_prediction.pl), we selected the following 14-mer peptides for synthesis, all of which are located near the N terminus of each protein group: Hwp1, QGETEEALIQKRSY; Eno1, DSRGNPTVEVDFTT; Pgk1, VPLDGKTITNNQRI; Gap1, NRSPSTGEQKSSGI; Fba, YGKDVKDLFDYAQE; and, Met6, PRIGGQRELKKITE. Synthetic peptides were produced commercially (GenScript) or synthesized in our laboratory by use of an automated peptide synthesizer.

Glycopeptides were synthesized by a conjugation procedure of which more details are presented in the SI and in reference ( 19). In brief, resin bound peptides were assembled with an automated synthesizer and after completion of the corresponding peptide sequence, S-trityl protected mercaptopropionic acid was coupled to install a sulfhydryl moiety to serve as attachment point for conjugation of the β-mannan trisaccharide epitope. The latter was synthesized as an allyl glycoside and subsequently reacted to provide a triethylene glycol tether terminated by an acrylate ester. The β-mannotriose-peptide (glycopeptide) conjugates were formed in high yield by reacting the deprotected oligosaccharide derivative with the thiol terminated peptide. All glycopeptides were purified by RP-HPLC, and their purity and identity confirmed by analytical HPLC, mass spectrometry and for selected examples by NMR. For ease of reference the conjugates were designated as β-(Man) ₃-Hwp1, β-(Man) ₃-Enol, β-(Man) ₃-Gap1, β-(Man) ₃-Pgk1, β-(Man) ₃-Fba, β-(Man) ₃-Met6. Details of the chemical synthesis and analytical data of the compounds are provided in the SI section.

Isolation and Culture of Dendritic Cells (DC) from Mouse Bone Marrow.

DCs were generated from bone marrow by a previously described method ( 38, 39), with some modifications. Briefly, bone marrow flushed with RPMI-1640 from the long bones of euthanized mice was gently pipetted and filtered through a 70- mm cell strainer to dissociate cell clusters. Red blood cells were lysed (ACK lysing buffer, 0.15 M NH ₄Cl, 1.0 mM KHCO ₃, 0.1 mM EDTA) and remaining bone marrow cells were suspended in complete medium [CM, RPMI-1640 supplemented with 10% FBS (FBS), 2 mM l-glutamine, 1% of nonessential amino acids and 100 units/ml penicillin and 100 mg/ml streptomycin], adjusted to 2 × 10 ⁵ cells per ml, plated in 6-well plates at 5 ml per well and cultured for up to 9 days in the presence of 40 ng/ml of rmGM-CSF and rmIL-4 (R&D Systems) at 37°C, 5% CO ₂. On days 4 and 7 of culture, the same amount of fresh GM-CSF and IL-4 was added to the wells.

To isolate the DC population, the cells were suspended in 2-4 ml of warm CM, overlaid onto the same volume of 14.5% (wt/vol) Nycodenz (Sigma) in CM and centrifuged at 1,200 × g for 20 min at 22°C. After centrifugation, cells in the interface were collected, washed with CM three times and subjected to phenotypic and functional analyses. Flow cytometric analysis and the use of MAbs specific for CD11c, CD11b, CD19 and CD3e (eBioscience) showed that >80% of the cells were DCs.

Immunogens and Immunizations.

DCs were pulsed in vitro with vaccines as described ( 40, 41) and with our modifications. Briefly, DCs in culture were pulsed with the antigen of choice (1 μM) on day 6. On day 7, PGE ₂ (10 −7-10 −9M) was added along with LPS (2 μg/ml, Sigma) for 24h. On day 9, antigen-pulsed DCs were washed extensively and 5 × 10 ⁵ in 200 μl DPBS were given i.p. (ip). The mice were boosted at day 14 with fresh antigen-pulsed DCs and boosted a second time at day 28 with antigen (10 μg) emulsified in complete Freund adjuvant (CFA).

Serological Assays.

Sera were ELISA analyzed for antibody titers. Control groups consisted of mice given DCs alone at the time of priming and first booster followed by DPBS at the time of the second booster, DPBS for the priming and first booster and CFA alone at the time of the second booster, DPBS alone for all three injections, or DCs alone for priming and first booster followed by CFA alone at the time of the second booster. Briefly, C. albicans PMP extract ( 13), which is composed mainly of mannan, but also 12−20% protein, was dissolved at 10 μg/ml in carbonate buffer (pH 9.6). The glycopeptide conjugates, β-(Man) ₃-BSA or synthetic peptides were dissolved at 10 μg/ml in PBS (pH 7.4). Each was used to coat 96-well ELISA plates for testing duplicate serial 2-fold dilutions of samples of each immune serum and control sera. Color development for each well was achieved by secondary antibody (goat anti-mouse polyvalent Ig-HRP, Sigma) and substrate ( O-phenylenediamine and H ₂O ₂) and OD determined at 492 nm.

Fungal Challenge and Assessment of Protection.

Two weeks after the second boost, immune and control mice were infected i.v. with a lethal dose of live C. albicans yeast cells (5 × 10 ⁵ in 0.1 ml of DPBS) as described in ref. 41. Passively immunized mice (below) also received the same challenge dose. Protection was evaluated by monitoring animal survival for up to 120 days and by quantifying the number of CFU (mean ± SE) per kidney pairs ( 5, 27, 42).

Protection Against Candida Infections by Passive Transfer.

Pooled serum from β-(Man) ₃-Fba immunized mice was ELISA titered as described above. Passive immunization was performed by single i.p. administration of 0.5 ml of the pooled antiserum, or antiserum preadsorbed with yeast as a control ( 5, 15, 27), 4 h before i.v. challenge with C. albicans. Infected mice were killed when they became moribund and their kidneys were assessed for CFUs.

Statistical Analysis.

Median survival times (MST) were statistically evaluated by Kaplan-Meier (GraphPad Prism, version 4). In all analyses, there were five mice per group ( n = 5) and a two-tailed t test was used.

Acknowledgments

The authors acknowledge C.J. Clancy and M.H. Nguyen for their critical review. We thank P. Lambert (J.E.C. laboratory) for technical assistance in providing PMP extracts of C. albicans. This work was supported by National Institutes of Health-National Institute of Allergy and Infectious Diseases Grants RO1 AI24912 and PO1 AI061537, and the Research Institute for Children. S.D. thanks the Deutscher Akademischer Austauschdienst and the Alberta Heritage Foundation for Medical Research for postdoctoral fellowships.

Footnotes

§To whom correspondence should be addressed at:
Research Institute for Children, Children's Hospital, 200 Henry Clay Avenue, New Orleans, LA 70118.
E-mail: jcutler{at}chnola-research.org

↵‡ Present address: Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.

Author contributions: H.X., S.D., D.R.B., and J.E.C. designed research; H.X., S.D., and J.E.C. performed research; S.D. contributed new reagents/analytic tools; H.X., D.R.B., and J.E.C. analyzed data; and H.X., S.D., D.R.B., and J.E.C. wrote the paper.

Conflict of interest statement: D.R.B. and J.E.C. are listed as coinventors on a pending patent filing that describes the chemical conjugation process.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0803195105/DCSupplemental.

# data[].attributes.attachment_link.title
Synthetic glycopeptide vaccines combining β-mannan and peptide epitopes induce protection against candidiasis

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The first fully synthetic glycopeptide vaccines against a fungal disease have been used to combat disseminated candidiasis in mice. Six T cell peptides found in Candida albicans cell wall proteins were selected by algorithm peptide epitope searches; each was synthesized and conjugated to the fungal cell wall β-mannan trisaccharide [β-(Man)3] by novel saccharide-peptide linker chemistry to create glycopeptide conjugates.

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Candidiasis: Predisposing Factors, Prevention, Diagnosis and Alternative Treatment

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Candidiasis: Predisposing Factors, Prevention, Diagnosis and Alternative Treatment

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Infections caused by Candida species have been increased dramatically worldwide due to the increase in immunocompromised patients. For the prevention and cure of candidiasis, several strategies have been adopted at clinical level. Candida infected patients are commonly treated with a variety of antifungal drugs such as fluconazole, amphotericin B, nystatin, and flucytosine.

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Candidiasis: A Fungal Infection- Current Challenges and Progress in Prevention and Treatment

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Candidiasis: A Fungal Infection- Current Challenges and Progress in Prevention and Treatment

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Current treatment landscape - Candidiasis

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Current treatment landscape - Candidiasis

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Abstract

Introduction

Invasive candidiasis (IC) comprises both bloodstream and other deep-seated invasive infections caused by Candida species, and represents a significant cause of morbidity and mortality. IC may affect many different patient populations cared for by a large number of physicians in varying types of hospital units. In recent years, there have been substantial changes in IC management and in the availability of antifungal agents, with greater complexity in prescribing suitable drugs and increasing interest in the development of stewardship programmes. The aim of this review is to provide an overview of the changing epidemiology of Candida species infection and to outline the current diagnostic and treatment options, with a specific focus on the role of stewardship programmes in optimizing management strategies.

The changing epidemiology of Candida infections

Over recent decades, the incidence of IC has shown stable or increasing rates in most regions, probably due to the growing complexity of surgical procedures, the existence of patient populations at higher risk of infection and the changes in patient demographic characteristics. In recent epidemiological studies, candidaemia has been described as the fourth to the tenth most common cause of bloodstream infection worldwide and its incidence has showed important geographical variation, ranging from 0.02 to 1.73 cases per 1000 hospital admissions.

The epidemiology of candidaemia has been studied extensively in large series focusing on specific high-risk patient populations, including neonates, patients with cancer and surgical and critically ill patients. In recent years, however, candidaemia has emerged as a rising problem in patients hospitalized in other settings, such as internal medicine wards. This setting can represent a significant reservoir for Candida infections, showing prevalence ranging from 24% to 57% and higher mortality rates compared with other wards.

During recent years, changes in species distribution have also occurred according to different geographical areas, local hospital-related factors, patient predisposing conditions and the types of antifungal agents received. Candida albicans used to represent the predominant pathogen among the Candida species; however, today this species accounts for only half the isolates detected. A shift towards an increasing prevalence of Candida glabrata and Candida parapsilosis species has been recently documented, especially among critically ill patients, probably due to an increase in oncology-haematological patients previously exposed to antifungal therapy. Changes in species distribution may impact treatment recommendations because of differences in susceptibility to azoles and echinocandins among these species. Previous exposure to antifungals is clearly associated with a shift in species distribution and MICs of available antifungal agents. In addition, inappropriate antifungal use has contributed to the global increase in antifungal resistance to both triazole and echinocandin antifungal drugs, and merits continued vigilance.

Current treatment options for Candida infections

During the past few years, new insights have substantially changed therapeutic strategies. The classes of antifungal agents that are mainly used in invasive Candida species infections include azoles, polyenes and echinocandins.

Azole antifungal agents target the 14-α-demethylase enzyme which mediates the conversion of lanosterol to ergosterol in the fungus wall. This class is metabolized by P450 cytochromes, which can result in drug-drug interactions. Fluconazole is used in the treatment of candidaemia as a de-escalation therapy, and for the treatment of non-critically ill patients without previous exposure to azoles and without evidence of colonization with a strain that has reduced susceptibility to azoles.

Amphotericin B is a polyene antifungal agent that binds to the ergosterol in the fungal membrane. Owing to its toxicity, amphotericin B deoxycholate has now been replaced by better-tolerated polyenes. Three lipid formulations of amphotericin B are currently approved for clinical use: liposomal amphotericin B (L-AmB), amphotericin B lipid complex (ABLC) and amphotericin B colloidal dispersion (ABCD). L-AmB is widely used and has favourable pharmacokinetics along with high intracellular penetration in the cerebral spinal fluid and in the eye. Both L-AmB and ABLC achieve therapeutically effective concentrations in the epithelial lining fluid of critically ill patients. L-AmB is used as a first-line therapy for disseminated forms of Candida species infection, and as a second-line therapy for IC.

Echinocandins target the fungal cell wall and act by inhibiting 1,3-β-d-glucan (BDG) synthesis, showing fungicidal activity against Candida species. As BDG is not part of human cells, echinocandins display strong tolerability and safety profiles. Other characteristics of these compounds include broad and strong fungicidal action with activity against biofilms, and few drug-drug interactions; the development of resistance is also rare. Currently, three echinocandins (anidulafungin, caspofungin and micafungin) are used for intravenous treatment and prevention of IC, although most data come from randomized controlled trials for candidaemia; very few data on other forms of IC are available. In addition, it should be taken into account that these drugs do not achieve therapeutically effective concentrations in some tissues (e.g. eyes, CNS, urine, endocardium and peritoneum) and their pharmacokinetic/pharmacodynamic properties are poorly known for critically ill patients. Moreover, CLSI clinical breakpoints have not yet been established for caspofungin due to significant inter-laboratory variation in MIC ranges. In general, isolates that are susceptible to anidulafungin as well as micafungin should be considered susceptible to caspofungin. Echinocandins are recommended as first-line therapy in the treatment of IC in critically ill patients and are preferred in non-critically ill patients with previous exposure to azoles and/or evidence of colonization with a Candida strain with reduced susceptibility to azoles.

Flucytosine (5-fluorocytosine) is an antifungal agent that interferes with both DNA and protein synthesis. Owing to the high incidence of primary and/or acquired resistance, use of flucytosine as monotherapy is significantly restricted. Nevertheless, it should be kept in mind that flucytosine has very good tissue distribution and should be used in combination with other antifungals in cases of endophthalmitis, endocarditis or CNS infections.

A few new classes of antifungal agents are currently in the pipeline. These include agents with mechanisms of action similar to clinically available classes, and novel mechanisms of action that are either fungus-specific or more selective for fungal targets than mammalian cells. Many of these agents have demonstrated potent in vitro and in vivo activity against a broad range of fungi, with some also maintaining activity against isolates known to be resistant to azoles and echinocandins.

Optimization of management of invasive candidiasis

The management of IC has changed markedly in the past decade. To minimize the negative impact of this infection, several management strategies have been proposed: prophylaxis, empirical therapy, pre-emptive therapy, and presumptive and culture-based targeted treatment. However, in this review, for practical purposes the term 'early' antifungal treatment refers to pre-emptive, presumptive or empirical therapy. The most important principles for the treatment of invasive Candida infections are summarized in Table 1. Each recommendation is discussed in turn below.

▪ Restrict antifungal prophylaxis

▪ Start prompt 'early' antifungal treatment based on risk factors

▪ Select an antifungal agent that the isolate is susceptible to

▪ Achieve adequate source control

▪ Use an adequate dose: low dose is associated with resistance

▪ Stop 'early' inappropriate therapy

▪ De-escalate whenever possible

▪ Check duration of therapy

Restrict antifungal prophylaxis to high-risk patients

Although the benefits of antifungal prophylaxis are well established in neutropenic patients (e.g. haematological patients), its utility in non-immunocompromised, critically ill patients with sepsis and no confirmed fungal infection is still controversial. Fluconazole prophylaxis in patients admitted to an ICU can reduce the incidence of IC; however, the patient population should be carefully considered before the use of prophylactic fluconazole is recommended. For example, in critically ill patients fluconazole can promote the emergence of non-albicans Candida species, some of which are less susceptible or resistant to fluconazole and its use prophylactically. We suggest restricting antifungal prophylaxis to surgical patients presenting with anastomotic leakage after abdominal surgery or re-operation of the digestive tract during the same hospitalization in order to prevent the development of IC (Figure 1). The optimal duration of prophylactic treatment is not known.

Start prompt 'early' antifungal treatment

IC has considerable clinical and economic consequences. The high morbidity and mortality associated with IC are, in part, related to the risk factors of the critically ill patient population, but challenges with diagnosis of the disease due to a lack of accurate diagnostic tools may also be a causative factor. Conventional mycological blood cultures are currently the gold standard for the diagnosis of candidaemia, but data suggest that these tools may fail to diagnose IC in up to 25%-50% of cases. It is therefore vital to investigate methods to improve the diagnostic accuracy of these tests. For example, in order to maximize the sensitivity of blood cultures, guidelines recommend filling the culture bottles with 10 mL of blood. The use of bottles containing adsorbing resins and bottles containing non-resin selective milieu also increases the positivity rate, especially in patients who have received antifungal therapy prior to blood culture. Furthermore, the time required for test results must also be considered. For example, time to positivity of blood cultures in patients with candidaemia can be 2-5 days from the time the blood culture is drawn, depending on the Candida species. This delay in diagnosis may cause a delay in the initiation of appropriate antifungal therapy if treatment is not initiated until a diagnosis of candidaemia is confirmed. Numerous studies have demonstrated that a delay in the initiation of antifungal therapy (e.g. >12 h after the first positive blood culture) is associated with significant increases in in-hospital mortality and cost of care for patients with candidaemia. Due to the challenges faced in rapidly diagnosing a fungal infection, the majority of treatment courses (∼75%) are currently used as part of a pre-emptive, presumptive or empirical strategy.

It is therefore vital to consider the value of predictive models. Predictive models of IC using the Candida Colonization Index (CCI) and corrected CCI (cCCI), or one of the available predictive scores (including the Candida score), are useful to stratify non-neutropenic critically ill patients at high risk; however, these are currently often indiscriminately applied. Recent studies comparing different clinical scores have demonstrated that the CCI has greater predictive ability for the diagnosis of intra-abdominal candidiasis in a highly selected group, whereas the Candida score is higher than the latter in non-selected (medical/surgical) patient populations with candidaemia. Although these prediction rules display high negative predictive values, insufficient positive predictive values (<15%) limit their use. The risks of toxicity, selection of resistance and treatment costs should also be factored in when considering the clinical application of these predictive scores.

In recent years, other non-culture-based tests for diagnosis and susceptibility have been advocated to offset the diagnostic challenges and improve patient management, but their availability is currently limited to reference mycology laboratories and is associated with high costs. BDG testing has shown high negative predictive value and can be useful to rule out IC; nevertheless, it is not specific for IC as it is considered a pan-fungal diagnostic method. False-positive results are frequently observed in the most severe critically ill patients undergoing renal replacement therapies or extracorporeal membrane oxygenation, and in patients who have received immunoglobulin therapy. Interestingly, in a recent prospective cohort study, BDG showed superiority to culture, Candida score, CCI and cCCI in predicting the diagnosis of blood culture negative intra-abdominal candidiasis in a highly selected population of patients with gastrointestinal post-operative leakage or necrotizing pancreatitis, and correlated with severity of illness and outcome.

Different commercially available Candida tests measuring mannan/antimannan antibodies in serum are specific diagnostic markers of Candida species, showing high negative predictive values in patients with IC and possibly being useful to detect early infections (∼6 days prior to blood culture). PCR detection of Candida DNA is a potentially powerful diagnostic tool to promptly identify Candida species but its diagnostic performance is still questioned because of the heterogeneity of the available results, the lack of reliable reference standards and differences in techniques used.

Antibody detection kits, such as the Candida albicans germ tube antibody test (CAGTA) are under evaluation, but there are limited data about their clinical accuracy. Procalcitonin (PCT) for detection of Candida species has a high negative predictive value by either blood culture or PCR and may be a useful tool to rule out the presence of candidaemia and guide antifungal treatment regimens in critically ill patients. Overall, a combination of non-culture-based techniques may be needed to optimize the diagnosis of Candida infection and reduce the incidence of undiagnosed patients.

Understanding the relationship between microbiological findings, clinical symptoms and serological and molecular biomarkers may help clinicians in the stratification of patients and to more accurately select the candidates suitable for prompt 'early' antifungal treatment.

The criteria to start prophylaxis and early antifungal therapy based on the authors' experience are listed in Figure 1. Treatment should be based on expert opinion with a careful assessment of: (i) individual risk factors (immune suppression, surgery, invasive procedures, previous use of broad-spectrum antibiotics, pancreatitis, total parenteral nutrition, haemodialysis, steroids and length of hospital stay); (ii) severity of infection (sepsis or organ failure); (iii) fungal history (multiple colonizations, previous candidaemia or proven IC); and (iv) non-culture tests (positivity for a BDG level >250 pg/mL or Candida PCR, very low or non-significant PCT).

Start adequate antifungal treatment and determine source control

The selection and optimization of an antifungal regimen in treating Candida bloodstream infection are based on multiple factors, including the knowledge of local epidemiological data to guide empirical therapy as well as both species identification and antifungal susceptibility testing.

In patients with IC the synergistic effect of optimal treatment and source control within 24−48 h has been advantageous and has marked a paradigm shift in the treatment of IC. Current guidelines from the ESCMID and the IDSA recommend the empirical use of echinocandins in critically ill patients, whereas azoles and lipid formulations of amphotericin B are considered to be alternative or second-line options (Table 2). Amphotericin B should also be reserved for end-organ infections such as meningitis, endocarditis or osteomyelitis, or for patients where co-infection with other fungal pathogens is documented or suspected. There is currently no evidence of significant benefit in combining antifungals for the treatment of IC.

New therapeutic practices may result in ecological shifts and the emergence of resistance, which could influence future recommendations for the management of patients suspected of having IC; this merits vigilance. A prospective multicentre study showed that both fluconazole and caspofungin, the two major drugs recommended for the first-line treatment of candidaemia and for early treatment in high-risk patients, were associated with changes in the epidemiology of Candida infection and with an overall decrease in susceptibility of the isolates to these drugs. In addition, the emergence of co-resistance to both azoles and echinocandins in clinical isolates of C. glabrata, a species that appears to be unique in its ability to sequentially acquire and express resistance mutations, has been documented over time.

Source control, which encompasses all measures to control invasive infection and restore optimal function of the affected area, has been shown to be an important determinant of outcome for patients with candidiasis. Although catheter removal in patients with candidaemia remains a controversial issue, based on data reported in expert recommendations and previous studies, central venous catheter and device withdrawal should be attempted, any identified collection should be drained and adequate surgical source control should be performed.

Use an adequate dose of antifungal therapy

Although in the past lower doses of antifungal therapy were considered useful to avoid side effects, adequate doses of treatment are now considered paramount to avoid resistance development. Fluconazole should be used at a 12 mg/kg loading dose followed by 6 mg/kg daily. Three lipid formulations of amphotericin B are currently approved: L-AmB, ABLC and ABCD, which are generally used at the following doses: 3 mg/kg daily, 5 mg/kg daily and 3-4 mg/kg daily, respectively. Three echinocandin agents are available: caspofungin (dose of 70 mg on day 1 followed by 50 mg daily), micafungin (dose of 100 mg daily) and anidulafungin (dose of 200 mg on day 1 followed by 100 mg daily thereafter).

Stop inappropriate therapy early

Early treatment is frequently used, although not proven to be effective, and its discontinuation should be carefully evaluated to stop 'inappropriate' therapy as soon as possible. Antifungals are usually maintained for an extended time (∼10 days in patients without IC) with the associated unnecessary risk of toxicity and additional costs. A recent study suggested that stopping empirical treatment early, within 5 days in the absence of documented candidiasis, did not modify the prognosis of ICU patients. The low sensitivity of blood cultures complicates the decision to stop antifungal treatment safely. It should be emphasized that blood cultures should be appropriately taken before any treatment decision is made, in order to improve their accuracy and allow successful early discontinuation. Importantly, the added use of combinations of different biomarkers (BDG, mannan/antimannan and the CAGTA test) could help as a decision-making tool for discontinuing unnecessary empirical therapy in patients with suspected candidaemia.

De-escalate antifungal therapy if possible

In cases of proven candidiasis or candidaemia, treatment should be given immediately; subsequently, early step-down therapy with a triazole should be considered. Antifungal susceptibility testing is not routinely performed at all institutions but can identify patients with resistant Candida species who are receiving an inappropriate agent and patients who would be candidates for de-escalation. De-escalation from an echinocandin to intravenous or oral fluconazole should be encouraged when the patient is clinically stable and the isolated strain is susceptible. Nevertheless, usually in daily practice a low proportion (around 20%-40%) of de-escalation from an echinocandin to fluconazole therapy in patients with fluconazole-susceptible isolates is performed. The exact timing for shifting to fluconazole is unknown and may vary from patient to patient, depending on patient- and pathogen-related factors. For IC, the ESCMID and IDSA guidelines recommend a de-escalation strategy (3 days in stabilized patients, as per IDSA, and 10 days overall, as per ESCMID) to limit the emergence of resistant strains and to reduce treatment costs. The safety of de-escalation (within 5 days) in the case of proven or probable IC has been recently suggested for non-neutropenic patients treated in the ICU, since it was not associated with increased mortality and led to a subsequent and significant decrease in antifungal use.

Determine an adequate duration of antifungal therapy

The duration of treatment depends on the extent of organ involvement. For uncomplicated candidaemia, duration of treatment is generally considered to be 14 days from the first negative blood culture, requiring daily blood cultures to be performed until negativity. It is important to highlight that this duration applies only to patients in whom disseminated disease, abscesses, or end-organ disease have been excluded. Therefore, it is recommended that all patients with candidaemia undergo diagnostic procedures to detect organ involvement, including transoesophageal echocardiography, fundoscopy and searching for a thrombus. Treatment duration might be much longer in deep-seated infections and endocarditis.

The role of Candida-specific stewardship programmes

The appropriate use of antifungal agents can improve efficacy, limit the potential for emergence of antifungal resistance and reduce treatment-related costs. The importance of care bundles has been described for the prevention and management of many bacterial infections. However, there is limited experience with antifungal stewardship (AFS) programmes targeting antifungal treatment bundles. We have analysed the most significant examples of AFS programmes that have been implemented to date, aiming to target antifungals active against invasive Candida infections. Table 3 includes the most recent publications.

Swoboda et al. demonstrated that the implementation of standardized practice guidelines for antifungal therapy in ICUs decreased the use of selected antifungal treatment bundles, resulting in a 50% reduction in costs. Nevertheless, quality indicators, including length of stay, readmission and mortality, did not change.

Apisarnthanarak et al. conducted a quasi-experimental study 18 months before and after the implementation of an AFS programme for IC. Measures included specific prescriptions, meetings with prescribers and the supervising team, and a systematic clinical assessment at three timepoints: initial (<48 h), between day 3 and day 7, and at the end of treatment. A 59% reduction in antifungal prescriptions (from 194 to 80 prescriptions per 1000 hospitalizations, P < 0.001) was reported after the intervention. The authors concluded that there was also a reduction in the inappropriate use of antifungals against Candida species from 71% to 24% ( P < 0.001) and that fluconazole use decreased from 242 to 117 DDDs per 1000 patient-days ( P < 0.0001). Notably, these measures had a positive impact on the species distribution, since the incidence C. glabrata and Candida krusei decreased significantly, whereas the rate of C. albicans infections tended to increase. Total cost savings were ∼US$ 30 000.

Standiford et al. described the implementation of a 7 year AFS programme in a large tertiary hospital. The duties of the team were to provide an active computer-assisted real-time review of antimicrobial orders to provide active intervention when necessary and the development of guidelines and policies. Before implementation, annual antifungal expenditure exceeded US$ 3.7 million, declining to US$ 1.3 million by the end of the programme. This study also looked at outcome measures such as hospital stay, readmissions and mortality, finding no significant changes before, during and after programme termination.

Lopez-Medrano et al. conducted an intervention study with a non-randomized uncontrolled 'before and after' methodology at a teaching hospital for 12 months. An infectious diseases (ID) specialist managed the supervision and was present in the hospital for 3 h per day, to provide advice for prescribers, and suggested rather than ordered therapies. A total of 662 antifungal therapies (51.7% of which targeted Candida species) were reviewed and a recommendation to change treatment was made in 29% of the cases, including a change from intravenous to oral treatment (15%), cessation of antifungal treatment (8%) and a change to fluconazole (6%). Expenditure on antifungals was reduced by US$ 370 000 (11.8% reduction), but use of antifungal therapy in DDDs was not affected by the intervention. The programme was not related to significant increases in the incidence of candidaemia, percentage of persistent or relapsing candidaemia cases and percentage of fluconazole-resistant Candida species.

A study by Guarascio et al. evaluated a care bundle on the use of antifungals aimed at decreasing the prescription of echinocandins in the ICU setting in a matched control analysis (36 patients after implementation of the AFS programme were compared with 72 paired controls). Clinicians documented five points of a daily protocol (based on initial diagnosis or indication, planned duration of therapy and the potential for de-escalation or discontinuation of therapy) and the renewal of the treatment depended on the answers. The authors found a significant reduction in median days of caspofungin therapy (4.00 versus 2.00, P = 0.001) in the bundle group. Nevertheless, most of this reduction in use was realized in the medical ICU ( P = 0.002) as opposed to the surgical ICU ( P  = 0.188), probably because of the higher compliance with the care bundle recommendations (79% versus 58%; not statistically significant). The average cost of treatment was decreased by approximately US$ 1000 per patient, compared with the previous period.

In their quasi-experimental investigation, Antworth et al. developed an AFS team that advocated prospective recommendations in accordance with care bundles, which included utilization of appropriate antifungal treatment with appropriate duration of use, removal of intravenous catheters, repeat blood cultures, monitoring of time until clearance of candidaemia, and performance of ophthalmological examinations. In this study, a significant increase in all candidaemia care bundle elements in the AFS programme group versus the control group was demonstrated (78.0% versus 40.5%, P = 0.0016). Of note, the implementation of the AFS programme significantly improved the proportion of patients receiving an ophthalmological consultation (97.6% versus 75.7%, P = 0.0108), the selection of appropriate definitive antifungals (100% versus 86.5%, P = 0.0488) and an appropriate duration of therapy (97.6% versus 67.7%, P  = 0.0012). Numerical increases were also seen for the other bundle components, such as removal of intravenous catheters and performance of repeat blood cultures, although they were not significantly different. Nevertheless, length of hospitalization, time until clearance of candidaemia, rate of persistent candidaemia and rate of recurrent candidaemia were similar in the AFS programme group versus the control group.

Mondain et al. performed a prospective observational study describing a multifaceted AFS programme in a French teaching tertiary-care hospital from 2005 to 2010, inclusive. Activities included the systematic evaluation of all costly antifungal prescriptions, education of staff, creation of a multidisciplinary antifungal management team, improvement of diagnostic tools, presence of systematic real-time ID advice for candidaemia, and creation of local guidelines on antifungal prophylaxis and the diagnosis and treatment of candidaemia. Advice was given for 54% of cases reviewed, with a compliance rate of 88%. Regarding candidaemia, optimal standards of care were achieved for the timing of antifungal treatment ( P = 0.0025), recommended first-line therapy ( P = 0.0025), duration of therapy ( P = 0.46) and the removal of central venous catheters ( P  = 0.27), compared with pre-AFS programme findings, even if statistical significance was not met for all process of care measures. Improvements were also noted in the performance of echocardiography and follow-up blood cultures, without reaching statistical significance. Total antifungal prescriptions in DDDs and their cost were contained between 2003 and 2010, corresponding to a 38% decrease in total antifungal use.

Alfandari et al. found that a permanent collaboration between haematologists and an ID physician could improve antifungal prescribing, since antifungal consumption decreased by 40% (from ∼1000 to 620 DDDs per 1000 hospitalization days), but invasive fungal infection incidence remained stable, during the study period (2003-12).

Micallef et al. performed an observational prospective 12 month study conducted on an AFS programme targeting the use of high-cost antifungals (specifically L-AmB, voriconazole and echinocandins) in a tertiary referral hospital. Clinical and financial interventions were performed. The authors found that nearly half of all treatments reviewed were stopped or changed (except for voriconazole) and that there was a crude cost saving of £180 000 in antifungal drugs over the year.

The future role of stewardship programmes in the management of Candida infection

The authors encourage further exploration in the use of care bundles in AFS programmes as part of a multifaceted approach to promote appropriate antifungal utilization and to optimize the management of patients with IC. These programmes cannot succeed without continuous exchange between clinicians managing the patients and a multidisciplinary team, including an ID specialist team, supported by a microbiologist and a pharmacist trained in ID quality information. Although long-term ecological benefits cannot yet be measured, data suggest that AFS programmes may decrease the rate of Candida strains resistant to fluconazole, without affecting the quality of care provided. As previous use of antimicrobial agents represents one of the major risk factors of IC, antifungals should be included in a global anti-infective stewardship programme. The integration of this programme with control of nosocomial infections and cross-transmission of multiresistant organisms is also a key point that must be considered.

Implementing, sustaining and assessing stewardship programmes require resources. Health professionals are now also tasked with the implementation and subsequent cost justification of new economic challenges. A report focusing on the cost analysis of such a programme demonstrated that the establishment of an AFS programme can be very cost-effective. Finally, it is important to emphasize that this stewardship work should be offered on an ongoing basis, as it has also been shown that the baseline prescribing culture returns shortly after an interruption. Further studies are needed and should explore the possibility of developing multicentre AFS programmes for extended periods of time.

Conclusions

There are several opportunities to optimize management strategies for Candida infections. The implementation of institutional AFS programmes has the potential to provide significant improvements in care for patients with Candida infections. However, a cultural change among healthcare providers and authorities will be needed to improve antifungal use worldwide.

Transparency declarations

M. B. serves on scientific advisory boards for AstraZeneca, Bayer, Gilead, Merck, Pfizer Inc., Paratek, Astellas Pharma Inc and Tetraphase and has received funding for travel or speaker honoraria from AstraZeneca, Gilead, Pfizer Inc., Merck, Novartis, Gilead Sciences, Angelini, Astellas Pharma Inc. and Vifor Pharma. J. F. T. serves on a scientific advisory board for Merck, has received research grants and/or speaker honoraria from Merck, Astellas, Pfizer and Gilead. M. P. has none to declare.

This article forms part of a Supplement sponsored and funded by Gilead Sciences Europe Ltd; editorial assistance was provided by Synergy Medical. The content of this Supplement is based on the sessions presented at the CARE VIII meeting, held in Madrid in November 2015.

References

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current treatment landscape: candidiasis | Journal of Antimicrobial Chemotherapy | Oxford Academic

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Invasive candidiasis (IC) comprises both bloodstream and other deep-seated invasive infections caused by Candida species, and represents a significant cause of morbidity and mortality. IC may affect many different patient populations cared for by a large number of physicians in varying types of hospital units.

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Candida infections of the mouth, throat, and esophagus

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Candida infections of the mouth, throat, and esophagus

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Definition

Candidiasis is an infection caused by a yeast (a type of fungus) called Candida. Candida normally lives in the digestive tract and on skin without causing any problems. Sometimes, Candida can multiply and cause an infection if the environment inside the mouth, throat, or esophagus changes in a way that encourages fungal growth.

Candidiasis in the mouth and throat is also called "thrush" or oropharyngeal candidiasis. Candidiasis in the esophagus (the tube that connects the throat to the stomach) is called esophageal candidiasis or Candida esophagitis. Esophageal candidiasis is one of the most common infections in people living with HIV/AIDS.

Symptoms

Candidiasis in the mouth and throat can have many different symptoms, including:

White patches on the inner cheeks, tongue, roof of the mouth, and throat (photo showing candidiasis in the mouth)

Redness or soreness

Cottony feeling in the mouth

Loss of taste

Pain while eating or swallowing

Cracking and redness at the corners of the mouth

Symptoms of candidiasis in the esophagus usually include pain when swallowing and difficulty swallowing. Contact your healthcare provider if you have symptoms that you think are related to candidiasis in the mouth, throat, or esophagus.

Risk & Prevention

Who gets candidiasis in the mouth, throat, or esophagus?

Candidiasis in the mouth, throat, or esophagus is uncommon in healthy adults. People who are at higher risk for getting candidiasis in the mouth and throat include babies, especially those younger than one month old, and people who:

Wear dentures

Have diabetes

Have cancer

Have HIV/AIDS

Take antibiotics or corticosteroids, including inhaled corticosteroids for conditions like asthma

Take medications that cause dry mouth or have medical conditions that cause dry mouth

Smoke

Most people who get candidiasis in the esophagus have weakened immune systems, meaning that their bodies don't fight infections well. This includes people living with HIV/AIDS and people who have blood cancers such as leukemia and lymphoma. People who get candidiasis in the esophagus often also have candidiasis in the mouth and throat.

How can I prevent candidiasis in the mouth, throat, or esophagus?

Ways to help prevent candidiasis in the mouth and throat include:

Maintain good oral health

Rinse your mouth or brush your teeth after using inhaled corticosteroids

Some studies have shown that chlorhexidine mouthwash may help to prevent oral candidiasis in people undergoing cancer treatment

Sources

Candida normally lives in the mouth, throat, and the rest of the digestive tract without causing any problems. Sometimes, Candida can multiply and cause an infection if the environment inside the mouth, throat, or esophagus changes in a way that encourages its growth. This can happen when a person's immune system becomes weakened, if antibiotics affect the natural balance of microbes in the body, or for a variety of other reasons in other groups of people.

Diagnosis & Testing

Healthcare providers can usually diagnose candidiasis in the mouth or throat simply by looking inside. Sometimes a healthcare provider will take a small sample from the mouth or throat. The sample is sent to a laboratory for testing, usually to be examined under a microscope.

Healthcare providers usually diagnose candidiasis in the esophagus by doing an endoscopy. An endoscopy is a procedure to examine the digestive tract using a tube with a light and a camera. A healthcare provider might prescribe antifungal medication without doing an endoscopy to see if the patient's symptoms get better.

Treatment

Candidiasis in the mouth, throat, or esophagus is usually treated with antifungal medicine. The treatment for mild to moderate infections in the mouth or throat is usually an antifungal medicine applied to the inside of the mouth for 7 to 14 days. These medications include clotrimazole, miconazole, or nystatin. For severe infections, the treatment is usually fluconazole or another type of antifungal medicine given by mouth or through a vein for people who don't get better after taking fluconazole. The treatment for candidiasis in the esophagus is usually fluconazole. Other types of prescription antifungal medicines can also be used for people who can't take fluconazole or who don't get better after taking fluconazole.

If you are a healthcare provider, click here to see the Infectious Diseases Society of America's Clinical Practice Guidelines for the Management of Candidiasis.

Statistics

The exact number of cases of candidiasis in the mouth, throat, and esophagus in the United States is difficult to determine because there is no national surveillance for these infections. The risk of these infections varies based on the presence of certain underlying medical conditions. For example, candidiasis in the mouth, throat, or esophagus is uncommon in healthy adults. However, they are some of the most common infections in people living with HIV/AIDS.In one study, approximately one-third of patients with advanced HIV infection had candidiasis in the mouth and throat.

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Candida infections of the mouth, throat, and esophagus

# data[].attributes.attachment_link.description
Candidiasis is an infection caused by a yeast (a type of fungus) called Candida. Candida normally lives in the digestive tract and on skin without causing any problems. Sometimes, Candida can multiply and cause an infection if the environment inside the mouth, throat, or esophagus changes in a way that encourages fungal growth.

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Estimation of the Burden of Serious Human Fungal Infections in Malaysia

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Estimation of the Burden of Serious Human Fungal Infections in Malaysia

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-Study showed that recurrent vaginal candidiasis (>4 episodes/year) was the most common of all cases with a diagnosis of candidiasis (n = 501,138). Oesophageal candidiasis (n = 5850) was most predominant among individuals with HIV infection.
-Candidemia incidence (n = 1533) was estimated in hospitalized individuals, some receiving treatment for cancer (n = 1073), and was detected also in individuals admitted to intensive care units (ICU) (n = 460).

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Estimation of the Burden of Serious Human Fungal Infections in Malaysia

Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia

Centre of Excellence for Research in AIDS (CERiA), Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia

Department of Microbiology, School of Basic & Applied Sciences, Central University of Tamil Nadu (CUTN), Thiruvarur 610 101, Tamil Nadu, India

Faculty of Biology, Medicine and Health, The University of Manchester and Manchester Academic Health Science Centre, Oxford Road, Manchester M13 9PL, UK

The National Aspergillosis Centre, Education and Research Centre, Wythenshawe Hospital, Manchester M23 9LT, UK

Author to whom correspondence should be addressed.

Received: 11 December 2017 / Accepted: 14 February 2018 / Published: 19 March 2018

AIDS; aspergillosis; cryptococcal meningitis; epidemiology; HIV infection; Malaysia

1. Introduction

Fungal infections (mycoses) lead to significant rates of morbidity and mortality worldwide, ranging from mild to invasive disease in humans, plants and animals [ 1]. Individuals with a dysfunctional immune system such as immunocompromised people with underlying HIV infection, very young children and the elderly are vulnerable to fungal infections [ 2]. Mycoses such as Pneumocystis pneumonia [ 3, 4, 5, 6], cryptococcal meningitis [ 7, 8, 9], disseminated histoplasmosis [ 10, 11, 12] and Talaromyces marneffei (formerly Penicillium marneffei) infection [ 1, 13] are reported commonly among patients with HIV/AIDS and may occur in those with cancer or in intensive care or with adult onset immunodeficiency [ 2, 14]. Further, individuals with chronic respiratory disease are susceptible to airborne fungal infections including allergic bronchopulmonary aspergillosis (ABPA) [ 15, 16], severe asthma with fungal sensitization [ 17, 18], chronic pulmonary and invasive aspergillosis [ 19]. With the advent of modern diagnostic non-culture-based technology, the number of individuals diagnosed with systemic fungal infections (SFIs) will rise as more patients are diagnosed, but in addition, modern immunosuppression also brings its risks [ 6, 20, 21] and cases may rise steadily over the coming years. Estimates from Brown et al., [ 21] reveal that the global prevalence of SFIs has risen to 4 for every 1000 individuals of the global population. Estimates also suggest that >90% of fungal infections resulting in death are attributed to by Candida spp., Cryptococcus spp., Aspergillus spp. and Pneumocystis jirovecii-together causing over two million life-threatening infections globally, each year [ 19, 21].

In most developing countries, national community-based surveillance programs are usually considered as the gold-standard for estimating disease prevalence and incidence. However, it is difficult for all countries to implement this uniformly as it is complex and can be very expensive. Together, these factors have contributed to the paucity of systemic data on the incidence and prevalence of human fungal infections in Malaysia. Many studies have employed sentinel surveillance to provide data at a much lower cost [ 22], although this approach has its own limitations, and therefore must be adjusted across urbanized and densely populated communities. Especially in Asia, where overlapping healthcare providers represent the norm [ 23], high quality epidemiological studies require considerable resources.

Several researchers have recently undertaken an actuarial approach to estimate the burden of fungal infections at the national level [ 1, 8, 16]. Subsequently, because of non-standardized data available on global fungal infections and the prevailing limited quantity of systematic data on the incidence and prevalence of fungal infections in Malaysia, we have aimed in this paper to try to estimate the burden of serious human fungal infections in Malaysia. The current estimation is expected to provide a better perspective of our understanding of the global distribution of serious human fungal infections, serve as a reference for future investigations, and will contribute to the improvement in the public health aspects of fungal infections in the region.

2. Material and Methods

The burden of serious fungal infections in Malaysia was estimated among both a general healthy population and individuals at risk including people living with HIV/AIDS, chronic obstructive pulmonary disease (COPD), asthma, cancer, tuberculosis (TB) as well as those in intensive care for treatment of chronic conditions. Since there is no specific data on serious fungal infections, the estimation of the burden of fungal infections was derived from worldwide data and based on the methodology of the Leading International Fungal Education (LIFE) program. Data on the Malaysian population were extracted from World Heath Statistic 2011 [ 24] based on age and gender. In addition, the epidemiology data were also extracted from various journals and articles obtained via PubMed and local journal database.

HIV population data including the number of HIV cases, proportion of HIV-diagnosed patients not receiving antiretroviral therapy (ART) and AIDS-related deaths were derived from WHO Progress Report 2011: Global HIV/AIDS Response [ 25] and the Jointed United Nations Programme on HIV/AIDS (UNAIDS) 2012 [ 26] as well as local reports for HIV. The data regarding the proportion of AIDS patients presenting with different fungal infections were derived from several epidemiological reports where cryptococcal meningitis and T. marneffei infection data were derived from General South East Asian rate [ 27, 28] and Vietnam data [ 1, 29], respectively. Pneumocystosis and histoplasmosis data were from Ratabasuwam (2005) [ 30] and Lian et al., (2007) [ 31], respectively. The incidence of oesophageal candidiasis has been estimated to occur in ~20% of HIV-infected patients without ART, and 5% of those receiving ART and so the incidence in Malaysia was estimated. The total annual incidence of pulmonary TB (PTB) was obtained from the WHO Global Tuberculosis Report [ 32]. Post-TB chronic pulmonary aspergillosis (CPA) was estimated based on the assumption that 22% of the patients with lung cavities and 2% of patients without cavities generally develop CPA [ 19, 33]. Data on COPD and asthma were derived from Lim et al., (2015) [ 31] and To et al., (2012) [ 34], respectively. The prevalence of COPD in all GOLD stages was obtained [ 34]. The prevalence of ABPA was estimated using a rate of 2.5% of adult asthmatics [ 15]. Further, the prevalence of severe asthma with fungal sensitization (SAFS) was estimated to be 33% among ~10% of the total adults with the most severe and poorly controlled asthma [ 17].

The estimation of candidemia was performed using a general rate of 5 per 100,000 population [ 33, 35]. We assumed that about a third of such cases occurred in intensive care, and Candida peritonitis (intra-abdominal candidiasis) which usually complicates complex surgery was also seen only in intensive care at a rate 50% of the candidaemia rate [ 36]. The number of cases of recurrent vaginal candidiasis was estimated based on the expected prevalence of 6% in women between 15-50 years old, derived from Sobel et al., (2007) [ 37]. The incidence of fungal keratitis was estimated based on the assumption of 10 cases per year in each of 40 hospitals. Data related to hematological malignancy and transplantation were collected depending on data availability.

3. Results

Malaysia has an estimated population of ~30 million people; ~29% are children and ~11.3 million are adult women, with 8% of women over 60 years of age [ 24]. The estimation of serious fungal infections based on the population is shown in Table 1, with the number of infections per underlying disorder and the rate per 100,000 population.

3.1. HIV/AIDS and AIDS-Defining Fungal Infections

According to the UNAIDS 2012 report, people living with HIV were estimated to be ~82,000, of which ~35,000 with CD4 <350 are not on any active ART regimen [ 25]. Opportunistic fungal infections are relatively common in HIV-infected people. Oesophageal candidiasis is a common serious fungal infection [ 14, 33, 38], and 5,850 cases of oesophageal candidiasis are estimated annually, (19 per 100,000). Cryptococcal meningitis is a common fungal infection in AIDS and among severely immunocompromised patients, usually caused by Cryptococcus neoformans [ 7, 8]. A total of 700 cases of cryptococcal meningitis in AIDS were estimated in Malaysia based on an annual rate of 8% in late stage HIV infection, and an additional 155 cases, some in non-immunocompromised patients. Cryptococcal meningitis has the highest incidence and mortality rates among subjects with advanced HIV disease stages and its global burden has recently been re-estimated in AIDS-508 cases and 456 deaths in Malaysia, similar to our estimate [ 8]. Similarly, Pneumocystis pneumonia (PCP) is also an AIDS-defining illness with an estimated 1286 cases, based on a 15% rate in newly presenting patient with AIDS. PCP is the second most common fungal infections diagnosed among HIV-infected patients in Malaysia [ 14]. Disseminated histoplasmosis [ 10, 14] and T. marneffei infection [ 13, 14] were also reported in patients with HIV/AIDS. Here, we estimate 175 cases of disseminated histoplasmosis and 350 cases with T. marneffei infection in those with advanced HIV infection.

3.2. Pulmonary Mycoses

The annual incidence of pulmonary TB was an estimated 18,923 cases reported in 2013 [ 32]. Among pulmonary TB survivors, CPA has an estimated annual incidence of 1211 and prevalence of 3817. Post-TB cavitation acts as a predisposing factor for the colonisation of Aspergillus spp., largely owing to the development of empty residual lung cavities following treatment for TB [ 19]. As CPA is also found in patients with many other pulmonary disorders, and occasionally as a primary illness [ 39], we have assumed a total prevalence of 7635 patients.

ABPA [ 15, 40] and SAFS [ 17, 41, 42] are the most common fungal infections among asthmatics with a notably high prevalence rate. According to a past study, the rates of asthma in adults are 5.51 per 100,000 population, and there are 1.2 million adults with asthma in Malaysia [ 43]. Among the estimated population of adult asthmatics, ABPA has been estimated in 30,062 (98/100,000) and SAFS in 39,682 cases (130/100,000). ABPA is rare in children and cystic fibrosis is rare in Malaysia. The prevalence and clinical impact of SAFS in children are yet to be described.

3.3. Invasive Fungal Infections

Invasive aspergillosis (IA) [ 44, 45] and candidemia [ 35, 46, 47, 48] are the major invasive fungal infections in modern medicine. The latter typically being a bloodstream infection, with far reaching implications for immunosuppressed individuals. Individuals with acute leukemia or transplantation are at high risk for the development of invasive fungal infections [ 49, 50]. Exacerbations of COPD and hospitalization, particularly to intensive care, are participating factors, as is chemotherapy for cancers and immunotherapy for transplantation.

COPD classification was done according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria. GOLD stage I, II, III, IV refer to COPD severity of mild, moderate, severe and very severe respectively. The prevalence of COPD across all GOLD stages in Malaysia was 5.1% in those 40 years and older, of whom 12.5% were of the severe symptomatic phenotype and 19% had been hospitalized [ 34]. A total of 1,018 cases of IA were estimated among immunocompromised patients, notably in ICU mostly with COPD as well as acute leukemia.

Meanwhile, 1073 cases of candidaemia were estimated among patients with haematological malignancies, on the medical wards or having undergone transplantation and another 460 cases were estimated to occur among subjects in ICU with an annual rate of 5 per 100,000 population [ 35]. This is likely to be a conservative rate, in the absence of population-based epidemiological data, and based on experience in other South East Asian countries [ 51]. We also estimated 230 patients with intra-abdominal candidiasis, and have not attempted to estimate Candida peritonitis occurring in those on chronic ambulatory peritoneal dialysis.

3.4. Fungal Keratitis

Fungal keratitis is a challenging ophthalmological problem leading to corneal blindness [ 52, 53, 54, 55]. It commonly occurs in developing countries, including Malaysia. Ocular trauma and use of contact lenses appear to be the common etiologies leading to fungal keratitis. Fusarium spp. are the most common aetiological agents for fungal keratitis followed by Aspergillus spp. and Candida spp. [ 53]. In the current study, we estimated a total of 400 cases of fungal keratitis, a prevalence of 1.3 per 100,000 population in Malaysia. This can be further supported by the 5-year retrospective review, which showed 46% of cases of fungal keratitis due to Fusarium spp., and higher incidence among males [ 55].

3.5. Recurrent Vaginal Candidiasis

Vaginal candidiasis is a common gynecological problem occurring among women globally, most commonly caused by Candida albicans, but occasionally by other Candida species [ 37, 56]. It is reported that ~70-75% of all women will experience at least one episode of vaginal candidiasis in their lifetime, especially during pregnancy [ 57]. Up to 40-55% of infected women will suffer from recurrent vaginal candidiasis [ 56]. Recurrent vaginal candidiasis is defined as at least four episodes of infection per year [ 37]. According to a global epidemiological review, ~5-9% women were estimated to be infected with recurrent vaginal candidiasis annually [ 37, 57, 58]. Our estimate shows that it is common in Malaysia where a total of 501,138 otherwise healthy women were estimated to be infected with recurrent vaginal candidiasis at a rate of 4800 per 100,000 females per year.

4. Discussion

Here, we report the estimated incidence and prevalence of certain serious fungal infections in Malaysia is approximately 2% of the population, equating to 590,214 of the total burden. Recurrent vaginal candidiasis has the highest burden of all the selected fungal infections. The second most common mycoses in Malaysia are asthma-associated fungal infections, ABPA and SAFS, although infrequently diagnosed or treated with antifungals. However, the actual cases of serious fungal infection may be higher than the current estimation as we have very little data on allergic fungal sinusitis, tinea capitis, histoplasmosis in non-AIDS patients, mucormycosis, sporotrichosis, mycetoma, chromoblastomycosis and phaeohyphomycosis. Nonetheless, this problem is serious enough to warrant more comprehensive epidemiological studies of fungal diseases in Malaysia. Further some estimates are likely significant underestimates, including candida bloodstream infection [ 51].

Serious fungal infections contribute to chronic illness and mortality across the world. In order to reduce the burden of fungal infection, a national reporting system on fungal infections and a fungal surveillance system should be implemented and improved to ascertain the impact of serious fungal infections, and promote public awareness of the seriousness of these infections. In addition, advanced laboratory diagnostic techniques and better surveillance within the healthcare system is likely to reduce the unnecessary burden of serious fungal infections in Malaysia.

Malaysia, complemented by the implementation of advanced laboratory diagnostic techniques, proper national reporting of fungal infections, and greater integration of fungal diseases management in Malaysia's health care system, can achieve better outcomes in reducing the impact and burden of fungal diseases [ 59].

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Estimation of the Burden of Serious Human Fungal Infections in Malaysia

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Fungal infections (mycoses) are likely to occur more frequently as ever-increasingly sophisticated healthcare systems create greater risk factors. There is a paucity of systematic data on the incidence and prevalence of human fungal infections in Malaysia. We conducted a comprehensive study to estimate the burden of serious fungal infections in Malaysia.

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Mechanism of pathogenicity of Candidiasis 

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Mechanism of pathogenicity of Candidiasis 

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Mechanism of pathogenicity of Candidiasis

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Mechanism of pathogenicity of Candidiasis

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-Total of 16 Candida species isolated from 34 392 clinical samples were from the oral cavity (oral swabs and throat swabs), blood, respiratory tract (sputum, tracheal secretions, nasopharyngeal aspirates, bronchoalveolar lavage), high vaginal swab, pus and urine.
-C. albicans (66.70%, 22 941/34 392), C. glabrata (11.71%, 4029/34 392), C. parapsilopsis (10.74%, 3692/34 392), C. tropicalis (9.19%, 3162/34 392) and C. krusei (1.15%, 396/34 392) were the five predominant .
-Candida species. C. albicans was the predominant species isolated from the oral cavity, respiratory tract and high vaginal swab; while the Candida species isolated from blood, urine and pus were predominant non‐albicans Candida.
-Uncommon Candida species, such as C. lusitaniae, C. haemulonii, C. humicola, Pichia ohmeri and C. ciferrii, were also isolated in this study.

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