Hot Topic

Fungal Molecular Diagnostics

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Published: February 2011

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Dr. Wengenack will discuss the latest technological advances in the molecular detection and identification of fungal organisms.

Presenter: Nancy L Wengenack, PhD, Director of the Mycology and Mycobacteriology Laboratories in the Division of Clinical Microbiology at Mayo Clinic

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Transcript

Introduction

Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.

Our presenter for this program is Dr. Nancy Wengenack, Director of the Mycology and Mycobacteriology Laboratories at Mayo Clinic. Dr. Wengenack will discuss the latest technological advances in the molecular detection and identification of fungal organisms.

Objectives

The objectives of this Hot Topic are: to describe the molecular methods that are routinely available in our laboratory for the detection and identification of fungi, to provide examples of how we utilize these molecular methods in our practice and finally, to discuss future directions in fungal molecular diagnostics.

Traditional Methods of Fungal Diagnostics

Before we begin discussing the use of molecular diagnostics, let us recall that until very recently, the methods used by mycologists for the identification of fungi in the laboratory have been largely unchanged for decades. Once received in the laboratory, a patient specimen will typically be plated onto several different types of media and these media will be incubated at a constant temperature for up to 4 weeks. Laboratory staff will periodically examine the plates for growth and when growth is noted, the staff will try to identify the fungus using both macroscopic and microscopic morphologies. Often, subculture of the fungus to other types of media, followed by additional incubation time may be required in order to induce sporulation or the development of characteristic structures that can be used in making a visual identification. If identification cannot be made based on morphology alone, there are a few biochemical tests that can sometimes be helpful in identification, most often for yeast.

Traditional Methods

One significant problem associated with traditional methods of fungal identification is that they can be very slow and may require up to 4 weeks for completion. They are often labor intensive requiring significant technologist hands-on times. Finally, traditional methods require a highly experienced laboratory staff who are well-versed in the visual recognition of fungal morphologies.

Potential Advantages of Fungal Molecular Diagnostics

The use of molecular diagnostics in the detection and identification of fungi is really in its infancy compared with other areas of microbiology such as virology and bacteriology. But molecular diagnostics offer mycologists many of the same advantages already enjoyed by our colleagues. One significant advantage is that molecular diagnostics are fast! Their use can shave days to weeks off of the time required to make a fungal identification. This, in turn, should lead to improved patient care by more rapidly providing clinicians with the information that they need to begin appropriate care and to select an appropriate antifungal agent. Further, the use of molecular diagnostics can contribute to improved safety for laboratory workers by reducing the need to manipulate growing cultures of particularly hazardous molds such as Coccidioides species.

Currently Available Molecular Diagnostics for Fungi

Currently there are a limited number of molecular diagnostic tests available for the detection and identification of fungi in the clinical laboratory. For fungal isolates growing in pure culture, nucleic acid hybridization probes, DNA sequencing, peptide nucleic acid fluorescence in situ hybridization (or PNA FISH) probes, and laboratory-developed polymerase chain reaction (or PCR) tests are available. Molecular methods used in the direct detection and identification of fungi from patient specimens are even further limited to a handful of laboratory-developed PCR tests targeting specific fungal agents. There are several diagnostics companies who have developed or who are developing PCR, array-based, and other molecular tests for fungi but, in large part, these have not yet entered into routine clinical laboratory use at this time.

Nucleic Acid Hybridization Probes

So let's talk in a bit more detail about some of the molecular tests that are available today beginning with the use of nucleic acid hybridization probes for the identification of fungi from pure cultures.

This slide provides an overview of how the nucleic acid hybridization probes function. The laboratorian obtains an RNA template from the organism to be identified by lysing a small amount of the organism after it is picked from a culture plate using an inoculating loop. A DNA-hybridization probe labeled with a chemiluminescent moiety is added to the solution containing the RNA template. If the hybridization probe sequence is complementary to the RNA target, it will bind tightly and specifically. Then, any unbound probe remaining in the solution is hydrolyzed to remove the potential for nonspecific signal and the DNA-RNA hybrid is detected by a chemiluminescence reaction.

Nucleic Acid Hybridization Probes for Fungi

Fungal nucleic acid hybridization probes are available for the identification of only Blastomyces dermatitidis, Coccidioides immitis, and Histoplasma capsulatum. These probes are FDA-approved products and they have excellent sensitivity and specificity as shown in the table on this slide. However, one should be mindful that the Blastomyces dermatitidis probe has the potential to cross-react with a few other fungi including Emmonsia species, Paracoccioides brasiliensis, and Gymnascella species. Generally, a review of fungal morphology and patient travel and clinical history will help to determine whether cross-reactivity is a potential issue. Of additional interest, the Coccidioides probe does not distinguish between the 2 recognized species, namely Coccidioides immitis and Coccidioides posadasii, but differentiation is typically of epidemiologic, rather than clinical, importance.

Nucleic Acid Hybridization Probes

So the nucleic acid hybridization probes are a very valuable molecular tool in the mycologist's toolbox for the identification of dimorphic pathogens. They are rapid with results available within approximately 2 hours after the growth of the organism in culture, they are highly sensitive and specific, and they are technically simple to utilize. The main limitation associated with the probes is that they are only available for the 3 dimorphic pathogens so other tools are needed for the identification of the majority of fungi seen in the clinical laboratory today.

DNA Sequencing for Fungi

When a fungus has grown in culture and it is not easily identified on the basis of macroscopic or microscopic morphology and it also does not a appear to be one of the dimorphic pathogens for which we have nucleic acid hybridization probe available, what else can be done to identify this organism? One option may be to try a series of biochemical and temperature tolerance tests but these are usually long and tedious to perform and sometimes do not provide any additional information. An alternative method that we make use of in the laboratory is DNA Sequencing.

Ribosomal Structure: Fungi

In this slide, I have depicted the ribosomal structural unit for fungi. The major RNA transcript is a repeating unit consisting of the small subunit 18S RNA gene, the 5.8S RNA gene, the large subunit 25-28S RNA gene, and finally the 5S RNA gene. The 18S, 5.8S, and 28S genes are separated by the internal transcribed spacer or ITS region. The most commonly used targets for fungal sequencing in the clinical laboratory today are the D1-D2 region of the 28S subunit and the ITS region. My laboratory has chosen to use the D2 region as our primary sequencing target for fungi.

Traditional Sequencing Workflow

The next slide depicts the typical workflow used for sequencing fungi in our laboratory. Using a biological safety cabinet, the organism to be identified is selected from a culture plate. Following lysis and a processing step that utilizes heat, the fungal DNA is amplified using a PCR reaction followed by a template cleaning step. Then a second PCR reaction is done to incorporate the dideoxynucleotides required for traditional Sanger sequencing. Following a second clean-up step to remove unincorporated nucleotides, the cycle sequencing is completed using a capillary electrophoresis instrument and the fungal DNA sequence is available for analysis by the laboratory staff.

Sequence Analysis

The DNA sequence from the patient-derived isolate is then compared to fungal sequence libraries containing well-characterized sequences. Libraries searched include the MicroSeq library from Applied Biosystems and a laboratory-specific custom library that we have developed over the last decade. Finally, if neither of these databases provides a good matching sequence, the NCBI GenBank database can be searched using the BLAST tool. Caution should be used when matches are achieved against the GenBank database since it is not curated and therefore may contain inaccurate sequences or organism identifications. Before using a GenBank sequence, we make certain the sequence is an exact match to the unknown isolate, that it is of a good length and quality score, that it was deposited by a reputable, experienced group or individual, that it has been published in a peer-reviewed journal, and that the phenotypic description in the journal matches the unknown isolate before considering reporting it as a match. The DNA sequence can be obtained in as little as 8 hours after an organism has grown in culture providing a very rapid turnaround time as compared with many phenotypic identification methods.

Current Applications of D2 Fungal Sequencing at the Mayo Clinic

Not every yeast or mold identification requires DNA sequencing. Many identifications are made quite easily and cost-effectively using traditional methods. Here at Mayo, we selectively utilize D2 DNA sequencing as a tool when traditional methods aren't providing a clear answer or when traditional methods require inordinate amounts of time. For example, it has been very helpful for some yeast that are difficult to identify using phenotypic methods such as the API20C assimilation test. Further, we have had great success using it to rapidly rule-out pathogens when fungi are persistently nonsporulating, as often occurs in the summer months. D2 sequencing has been very helpful for several cases of difficult to identify molds. And finally, D2 sequencing is extremely useful to confirm questionable nucleic acid hybridization probe results in instances where one may be considering a false-positive probe due to a cross-reacting organism. Where necessary, dermatophyte identification can be achieved an average of 9 days faster using sequencing as opposed to phenotypic methods.

Rapid, Direct Identification of Yeast from Blood Cultures

Next I will discuss the use of peptide nucleic acid fluorescence in situ hybridization, or PNA FISH, probes for the direct identification of yeast from positive blood culture bottles.

PNA-FISH Basics

This slide highlights the PNA FISH Yeast Traffic Light Probe method. The probes are single stranded peptide nucleic acids labeled with a fluorophore such as fluorescein or rhodamine. When a blood culture bottle signals as positive in the laboratory on an automated blood culture instrument, the technologist performs a Gram stain. If the Gram stain indicates that yeast are present, a small amount of the blood is then fixed to a fresh slide and the PNA FISH probes targeting unique rRNA sequences are added to the blood smear. The smear and probe combination is allowed to hybridize at 55°C for a specified amount of time and the slide is then washed to removed unincorporated probes. Finally the smears are mounted with a mounting medium and are examined by fluorescence microscopy.

PNA FISH for Candida spp—Direct Detection from Blood

After the Gram stain and the hybridization process is complete, Candida albicans and Candida parapsilosis are identified microscopically as bright green fluorescing cells, Candida tropicalis fluoresces bright yellow, and Candida glabrata and Candida krusei fluoresce bright red. Other yeast do not fluoresce. In addition to providing identification of the 5 most common species of Candida, the Traffic Light probes also provide an indication about the potential utility of fluconazole in these patients since Candida albicans and Candida parapsilosis are generally susceptible to fluconazole, Candida glabrata can be resistant to fluconazole and Candida krusei is intrinsically resistant to fluconazole. Therefore, a green fluorescent signal can serve as a "go" indication for the clinician to use fluconazole, while a red fluorescent signal serves as a "stop" warning sign because fluconazole may not be a good choice for this patient. Finally, the yellow signal produced by Candida tropicalis indicates that caution should be used since fluconazole susceptibility is variable for this organism.

Yeast Identification Timeline from Positive Blood Bottle

Therefore, within 2 to 3 hours of the blood culture bottle signaling positive in the laboratory, the PNA FISH Traffic Light probes provide the clinician with an indication of both the identity of the Candida species present and the potential utility of fluconazole treatment for their patient. This represents a major time savings over traditional identification methods, which generally require subculture of the blood to a fungal medium, waiting for growth, and morphologic and biochemical identification, all of which can require up to 3 or more days after the blood bottle becomes positive.

Potential Advantages of Direct Detection

Direct detection of Candida species from blood is obviously advantageous for the patient. Rapid identification of the 5 commonly encountered species should lead to improved patient safety by permitting the physician to select the most effective drug (for example, fluconazole versus an echinocandin) and to begin appropriate treatment as rapidly as possible, thus potentially decreasing the hospital length of stay. Decreasing the length of stay has an obvious impact on cost, but so does selecting the best drug even before traditional drug susceptibility results become available because the daily cost of fluconazole is much less than that of an echinocandin. Therefore, knowing that fluconazole should be effective within 2 to 3 hours of a positive blood culture can contribute to reduced healthcare costs.

Fungal PCR Assays at Mayo Clinic

Now that we have discussed the utility of nucleic acid hybridization probes, DNA sequencing, and PNA FISH probes for assisting in identification of fungi growing in culture, let’s switch gears and discuss molecular methods available for the direct detection of fungi in clinical specimens without the need for culture. Currently this is a very, very short list of methods consisting of a handful of laboratory-developed PCR assays that target specific fungi. In our laboratory, we have 2 of these real-time PCR assays that have been used routinely for several years. The first assay provides for the direct detection of Pneumocystis jiroveci from respiratory specimens such as bronchoalveolar lavage fluid and respiratory tissues. The second assay allows for the direct detection of Coccidioides immitis and Coccidioides posadasii from respiratory specimens, body fluids, and tissues. Within the next few months, we will begin offering a third real-time PCR assay that provides a single tube assay for detection and differentiation of Histoplasma capsulatum and Blastomyces dermatitidis from respiratory specimens, body fluids, and tissue.

Real-time PCR Workflow

The workflow for all of our real-time PCR assays is the same and is depicted in this slide. We begin with the patient specimen, which undergoes a processing step to lyse and inactivate the organism. Next the specimen is extracted to isolate the fungal nucleic acids and the nucleic acid is amplified using real-time PCR. Sequence-specific detection is achieved using fluorescence resonance energy transfer (or FRET) probes. The entire process requires about 4 hours from start to finish and, therefore, the result can be available the same day that the specimen is submitted.

Pneumocystis PCR

In the next few slides, I will briefly describe our real-time PCR assays for fungal pathogens and will highlight a couple of examples of how they have been useful in our practice. The first assay that I will discuss is the Pneumocystis jirovecii assay. A full description of this assay has been published in Diagnostic Microbiology and Infectious Diseases and I have provided the reference in this slide if you are interested in reading more of the details. So why did we choose to develop a Pneumocystis PCR assay? One reason was that this organism is not easily cultured in the clinical laboratory. Stains and immunofluorescence assays have, therefore, been utilized, but they are often insensitive, subjective, and require experienced readers. Our goal with the PCR assay was to develop a sensitive and objective method to detect this organism. The target selected is a 162-base pair region of the cyclin-dependent kinase or cdc-2 gene in Pneumocystis jiroveci. Cdc-2 is involved in Pneumocystis cell cycle regulation and, therefore, this gene is highly conserved. Following the workflow described on the previous slide, we use cdc-2 sequence-specific FRET hybridization probes to detect any Pneumocystis jirovecii DNA present in the patient’s specimen. Acceptable sources for this assay are respiratory specimens such as bronchoalveolar lavage (BAL) fluid and sputum.

Pneumocystis LC-PCR Melt Curves

This slide provides an example of the melt curve analysis used to detect Pneumocystis jiroveci. A sequence-specific melt peak occurs at approximately 61ºC with no cross-reactions occurring with any other respiratory pathogens including bacteria, viruses, and other fungi.

Concordance of the Pneumocystis LightCycler PCR Assay and Calcofluor White Stain for BAL Fluid

Verification of the sensitivity and specificity of the Pneumocystis PCR assay against a calcofluor white stain method that has been used for many years in our laboratory, indicated that the PCR assay had a 22% increase in sensitivity relative to the stain, with the PCR assay detecting more positives from BAL fluid than did the stain.

Coccidioides species Real-time PCR

Another PCR assay that has been very useful in our laboratory is the Coccidioides species real-time PCR assay, which can detect both Coccidioides immitis and Coccidioides posadasii in respiratory specimens and tissue. This assay was developed to decrease the time required for detection the organism relative to the time required for growth of the organism in culture, which can be 10 or more days. The PCR assay is rapid compared with culture, is more sensitive and specific than serology, and has the additional advantage of providing improved safety for laboratory workers since they spend less time manipulating growing cultures of this highly infectious mold. We utilize the Coccidioides PCR assay in several ways. It is used for the rapid identification of Coccidioides from organisms grown in culture. This provides us with another method of identification should the nucleic acid hybridization probe ever experience availability problems, plus it obviates the need for the lab to keep a growing culture of Coccidioides in the laboratory as a positive probe control. Continuous culture of Coccidioides is a problem for laboratories because not only is it a safety hazard for lab personnel, but it is a regulatory headache since Coccidioides species are listed as Select Agents by the Centers for Disease Control and Prevention Select Agent program. Finally, the PCR assay has been extremely useful for the direct detection of Coccidioides species from patient respiratory, body fluid, and tissue specimens where it can provide a rapid turnaround time without the need to wait for a culture to grow.

Melt-curve Analysis of Coccidioides spp Grown in Culture

Similar to the Pneumocystis assay, the Coccidioides PCR assay utilizes sequence-specific FRET probes and melt-curve analysis for detection. A melt peak occurs at approximately 60ºC with no cross-reactions occurring with any other respiratory pathogens including bacteria, viruses, and other fungi.

Utility of Coccidioides PCR Testing in the Clinical Setting

Since we now have several years of experience utilizing the Coccidioides real-time PCR assay, we have been able to do several studies evaluating the clinical utility of this approach. Two manuscripts have been recently accepted and are currently in press in the journal Mycopathologia. I have provided the reference information if you would like to learn more. In the first study, we reviewed the medical charts of 145 patients with suspected coccidiomycosis and found that the real-time PCR assay was superior to fungal culture for the diagnosis of pulmonary disease, but I should caution that the "n" was small for this study. The PCR assay was also found to be highly useful in rapidly ruling out coccidiomycosis with a greater than 99% negative predictive value.

Meningeal Coccidioidomycosis Diagnosed by Real-Time PCR Analysis of Cerebrospinal Fluid

In the second study, we described 2 cases of coccidioides meningitis that were rapidly diagnosed using the real-time PCR assay. The PCR results were available several days before culture or serologic confirmation was achieved. Laboratory studies on spinal fluid (CSF) are often difficult due to the paucity of organism present in the specimen, but these 2 cases serve to highlight the potential utility of molecular methods in the diagnosis of meningeal coccidiomycosis.

Histoplasma capsulatum/Blastomyces dermatitidis Real-Time PCR

The last PCR assay that I will discuss briefly is a new one that will be introduced this year. That assay allows for the detection and identification of Histoplasma capsulatum and Blastomyces dermatitidis in a single tube either from culture isolates or directly from patient respiratory or tissue specimens. We recently submitted a manuscript describing the assay for peer-review evaluation. The use of distinctly labeled fluorophores on the FRET probes allows for the detection and differentiation of Histoplasma capsulatum and Blastomyces dermatitidis from a single tube using different detection wavelengths. We are very excited about this assay since we will be able to rapidly detect and distinguish between these 2 dimorphic pathogens that have overlapping areas of endemicity and that often can have overlapping clinical pictures.

What lies in the future for fungal molecular diagnostics?

So let's close by thinking for a minute about what might the future hold in the area of fungal molecular diagnostics.

Mass Spectrometric Identification Of Fungi

One technology that is rapidly entering the clinical diagnostic arena today is mass spectrometry. Once the purview of chemists, mass spectrometry is rapidly finding in-roads into microbiology in the areas of bacteria and even fungal identification. In a recent publication by Stevenson et al at the National Institutes of Health, matrix-assisted, laser desorption ionization coupled with time of flight detection (or MALDI-TOF) mass spectrometry was utilized to identify 23 species of yeast from 6 different genera. Why would you want to throw a "big gun" like MALDI-TOF at yeast identification? Well, for one thing, MALDI-TOF uses very few reagents and consumables so it's a very "green" technology that can be done quite cheaply in terms of reagent and FTE costs. Equipment cost is also reasonable. It's rapid to perform, requiring only a few minutes from isolate to answer and it's technically simple for the technologist. Further, it appears to have wide applicability as a number of groups have reported success with bacterial identification using this approach.

We recently evaluated the ability of mass spectrometry to rapidly identify yeast in our laboratory and concur with the findings of Stephenson et al. We anticipate that MALDI-TOF will allow us to replace several slower phenotypic tests that we currently use for yeast identification at a comparable overall cost. What else does the future hold? It's exciting to think about the possibilities offered by technologies such as next generation sequencing but, at this time, the technology is too expensive and lacks the data analysis tools necessary to make it routinely applicable in clinical microbiology. Time will tell if this or other molecular methods continue to make in-roads into the highly traditional field of diagnostic mycology.

Summary

So in summary, I conclude by reiterating that molecular diagnostics are enabling mycologists to significantly reduce the turnaround time for detection and identification of many fungi in the clinical laboratory. However, there remains room for improvement, with most of the molecular methods available presently targeting identification of fungi after growth in culture. Additional challenges and opportunities remain in the direct detection of fungi from clinical specimens, but the potential to positively impact patient care comes with every addition of new molecular tools to our toolbox. Thank you very much for taking the time to listen to this Hot Topic presentation.

Disclosures

My disclosures for the program are the receipt of royalties from Roche Diagnostics and TIB MOLBIOL.

 


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