Fungal Infections

Molecular mycology

A longstanding diagnostic challenge for fungal infections is to identify an infecting fungal pathogen early in the infection process and rapidly assess its susceptibility to antifungal drugs to facilitate appropriate therapy.  Conventional culture-based approaches are too slow, often requiring several days, and insensitive which has led to the development of modern molecular approaches. The molecular revolution in biology has allowed some impressive developments in mycology diagnostics. The principal uses of molecular methods in the diagnostic mycology lab include:

  1. Direct detection of fungal pathogen nucleic acid for diagnosis
  2. Identification of positive cultures to genus or species level
  3. Molecular typing and phylogeny for outbreak and cluster analysis
  4. Rapidly identify the mechanisms of antifungal resistance

The last 2 methodologies require highly specialised labs, and so are not covered here, although valuable and important to understand disease transmission and selecting appropriate therapy.

Polymerase chain reaction (PCR) and real-time detection is an important molecular method for diagnosis that amplifies and accurately identifies fungal-specific nucleic acid for diagnosis, but sample type, sample preparation and carefully controlled laboratory conditions are essential for optimal results and to prevent both false positive and negative results. Test performance and cut-off values vary between different patient types and samples. Some tests are commercially available and partially or completely validated. Some are offered as a service and have usually been analytically validated but not always clinically validated. Clinical validation is often challenging and time consuming as each patient group and sample type needs testing, and the current methods for confirming the diagnosis lack precision.

Some drugs and other substances can inhibit PCR reactions and so all diagnostic assays should include an amplification control to exclude a failed (false negative) reaction due to inhibition. This is standard practice for commercial assays but not for some in house assays. Negative control assays are also important to rule out both environmental and amplicon (ie previous PCR assay) contamination, giving rise to false positive results.

Direct detection of dermatophyte and cutaneous fungal DNA

Microscopy and culture of nail, skin and hair samples is the usual labour intensive and slow means of establishing the diagnosis of a dermatophyte infection. Molecular diagnosis using kits for dermatophytes and Trichophyton rubrum generate an answer faster and are 4-18% more sensitive than conventional diagnosis. Time from sample to receipt and result is can be as little as 5 hours, but is generally 24 hours.

  • CE-marked PCR kit developed at the Serum Statens Institut (SSI Diagnostica) detects all dermatophytes and T. rubrum, the most common cause of skin and nail infections. Simple nail dissolution method.
  • MycoDerm, another CE-marked kit sold by Biotype Diagnostic GmbH detects 21 dermatophytes, yeasts and moulds from clinical specimens.  Simple nail dissolution method.
  • CE-marked real-time PCR kit from Fast Track Diagnostics detects and identifies 4 Trichophyton species and 3 Microsporum species directly
  • Statens Serum Institut Diagnostica. The RT-PCR molecular diagnostic assay is a new, rapid procedure which reliably detects pathogen infections in only 3 hours. 
  • DermaGenius 2.0 from Pathonostics identifies the most prevalent fungal infections within 3 hours. Assays are separately available as Nail or Complete multiplex products.

Direct detection of Pneumocystis jirovecii nucleic acid for diagnosis

P. jirovecii cannot be routinely cultured, so molecular detection is useful and substantially more sensitive than routine stains and slightly more sensitive than immunofluorescence.

Several DNA regions unique to P. jirovecii have been used as target regions for DNA amplification. The 2 most published are the mitochondrial large subunit rRNA (mt LSU rRNA) and the major surface glycoprotein gene family. Others gene targets include the 5S rRNA region, dihydropteroate synthase (DHPS), dehydrofolate reductase (DHFR), the internal transcribed (ITS) region, heat shock protein 70 (HSP70) and serine endoprotease encoding Kex-1. Commercially available assays include: multiplex real-time PCR against mtLSU and DHPS (Pathonostics); real-time PCR Pneumocystis jirovecii (Bio-Evolution); Pneumocystis carinii Real Time PCR Kit (Shanghai ZJ Bio-Tech, BioProducts AT, Clongen or Vacunek); AmpliSens Pneumocystis jirovecii (carinii) FRT PCR; Pneumocystis carinii Real Time PCR; RIDA-GENE Pneumocystis jirovecii (R-Biopharm AG); Pneumocystis jirovecii FTD (Fast-Track Diagnostics); Progenie kits (Progenie Molecular); reagents for BD MAXTM system Pneumocystis jirovecii (BioGX). Also available are non-approved kits such as LightMix Kit Pneumocystis jirovecii (TIB MOLBIOL); MycoReal Pneumocystis (ingenetix GmBH) and Pneumocystis jirovecii Path-P (PrimerDesign Genesig). The Bio-evolution kit appears to be substantially less sensitive than the AmpliSens kit.

The mt LSU rRNA was first shown in 1990 to be an excellent molecular target for rapid and sensitive PCP diagnosis in BAL and sputum samples which now yields diagnostic sensitivities ranging from 90 to 100%. Real-time, quantitative PCR has shortened the time of detection to three hours comparable with conventional techniques. Quantitation may be important in distinguishing between asymptomatic carriers and those with clinically relevant PCP (see below). The high sensitivity and specificity of the molecular technology have led to it being classified as the ‘gold standard’ for PCP diagnosis by the Laboratory Identification of Parasites of Public Health Concern arm of the Centers for Disease Control (Center for Global Health, 2009).

PCP may be detected in several different respiratory samples including bronchoalveolar lavage, bronchial aspiration, endotracheal aspirates, sputum and induced sputum. Oral washings may be useful, but signal degradation is rapid, unless transported on ice until processing. Occasional reports have shown P. jirovecii detected in blood and other samples such as vitreous fluid from the eye, and tissue.

Signal strength of PCP PCR in AIDS patients is usually much stronger than that from non-AIDS patients. The stronger the signal the greater the confidence that the patient has PCP and is not colonized. Low level positive samples can be difficult to interpret, especially in non-AIDS patients, but should be reported to clinicians, allowing a clinical judgment to be made. There is no single definitive cut-off separating infection from colonization. Signal strength falls with treatment, but not immediately. The earlier in treatment the sample is obtained, the greater the confidence that low level signals are significant. Positive results can occur in at risk patients without PCP, who then develop it later.

Direct detection of Aspergillus spp. nucleic acid for diagnosis

While Aspergillus spp. can sometimes be cultured from respiratory secretions in patients with invasive, chronic and allergic aspergillosis, the yield is poor and from blood it is rare with a frequency of <1%. Molecular diagnosis offers a more sensitive and potentially faster means of detecting Aspergillus. Subsequent sequencing of positive samples may provide species information, which is lacking with other biomarkers such as galactomannan and beta 1,3-glucan. The main challenge with achieving sufficient sensitivity to detect Aspergillus (and other filamentous moulds) is getting a high quality sample, and efficient DNA extraction system and a highly sensitive real-time PCR reaction. Preventing false positive results because of contamination in the sample container or extraction reagent Aspergillus environmental DNA contamination is the major difficulty with getting a fully operational and useful system in the clinical microbiology laboratory.

The most common regions for detection are the 18S rRNA, 28S rRNA and ITS regions. All are multicopy genes with 35-90 copies per nuclear genome. These targets provide natural amplification, improving sensitivity of detection. However some primer and probe selections exclude some common Aspergillus species, or only include A. fumigatus. Many systems cannot distinguish the very closely related Penicillium spp. slightly impairing specificity. Commercially available assays (2012) include MycAssay Aspergillus (Microgen Bioproducts Ltd), Septifast (Roche), VetPCR ASP.FUM Detection (Veterinary) (BioinGentech), MycoReal Aspergillus (Ingenetix GmbH), RenDx multiplex Aspergillus spp & Candida spp (whole blood, plasma & serum), AsperGenius (Pathonostics), Mycogenie (Ademtech), and Goldstream (Era Biology).  In a modest direct comparison, MycAssay Aspergillus was superior to Aspergillus spp. Q-PCR Alert. Other comparisons are lacking. Some kits are designed only for blood, others for respiratory or other samples, primarily because of differences in sample fungal DNA extraction, necessary for clinical validation. An international PCR standard is available for internal development of a molecular assay for Aspergillus spp (Lyon et al. 2013)

On blood (and there are many different ways of extracting DNA from whole blood, clot, serum and plasma) in haematology patients, meta-analyses or real-time PCR suggest a sensitivity of ~75% and negative predictive value (NPV) over 95% for 2 positive or negative samples. Sensitivity is much lower in non-haematology patients. These performance characteristics compare well with galactomannan. An area of uncertain is the relative performance with and without antifungal prophylaxis; some infections can be diagnosed despite itraconazole prophylaxis and empiric therapy with PCR. Furthermore, the application of PCR following therapy to assess disease burden is uncertain.

For respiratory samples, PCR is more sensitive than culture in the context of multiple disease types. Sample source matters and very dilute bronchoalveloar lavage (BAL) samples have lower fungal loads than upper airway and sputum specimens. Cystic fibrosis samples require liquefaction to optimize yield since respiratory samples are highly viscous and have a complex mix of host and bacterial nucleic acid.

Tissue samples can also be analysed by real-time PCR. Fresh tissue is easier to extract than fixed tissue but both yield satisfactory nucleic acid for analysis. Probably other samples are also useful such as corneal scrapings and vitreous, but there is limited published experience. Given the poor sensitivity of culture, it is likely that PCR will become standard practice for these special samples.

Direct detection of Candida spp. nucleic acid for diagnosis

As blood cultures are ~38% sensitive for the detection of invasive and disseminated candidiasis, and take ~2 days of incubation, there has been much interest in direct molecular detection of candida with PCR or other molecular methods. Over 4,500 patients have been included in more than 50 studies of PCR detection of Candida spp. in blood. Remarkably given the various blood volumes, extraction methods, PCR design and endpoint determinations, the sensitivity and specificity for candidaemia using whole blood was 100% (Avni, 2011). For tissue invasive candidaemia, PCR positivity rates were 85% compared with 38% for blood culture. As Candida PCR can be performed within hours, certainly within 24 hours, if required, these data are very supportive of routine application of Candida PCR for ill patients at risk of candidiasis.

Diagram from Ngyuen et al, Clin Infect Dis  2012;54:1240

Three commercial PCR system for detection of Candida spp in blood are currently available, namely SeptiFast (Roche) but inadequate numbers of patients with candidaemia have been studied to be confident of the real performance of this system for candidaemia and invasive candidiasis, partly because most studies have focused on detection of all bloodstream pathogens.  The RenDx (Renshaw Diagnostics) is a multiplex assay system which can detect Candida spp in blood, plasma and serum (Link). The T2Candida magnetic resonance PCR system is a triplex assay detection C. albicans and C. tropicalis together (detection limit 1-2 CFU/mL), C. parapsilosis complex (detection limit 3 CFU/mL) and C. glabrata and C. krusei (detection limit 1-2 CFU/mL). The published performance is a sensitivity of 99% and specificity 94%. (link). A different triplex PCR system has been developed by Tianjin Era Biology, with a combined Candida spp., Cryptococcus spp. and Aspergillus PCR.

Direct detection of all fungal DNA (pan-fungal) for diagnosis

In certain clinical situations, the likelihood of a specific fungal infection is high and a directed molecular test is useful, as for example detecting P. jirovecii. However in numerous other situations, there is a wide differential diagnosis with regard to which fungus, if any, is responsible. For this reason there have been several reports of using panfungal detection, attempting to pick up all possible fungi responsible. As the DNA databases have expanded with additional sequences being deposited in GeneBank and other repositories, so the precise specificity of primers and probes can be better interrogated. The sensitivity of any panfungal assay is likely to vary somewhat according to the fungus being detected (with slightly different reaction kinetics for different fungi), as well as extraction and internal copy number differences between different genera, species and strains.  Overall a panfungal assay could have utility if it has a high negative predictive value to exclude a fungal infection, or as a ‘capture’ assay so that the precise fungus responsible can be identified by second step sequencing. One very specific problem with panfungal assays is the contamination issues for all reagents in the assay and all plasticware. False positives are problematic in frequency.

Only one commercial panfungal assay is available (2012), namely MycoReal Fungi (Panfungal PCR; Ingenetix GmbH, Vienna). Nothing is published about this assay.

Identification of cultures or tissue for identification

Several molecular identification kits are available (2012) for identifying cultures. These include MicroSeq D2 LSU rDNA Fungal Identification Kit (Applied Biosystems), BlackLight® Fungal ID Kit (BlackBio), Pyrosequencing (Qiagen), AccuProbe kits for the identification of Blastomyces dermatitidis, Histoplasma capsulatum and Coccidioides immitis. PNA FISH methodology (AdvanDx) provides a partial indication of Candida species in blood culture.

Sometimes a fungus is seen in a specimen or tissue and not cultured. It is important to identify such fungi to genus and preferably species level.  Fresh non-embedded tissues have shown that sensitivity for PCR detection of fungi exceeds 95%, while the sensitivity of paraffin-embedded samples is currently ~60%. If fungal infection is strongly suspected prior to biopsy or resection, retention of some of the sample fresh (ie not placed in formalin) may facilitate aetiological diagnosis. The fungal DNA extracted from FFPE specimens can be degraded and in low concentration, and it often contains substances that inhibit protein digestion or DNA amplification. However, when fungal elements are detected in FFPE tissue sections and fungus culture is not available, PCR can in some cases determine the organism that is causing the infection.

Only for Aspergillus spp. is a commercially available technique to determine the genus of fungi found in tissue sections published, but this method does not separate species of Aspergillus.  The majority of the published assays target specific rRNA genes (18S or D1-2 of 28S) or the intervening internal transcribed spacer (ITS1 and ITS2).

A common strategy is to amplify up one or two discriminatory regions (such as ITS1 and D1-2 of 28S), and sequence these. For some species, other single copy genes appear to more discriminatory for determining species, such as calmodulin, but this approach has rarely been applied to tissue or microscopy positive samples.

One of the major challenges currently has been the relatively low quality of the databases for bioinformatic comparison. Recently this has been addressed by the International Society for Human and Animal Mycology (ISHAM) and a database is now on line. The database currently contains more than 3200 sequences representing 524 human/animal pathogenic fungal species. The naming of organisms is also up to date and avoids the problem of old nomenclature, which is a major problem with GeneBank. When reporting identification, always use the currently accepted name and the most recognized by clinicians so that they can link the finding with the literature

External quality control

In order to maintain high standards and fulfil requirements for laboratory validation, participation and good performance in external quality control schemes is important. For molecular diagnostic assays for fungi, one scheme exists at http://www.qcmd.org. External quality assurance programs are run for Candida spp., for Aspergillus spp. and Pneumocystis jirovecii, as well as numerous other non-fungal pathogens.

PCR testing

PCR machine

References

1. Avni T, Leibovici L, Paul M. PCR diagnosis of invasive candidiasis: systematic review and meta-analysis. J Clin Microbiol 2011;49:665-70.

2. Lyon GM, Abdul-Ali D, Loeffler J, White PL, Wickes B, Herrera ML, Alexander BD, Baden LR ... Callendo AM. Development and Evaluation of a Calibrator Material for Nucleic Acid-Based Assays for Diagnosing Aspergillosis. J Clin Microbiol 2013;51(7):2403-2405. (available here)

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