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Future Microbiology
Review
Enhancing molecular approaches for diagnosis of fungal infections Sean X Zhang Division of Medical Microbiology, Department of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, 600 Wolfe Street, Meyer B1-193, Baltimore 21287, MD, USA n Tel.: +1 410 955 5077 n Fax: +1 410 614 8087 n [email protected]
Molecular tests can improve the diagnosis of fungal infections. Despite the increasing application for fungal detection, molecular tests are still not accepted as a diagnostic criterion to define invasive fungal diseases. This limitation is largely due to a lack of a standardized method. Method standardization can be achieved by following a consensus protocol developed by a working group, by performing a molecular test in a centralized laboratory or by using a commercial assay that provides a standardized method and quality-controlled reagents. Forming a consortium or a working group consisting of large-scale diagnostic mycology laboratories can accelerate the process of validating and implementing a commercial molecular assay for clinical use through a joint effort between industry partners and clinicians. Development of molecular tests not only for the detection of fungi but also for the identification of antifungal drug resistance directly in blood, bronchoalveolar lavage fluid, cerebrospinal fluid, and formalin-fixed and paraffin-embedded tissues greatly enhances fungal diagnostic capacities. Advances of developing quantitative assays and RNA detection platforms may provide another avenue to further improve fungal diagnostics.
Fungi are eukaryotic organisms and are ubiquitous in our living environment. The incidence of human infections caused by fungi has increased dramatically over the past two to three decades, owing to an enlarging immunocompromised patient population [1–5]. Recent epidemiology data indicate that fungal infection has become one of the most common infection-associated mortalities in the USA [6,7]. The laboratory diagnosis of fungal infections is still largely dependent on microscopic and culture-based methods, and these methods do not always meet clinical needs owing to poor sensitivity and lengthy incubation time. Therefore, the area holding the most promise for fungal diagnostics is the development of nucleic acid amplification and detection platforms because they offer ultra sensitive and rapid detection of fungal pathogens directly in clinical samples [8]. These molecular assays are widely used by clinical laboratories to aid in diagnosis of viral and bacterial infections; however, their application for diagnosis of fungal disease in clinical settings is still limited. In fact, none of the molecular assays are even included in the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Disease Mycoses Study Group (EORTC/MSG)’s criteria to define invasive fungal disease [9]. In this article, the current status of applying molecular assays for diagnosis of fungal infections is reviewed and the future 10.2217/FMB.13.120 © 2013 Future Medicine Ltd
direction of enhancing molecular approaches for fungal diagnostics is elaborated. Performance of molecular tests on fungal detection
Khot and Fredricks searched PubMed for any publication related to PCR-based diagnosis of human fungal infections over a period of 10 years (from year 1999 to 2009) [10]. Their search using a set of keywords related to the name of fungal pathogens and the diagnostic method of PCR resulted in 262 publications. Using selection criteria based on the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines [11], they included 68 publications that provided study parameters (e.g., PCR format, fraction and volume of sample extraction, positive and negative controls, analytical sensitivity and specificity, and limit of detection analysis, among others) for further analysis. Most of the molecular assays reported in those papers were developed inhouse and their performance (analytical sensitivity and specificity) showed a wide range [10]. For example, a range of sensitivity of 55–100% and specificity of 63.5–100% was reported in 33 papers describing molecular tests for diagnosis of invasive aspergillosis. In six papers evaluating molecular tests for the diagnosis of invasive candidiasis, a range of sensitivity of 77–100% and specificity of 66–100% was reported. In 13 papers assessing molecular tests for diagnosis of Pneumocystis pneumonia, a range of sensitivity of 40–100% and specificity Future Microbiol. (2013) 8(12), 1599–1611
Keywords commercial n diagnosis fungal infections n fungi n implementation n molecular assays n multiplex PCR n outcome n standardization n validation n n
part of
ISSN 1746-0913
1599
Review
Zhang
of 59–100% was reported. For diagnosis of other invasive fungal infections by molecular tests, a range of sensitivity of 75–100% and specificity of 70–98% was reported in 14 papers. An additional search in PubMed extended to the year 2012 following the same keywords used in Khot and Fredricks’ paper resulted in 512 publications. This number reflected a sharp increase of publications from 2009 to 2012 in the area of applying molecular tests for diagnosis of fungal infections. Mengoli et al. conducted a meta-analysis of the use of PCR tests for the diagnosis of invasive aspergillosis in retrieved data from 16 studies containing over 10,000 blood, serum or plasma samples obtained from 1618 patients at risk of fungal infections [12]. The overall sensitivity and specificity of PCR (for a single positive sample) were 88 (95% CI: 75–94%) and 75% (95% CI: 63–84%). Likewise, Avni et al. conducted a systematic review assessing diagnostic accuracy of PCR assays performed in bronchoalveolar lavage fluid (BAL) for diagnosis of invasive pulmonary aspergillosis [13]. From the 19 publications between 1993 and 2012 included for review, they found the pooled diagnostic sensitivity and specificity of the PCR assays were 90.2 (95% CI: 77.2–96.1%) and 96.4% (95% CI: 93.3–98.1%), respectively. In another systematic meta-analysis evaluating PCR assays performed in whole blood for diagnosis of invasive candidiasis in 54 studies with almost 5000 patients, the pooled sensitivity and specificity of the PCR assays for the diagnosis of candidemia were 95 (95% CI: 88–98%) and 92% (95% CI: 88–95%), respectively [14]. It is notable that one study showed that the sensitivity of PCR for diagnosing candidemia was significantly lower than deep-seated candidiasis [15], probably due to the fact that Candida DNA may be continuously released into the bloodstream during deep-seated candidiasis, whereas transient or catheter-associated candidemia may have been resolved spontaneously or as a result of catheter removal. Outcome data of molecular tests
Determination of an assay’s diagnostic value is often dependent on the improvement of patients’ clinical outcome. Despite the increased number of publications, only few papers (
Future Microbiology
Review
Enhancing molecular approaches for diagnosis of fungal infections Sean X Zhang Division of Medical Microbiology, Department of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, 600 Wolfe Street, Meyer B1-193, Baltimore 21287, MD, USA n Tel.: +1 410 955 5077 n Fax: +1 410 614 8087 n [email protected]
Molecular tests can improve the diagnosis of fungal infections. Despite the increasing application for fungal detection, molecular tests are still not accepted as a diagnostic criterion to define invasive fungal diseases. This limitation is largely due to a lack of a standardized method. Method standardization can be achieved by following a consensus protocol developed by a working group, by performing a molecular test in a centralized laboratory or by using a commercial assay that provides a standardized method and quality-controlled reagents. Forming a consortium or a working group consisting of large-scale diagnostic mycology laboratories can accelerate the process of validating and implementing a commercial molecular assay for clinical use through a joint effort between industry partners and clinicians. Development of molecular tests not only for the detection of fungi but also for the identification of antifungal drug resistance directly in blood, bronchoalveolar lavage fluid, cerebrospinal fluid, and formalin-fixed and paraffin-embedded tissues greatly enhances fungal diagnostic capacities. Advances of developing quantitative assays and RNA detection platforms may provide another avenue to further improve fungal diagnostics.
Fungi are eukaryotic organisms and are ubiquitous in our living environment. The incidence of human infections caused by fungi has increased dramatically over the past two to three decades, owing to an enlarging immunocompromised patient population [1–5]. Recent epidemiology data indicate that fungal infection has become one of the most common infection-associated mortalities in the USA [6,7]. The laboratory diagnosis of fungal infections is still largely dependent on microscopic and culture-based methods, and these methods do not always meet clinical needs owing to poor sensitivity and lengthy incubation time. Therefore, the area holding the most promise for fungal diagnostics is the development of nucleic acid amplification and detection platforms because they offer ultra sensitive and rapid detection of fungal pathogens directly in clinical samples [8]. These molecular assays are widely used by clinical laboratories to aid in diagnosis of viral and bacterial infections; however, their application for diagnosis of fungal disease in clinical settings is still limited. In fact, none of the molecular assays are even included in the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Disease Mycoses Study Group (EORTC/MSG)’s criteria to define invasive fungal disease [9]. In this article, the current status of applying molecular assays for diagnosis of fungal infections is reviewed and the future 10.2217/FMB.13.120 © 2013 Future Medicine Ltd
direction of enhancing molecular approaches for fungal diagnostics is elaborated. Performance of molecular tests on fungal detection
Khot and Fredricks searched PubMed for any publication related to PCR-based diagnosis of human fungal infections over a period of 10 years (from year 1999 to 2009) [10]. Their search using a set of keywords related to the name of fungal pathogens and the diagnostic method of PCR resulted in 262 publications. Using selection criteria based on the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines [11], they included 68 publications that provided study parameters (e.g., PCR format, fraction and volume of sample extraction, positive and negative controls, analytical sensitivity and specificity, and limit of detection analysis, among others) for further analysis. Most of the molecular assays reported in those papers were developed inhouse and their performance (analytical sensitivity and specificity) showed a wide range [10]. For example, a range of sensitivity of 55–100% and specificity of 63.5–100% was reported in 33 papers describing molecular tests for diagnosis of invasive aspergillosis. In six papers evaluating molecular tests for the diagnosis of invasive candidiasis, a range of sensitivity of 77–100% and specificity of 66–100% was reported. In 13 papers assessing molecular tests for diagnosis of Pneumocystis pneumonia, a range of sensitivity of 40–100% and specificity Future Microbiol. (2013) 8(12), 1599–1611
Keywords commercial n diagnosis fungal infections n fungi n implementation n molecular assays n multiplex PCR n outcome n standardization n validation n n
part of
ISSN 1746-0913
1599
Review
Zhang
of 59–100% was reported. For diagnosis of other invasive fungal infections by molecular tests, a range of sensitivity of 75–100% and specificity of 70–98% was reported in 14 papers. An additional search in PubMed extended to the year 2012 following the same keywords used in Khot and Fredricks’ paper resulted in 512 publications. This number reflected a sharp increase of publications from 2009 to 2012 in the area of applying molecular tests for diagnosis of fungal infections. Mengoli et al. conducted a meta-analysis of the use of PCR tests for the diagnosis of invasive aspergillosis in retrieved data from 16 studies containing over 10,000 blood, serum or plasma samples obtained from 1618 patients at risk of fungal infections [12]. The overall sensitivity and specificity of PCR (for a single positive sample) were 88 (95% CI: 75–94%) and 75% (95% CI: 63–84%). Likewise, Avni et al. conducted a systematic review assessing diagnostic accuracy of PCR assays performed in bronchoalveolar lavage fluid (BAL) for diagnosis of invasive pulmonary aspergillosis [13]. From the 19 publications between 1993 and 2012 included for review, they found the pooled diagnostic sensitivity and specificity of the PCR assays were 90.2 (95% CI: 77.2–96.1%) and 96.4% (95% CI: 93.3–98.1%), respectively. In another systematic meta-analysis evaluating PCR assays performed in whole blood for diagnosis of invasive candidiasis in 54 studies with almost 5000 patients, the pooled sensitivity and specificity of the PCR assays for the diagnosis of candidemia were 95 (95% CI: 88–98%) and 92% (95% CI: 88–95%), respectively [14]. It is notable that one study showed that the sensitivity of PCR for diagnosing candidemia was significantly lower than deep-seated candidiasis [15], probably due to the fact that Candida DNA may be continuously released into the bloodstream during deep-seated candidiasis, whereas transient or catheter-associated candidemia may have been resolved spontaneously or as a result of catheter removal. Outcome data of molecular tests
Determination of an assay’s diagnostic value is often dependent on the improvement of patients’ clinical outcome. Despite the increased number of publications, only few papers (
