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 ~ Introduction
 ~  Immunological As...
 ~ Conclusions
 ~  References
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REVIEW ARTICLE
Year : 2012  |  Volume : 30  |  Issue : 3  |  Page : 264-269
 

Invasive candidiasis: A review of nonculture-based laboratory diagnostic methods


Department of Microbiology, Medicine, Kuwait University, Safat 13110, Kuwait

Date of Submission15-Jan-2012
Date of Acceptance09-Apr-2012
Date of Web Publication8-Aug-2012

Correspondence Address:
Z Khan
Department of Microbiology, Medicine, Kuwait University, Safat 13110
Kuwait
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.99482

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 ~ Abstract 

Invasive candidiasis is a life-threatening complication of critically ill immunocompromised patients with high attributable mortality. Due to non-specific clinical presentation, early detection of candidemia and accurate identification of Candida species are essential pre-requisites for improved therapeutic outcome. Since blood culture-based methods lack sensitivity and species-specific identification by conventional methods is time-consuming, detection of immunological and molecular markers has provided an alternative for early diagnosis of invasive candidiasis. Additionally, serial estimations of these biomarkers have provided opportunities to monitor response to therapy and initiate pre-emptive therapy in suspected patients before clinical signs appear. Antigen-based methods include detection of β-D-glucan, a panfungal marker, and Candida mannan, a genus-specific marker. Although both these markers have moderate sensitivity, they provide a useful adjunct to the diagnosis if performed in select patient population in parallel for exclusion of false positive/negative results. A negative β-D-glucan test on at least two occasions has a high negative predictive value. Concomitant detection of Candida mannan and anti-mannan antibodies has sensitivity of ~70% before blood cultures become positive. Significant advances have also been made in nucleic acid-based detection methods, including a commercial real-time PCR assay (SeptiFast) for detection of five major clinically important Candida spp. in blood specimens within 6 h. Furthermore, matrix-assisted laser desorption ionization time-of-flight mass spectrometry enables species-specific identification of yeast isolates within an hour. While these immunological and molecular tools mark a significant advance towards early and specific diagnosis of candidemia and invasive candidiasis, further evaluation of these approaches in different clinical settings is warranted.


Keywords: β -D-glucan, candidemia, Candida mannan, candida DNA, diagnosis, invasive candidiasis


How to cite this article:
Ahmad S, Khan Z. Invasive candidiasis: A review of nonculture-based laboratory diagnostic methods. Indian J Med Microbiol 2012;30:264-9

How to cite this URL:
Ahmad S, Khan Z. Invasive candidiasis: A review of nonculture-based laboratory diagnostic methods. Indian J Med Microbiol [serial online] 2012 [cited 2015 Sep 3];30:264-9. Available from: http://www.ijmm.org/text.asp?2012/30/3/264/99482



 ~ Introduction Top


Invasive fungal infections have recently emerged as a major cause of morbidity and mortality in immunocompromised individuals and Candida spp. are among important pathogens for patients admitted to intensive care units (ICUs). [1] Candida spp. are now among the four most common causes of hospital-associated infections and catheter-associated urinary tract and bloodstream infections. [2] The vast majority of invasive Candida infections are caused by only four species which include Candida albicans, Candida glabrata, Candida parapsilosis and Candida tropicalis. [3] The role of other Candida spp. is minor. However, mini outbreaks caused by some species have occasionally been recorded among select patient populations. [3],[4]

Nosocomial Candida infections could be caused by the patient's own flora or could be acquired from exogenously sources or hands of healthcare staff. The most common pathogenic Candida species is C. albicans which is also a major component of human microbiota, occurring normally on skin and mucosal surfaces. Bloodstream infections caused by other Candida spp. have also increased in recent years. [5],[6] In multicenter surveys originating from Asia-pacific region, the relative distribution of these non-albicans Candida spp. among bloodstream isolates was as follows: C. parapsilosis, 16%, C. tropicalis 14%, C. glabrata 10% and other Candida spp. <4%. [5] In a prospective 4-month study in a tertiary care centre, Chakrabarti et al. [7] reported 140 cases of fungemia, with a majority (73%) of episodes occurring in paediatric patients. A notable finding of the study was the unusual preponderance of C. guilliermondii (30%) and C. pelliculosa (18%) in paediatric patients. Furthermore, 6 of 10 fluconazole-resistant isolates belonged to C. tropicalis. Although this study was limited to a single centre, nonetheless, it points towards changing epidemiology of candidemia with emergence of fluconazole resistance in a particular clinical setting in North-western India.

The vast majority of C. albicans isolates remain susceptible to fluconazole despite widespread use of this drug in clinical practice. However, there is a widely held view that prior exposure to fluconazole is the single most important factor leading to fungemia caused by less susceptible non-albicans Candida species, such as C. glabrata and C. krusei. [8] Since fluconazole is the mainstay of anti-Candida therapy and prophylaxis in resource-limited countries of Asia and Africa, the threat of emergence of resistance is a real possibility. The introduction of echinocandins has proven to be highly effective against Candida spp. isolates with reduced susceptibility to azoles. [9] Although some reports have suggested that C. parapsilosis complex species are relatively less susceptible than other Candida spp. in in vitro studies, all the three echinocandins have been used for the treatment of mucosal and invasive candidiasis with favourable outcomes and with few side effects. However, recent recognition of a C. glabrata phenotype resistant to azoles and echinocandins occurring in ICU and non-ICU settings is a worrisome development in this context. [10]

In addition to the differences that exist in antifungal susceptibilities of individual Candida species, there are also host-related preferences. C. parapsilosis is the second most important cause of candidemia in East/Southeast Asia and Middle East, particularly affecting paediatric patients in association with intravenous catheters. On the other hand, C. tropicalis may be seen more frequently among neutropenic patients with prolonged stay in ICU while C. glabrata and C. krusei may occur more commonly among elderly hematologic patients receiving fluconazole prophylaxis. [11],[12],[13] Likewise, attributable mortality in candidemia is also variable, being lowest for cases caused by C. parapsilosis (30%) and highest for cases caused by C. krusei (59%). [3],[6]

Rapid diagnosis of candidemia and invasive candidiasis and species-specific identification are crucial for timely administration of appropriate antifungal treatment. [9] Blood culture is the gold standard for the diagnosis of candidemia; however, it takes a minimum of 24-48 h to become positive. [14] Moreover, species-specific identification of blood culture isolates of Candida spp. by conventional methods is time-consuming as these methods principally rely on phenotypic characteristics which lack sensitivity. Thus many closely related species (such as C. albicans and C. dubliniensis) may be misidentified due to indistinguishable physiological characteristics. [15],[16]

It has been shown that a delay of each day in initiating antifungal (fluconazole) therapy after first blood culture becomes positive increases the risk of mortality significantly. Thus, the risk of mortality was 15% if antifungal treatment was started on the same day when blood cultures became positive, which increased to 24%, 37%, and 40% with initiation of treatment on days 1, 2, and ≥3, respectively. [17] In this context, the non-culture-based methods offer an attractive alternative. While these methods have the potential to overcome limitations of low blood culture positivity (<50% even in autopsy-proven cases of candidiasis), they also provide a window of opportunity to initiate pre-emptive antifungal therapy even before signs and symptoms of clinical disease appear. [18],[19] While these non-culture-based methods hold the promise to simultaneously detect and/or identify causative Candida species using genus and species-specific biomarkers in blood specimens, they can also be helpful in monitoring therapeutic response to therapy. Here, we present an overview of the advances that have been made in this direction in recent years using immunological and molecular approaches.


 ~ Immunological Assays for Detection of Candidemia Top


Antigen detection

Two antigen-based tests are currently available for predicting the onset of candidemia. Mannan is a major component of Candida cell wall, accounting for up to 7% of total dry cell weight and is released in blood circulation during infection. The Platelia Candida Ag test (Bio-Rad Laboratories, Marnes-la-Coquette, France) detects the presence of mannan in blood (serum) samples in ELISA format. [19] Several retrospective and prospective studies have evaluated the utility of mannan detection for the diagnosis of invasive candidiasis in haematological and ICU patients with an overall sensitivity of 58% and specificity of 93% [Table 1]. [19],[20],[21],[22] The sensitivity of the test seems to vary with the infecting Candida species, being highest for C. albicans. [21] A positive test has been recorded several days before radiological detection of hepatosplenic candidiasis or positive blood cultures. [21] In high risk patients, the test is recommended to be carried out two to three times per week since the presence of mannan in blood is short-lived due to rapid clearance during each episode of candidemia with concomitant appearance of anti-mannan antibodies. Recent studies have demonstrated an increased sensitivity of this test when combined with detection of anti-mannan antibodies in critical but not in immunocompromised patients (described below).
Table 1: Performance of commercially available antigen- and antibody-based assays for the diagnosis of candidemia and invasive candidiasis

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Another antigen-based test detects the presence of 1,3-β-D-glucan (BDG), another important component of the cell wall of most fungi, in serum samples and is approved by Food and Drug Administration (FDA) of the United States of America. [14],[18] The test is commercially available from at least four manufacturers with the Fungitell (associates of Cape Cod Inc., East Falmouth, MA, USA) assay being more popular. The test has been evaluated, mostly in ICU patients with an overall sensitivity of 77% and specificity of 85% for subjects with proven or probable invasive fungal infections [Table 1]. [22],[23],[24] The test is applied twice weekly and a single positive test is indicative of infection. True positive cases usually show falling BDG titres that eventually become negative in patients responding to antifungal treatment, a trend that is not observed in patients not responding to therapy. False positive results due to several conditions, such as haemodialysis, abdominal surgery, treatment with β-lactam antibiotics and concomitant presence of lipopolysaccharide due to Gram-negative bacteremia make its application in clinical setting rather difficult. [22],[23],[24] However, based on excellent negative predictive value of this test (nearly 100%), lack of BDG detection is most useful for excluding invasive fungal infection. [22],[23],[24] Also, colonization of individuals with Candida spp. has no apparent effect on the diagnostic performance of the BDG test. [18],[20],[22],[25]

Antibody-based assays

Two antibody-based tests are available. An ELISA-based test that detects anti-mannan antibodies which develop in patients when mannan disappears after an episode of candidemia, is marketed, as Platelia Candida Antibody (Bio-Rad Laboratories, Marnes-la-Coquette, France). The overall sensitivity of the anti-mannan antibody test in several studies was reported as 59% with an overall specificity of 83% [Table 1]. However, when the test was combined with simultaneous mannan detection, the sensitivity and specificity values improved considerably (83% and 86%, respectively), both in haematological and ICU patients with candidemia. [19],[20],[21] Studies have shown that detection of mannan and anti-mannan antibodies offer a useful diagnostic aid in patients with invasive candidiasis than either test alone, particularly in hepato-oncologic patients where microbiological documentation of infection is more difficult. Recently, newer versions of Candida mannan and anti-mannan antibody tests have been introduced (Platelia Candida Ag Plus and Platelia Candida Antibody Plus) with revised recommended cut-off values for a positive test and their performance in clinical studies is under evaluation.

An indirect immunofluorescence assay (C. albicans IFA IgG; Virvell Laboratories, Spain) has recently been developed by a Spanish group that detects antibodies against C. albicans germ tubes (CAGTA). [26] The overall sensitivity and specificity of CAGTA have been reported to vary from 77% to 89% and 91% to 100%, respectively. [26] More studies are needed to validate this assay for the diagnosis of invasive candidiasis in critical care settings.

Nucleic acid amplification-based tests

The nucleic acid amplification techniques offer an attractive alternative to improve timely diagnosis of invasive candidiasis in high risk patients. Different formats of both in-house developed and commercial molecular tests are available for qualitative and/or quantitative detection of Candida species-specific DNA and many studies have yielded encouraging results. Commonly used techniques include conventional, semi-nested and nested PCR, [27],[28] PCR-enzyme immunoassay, [29] different variations of real-time PCR, [28],[30] multiplex PCR followed by DNA sequencing or pyrosequencing [Table 2]. [31],[32] Despite high potential of PCR-based tests, detection of nucleic acid in body fluids is challenging due to low pathogen burden and tough fungal cell walls which impede efficient lysis and liberation of DNA, leading to false-negative results. The presence of PCR inhibitors may also lead to false-negative results. False-positive results may also arise due to airborne-contamination of specimens, particularly if panfungal primers, targeting highly conserved rRNA or other genes, are used. Furthermore, as many Candida spp. are human colonizers of skin, mucosal surfaces or other anatomic sites, detection of target nucleic acids corresponding to few Candida cells in the clinical samples causes difficulty in distinguishing colonization from infection. [27],[28]
Table 2: Performance of molecular assays for the diagnosis of candidemia and invasive candidiasis from selected studies

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Clinical evaluation studies of quantitative real-time PCR assays have reported sensitivities and specificities of >90% for the detection of Candida species in serum samples from ICU patients [Table 2]. [28],[30] Despite these advances, clinical applicability of PCR-based diagnosis of candidemia has been limited by lack of optimal DNA extraction techniques, selection of optimal specimen type and absence of a standardized and validated commercial test. Automated DNA extraction procedures and standardized molecular technology platforms that meet accepted regulatory standards for clinical diagnosis have recently become available. The first commercially available real-time PCR method (SeptiFast) designed to detect 25 most frequently isolated bloodstream microbial pathogens, including five Candida species, was introduced by Roche Diagnostics. [33] The test is performed directly on blood sample drawn at the same time as for culture. Extraction of both human and pathogen DNA involves mechanical lysis and manual spin columns under a contamination-controlled workflow. The extracted DNA is amplified in three separate real-time PCR assays, one each for Gram-positive, Gram-negative and fungal pathogens. The amplicons are hybridized to species-specific fluorescent probes for identification of the pathogens and the test is completed in ~6 h. [33]

Initial evaluation of the SeptiFast assay in ICU patients with haematological malignancies was promising as it exhibited 83% concordance with blood culture results and the discrepant results were in favour of SeptiFast as many samples from clinically suspected patients were PCR-positive but tested negative by culture. Subsequent evaluations, employing larger and more diverse patient populations, including neonates and children, have yielded similar results [Table 2]. [34],[35],[36] In some studies, the SeptiFast-positive, culture-negative cases were frequently recognized as clinically significant based on clinical data, disease severity and analytical evidence of infection. However, some cases, particularly those caused by C. glabrata were detected more often by culture than by SeptiFast, possibly due to low Candida count in bloodstream and smaller blood volume used for DNA extraction and subsequent analysis. Also, some SeptiFast-negative, culture-positive or SeptiFast-negative, culture-negative samples could be due to organisms that are not identified by the assay or unculturable organisms, respectively. [37]

Another commercial test (SeptiTest) employs a unique DNA extraction procedure that reduces the burden of human DNA, a major source of inhibition in PCR-based diagnostic methods. Multiplex PCR on extracted DNA followed by sequencing or other manipulations are performed to identify pathogens in blood samples. [38] Freely circulating pathogen DNA released into bloodstream from deep-seated infection sites is, however, also lost along with human DNA and may cause false-negative results.

Rapid identification of blood culture isolates of candida species

Molecular methods offer rapid species-specific identification of blood culture isolates, while phenotypic tests based on macro- and microscopic colony features and evidence of assimilation/fermentation of sugars/other compounds by using automated/semiautomated systems such as Vitek 2 or ID32C yeast identification systems may take one to several days. [39],[40] Conventional PCR with panfungal primers followed by species-specific detection of amplicons by enzyme immunoassay or uniplex/multiplex PCR with species-specific primers followed by gel electrophoresis is simple and cost-effective for identifying most frequently isolated Candida species. [16],[27],[29],[41],[42],[43] Panfungal real-time PCR assays, using species-specific probe primers, yield faster results in a single step. However, practical application of using distinct probe primers in a single reaction is limited. [28] Panfungal PCR followed by sequencing of species-specific regions or hybridization with specific probe primers on a microarray is suitable for rapid detection of yeast and yeast-like fungi. [31],[32],[44],[45],[46],[47],[48]

Other novel methods have also been exploited recently for rapid identification of Candida spp. isolates. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), based on protein fingerprints, has been used for the identification of clinical Candida spp. isolates. [49] Even closely related species/species complexes have been identified by MALDI-TOF MS within a few minutes. Precise base composition determination of amplicons, generated by broad-range PCR amplification of loci containing species-specific sequences (such as 18S, 5.8S or 28S rRNA genes for fungal pathogens), by electrospray ionization/mass spectrometry (PCR/ESI-MS) has also been developed. The base composition is compared with signature reference standards (base compositions of amplicons of known organisms previously determined with PCR/ESI-MS) for identification. The method has been used for identification of eight medically important Candida spp. and a recent study has reported excellent concordance between PCR/ESI-MS and repetitive sequence PCR for well characterized reference strains of several Candida spp. [50]


 ~ Conclusions Top


The absence of specific signs and symptoms and low blood culture positivity even in proven cases of invasive candidiasis have always posed a diagnostic challenge to clinicians and microbiologists alike. However, sustained efforts by investigators in recent years have led to the development of new antigen and molecular/DNA-based approaches which have proved useful adjuncts to the early diagnosis of candidemia/invasive candidiasis and species-specific identification of Candida species. Two antigen-based tests, β-D-glucan, a panfungal marker and Candida mannan, a genus-specific marker are now commercially available and have been evaluated with variable sensitivities and specificities. Due to high negative predictive value of these tests, serial monitoring of biomarkers in select category of high-risk patients allows clinicians to withhold antifungal agents as long as they remain negative and to use them pre-emptively to prevent full blown disease when they turn positive. Concomitant detection of two or more circulating biomarkers improves sensitivity and/or specificity. Significant progress has also been made in developing nucleic acid-based tests. A commercial, real-time PCR-based test (Septi-Fast) can detect five most frequently isolated Candida spp. in blood specimens from suspected patients within 6 h. Novel technologies such as high throughput sequencing and MALDI-TOF MS have also been developed recently to reduce the time required for species-specific identification of clinical yeast isolates.

 
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    Tables

  [Table 1], [Table 2]

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