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Update on the Identification of Coccidioides immitis and Coccidioides posadasii

Rapid Detection using PCR

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Published: August 2012

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Coccidioides species are dimorphic fungal pathogens. Coccidioides immitis is endemic to the San Joaquin valley region of California and coccidioidomycosis is sometimes referred to as Valley Fever. Coccidioides posadasii is found in the desert regions of the southwestern United States including Arizona, Utah, New Mexico, and West Texas and also in parts of Mexico, Argentina, Paraguay and Central America. Dr. Wengenack provides an overview of Coccidioides infection and describes transmission, epidemiology, and pathogenesis. She also discusses the laboratory methods that are useful for the detection and identification of Coccidioides species with a particular emphasis on rapid detection using PCR

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

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Welcome to Mayo Medical Laboratories Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice. Our speaker for this program is Dr. Nancy Wengenack, Director of the Mycology and Mycobacteriology Laboratories and Associate Professor of Laboratory Medicine and Pathology in the Division of Clinical Microbiology at Mayo Clinic in Rochester, Minnesota. Dr. Wengenack presents an update on the epidemiology and pathogenesis of Coccidioides infection and laboratory methods of detection. Thank you, Dr. Wengenack. Thank you, Sarah. Today, I will be discussing the use of real-time PCR for the rapid detection and identification of the dimorphic pathogens Coccidioides immitis and Coccidioides posadasii.

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

The objectives of this Hot Topic are: to describe the epidemiology of coccidioidomycosis, to describe the pathogenesis of Coccidioides species, and finally, to discuss the laboratory methods that are useful for the detection and identification of Coccidioides species with a particular emphasis on rapid detection using PCR.

Coccidioides species are dimorphic fungal pathogens and for this particular genus that means that the organism grows as a filamentous mold in the environment at ambient temperature and exists as a spherule containing endospores in the body at 37ºC. There are 2 species within the genus Coccidioides with each species occupying a different geographic niche. Coccidioides immitis is endemic to the San Joaquin valley region of California and this is why coccidioidomycosis is sometimes referred to as Valley Fever. The second species, Coccidioides posadasii, is found in the desert regions of the southwestern United States including Arizona, Utah, New Mexico, and West Texas and also in parts of Mexico, Argentina, Paraguay and Central America. There is very little difference in morphology or clinical presentations between the 2 species. At this time, separation of the 2 species is important for epidemiologic purposes but not for clinical purposes.

Here on the map, I pictorially show the areas of the world where Coccidioides species are endemic including the United States, Mexico, and parts of Central and South America. The larger map of the United States on the right further defines the states within the desert southwest where the organism is commonly found, classically southern California, Arizona and New Mexico with reach into adjoining states.

Coccidioides species are associated with soil and dust in their endemic areas. Arthroconidida are the infectious form of the agent and they can disassociate and be carried by the wind for many miles. Cases of coccidioidomycosis increase when there are rainy summers followed by dry winters, after earthquakes, or after humans disturb the soil in some way such as during construction projects or archeological digs.

So how does one acquire coccidioidomycosis? As mentioned earlier, the arthroconidia are the infectious form. They are inhaled into the lungs where they then begin to transform into multinucleated spherical structures containing hundreds of endospores. The majority of persons have a mild or asymptomatic pulmonary infection which resolves without intervention. Evidence of this may be seen later on chest X-ray in the form of a coin lesion. Some people who are infected will present to their physician with an apparent community-acquired pneumonia which often has an associated rash and arthralgias. The incubation time for this is about 2 weeks after exposure. Often these pneumonias resolve and only a few will progress to chronic pulmonary disease which can present as nodular or cavitary disease. Of these cases, even fewer will progress to systemic or CNS coccidioidomycosis but these forms are quite serious and are associated with heightened morbidity and mortality.

Common sites of systemic dissemination for Coccidioidies species are the lymph nodes, bones, and joints. Also skin manifestations are common and these can occur through dissemination or through direct inoculation. Finally, CNS dissemination can occur although it typically occurs in less than 1% of patients with coccidioidomycosis but when it does occur, it is often associated with a poor outcome.

This slide provides a schematic of the life cycle of Coccidioides species. Beginning at the bottom of the figure, an arthroconidia in the environment will germinate and begin to differentiate into a tubular hypha. Over time, the hypha segments into arthroconidia with empty disjunctor cells separating the arthroconidia. When the individual arthroconidia separate from the disjunctor cells, they can easily become airborne and some of the arthroconidia return to the soil to repeat the entire soil life-cycle process. Some of the arthroconidia, however, can be inhaled by humans which are a dead end host for this fungus. Once inside the body, the inhaled arthroconidia begin to enlarge and develop in to a thick-walled spherule. Spherules in tissue can have a variety of sizes ranging from 10 to 100 microns in diameter. Inside the spherules, endospores develop which are approximately 2 to 5 microns in diameter and there can be hundreds of endospores inside a single spherule. After the spherule matures, it can rupture, releasing its endospores in the tissue where they can repeat the cycle and develop into new spherules.

This slide shows a KOH/Calcofluor white stain of Coccidioides species in tissue. As you can see in the picture on the left, there is a mature spherule at the top of the frame with the smaller, round endospores spilling out. In the right-hand photo, you can see several smaller-sized spherules, 2 of which are broken and have already lost many of their endospores but you can still see several endospores inside the spherules.

There are several groups of individuals who are at an increased risk for progression to extrapulmonary or disseminated coccidioidomycosis and those are persons of African American or Filipino descent, pregnant women (especially those in their 3rd trimester) and patients with immunosuppressive conditions such as malignancies, transplant patients, and those with HIV or diabetes.

This slide lists the laboratory methods currently available for the detection and identification of Coccidioides species. These include direct microscopic examinations, serologic testing, culture followed by identification using either microscopic morphology, nucleic acid hybridization probes, real-time PCR or DNA sequencing, and finally, direct detection using PCR. Direct PCR is the method that I will highlight later in this presentation because of its utility from a variety of specimen types and because it obviates the need to wait for growth of the organism in culture. This can be very important in cases of disseminated disease, especially for meningitis cases where time is critical for providing a good outcome for the patient.

In this slide, the identification of Coccidioides species on a direct smear is highlighted. As mentioned earlier, spherules are typically large with a size of approximately 10 to 100 microns and they contain multiple endospores which are 2 to 5 microns in diameter. Spherules can, at times, be visually confused with other large fungi such as Rhinosporidium seeberi by an inexperienced reader. In addition, some of the spherule walls can be ruptured and the endospores may have spilled out which can also be a source of confusion to a laboratorian performing a direct examination. The endospores themselves can be easily confused with fungi of a similar small size including Candida glabrata, the yeast forms of Blastomyces dermatitidis and Histoplasma capsulatum as well as with the cysts of Pneumocystis jirovecii. Finally, active lesions may contain organisms but resolving lesions may contain fewer organisms making diagnosis using a direct exam difficult at times. Occassionally, mycelia may be seen in a lung cavity or in tissue from a skin lesion.

Serology is also available for the detection of antibodies against Cocciodioides species. An immunodiffusion method can be used to measure both IgM and IgG class antibodies against Coccidioides. Detection of IgM indicates a recent active infection with IgM appearing within several days of infection and reverting to negative within a few months. Falsely positive IgM results can also occur and some publications suggest that the rate can be as high as 15% especially in certain types of patients such as those with cystic fibrosis.

Detection of IgG using immunodiffusion typically indicates an active or recent past infection. The IgG antibody is usually positive within 4 weeks after infection and stays positive throughout clinically active disease. Falsely positive results due to cross-reacting antibodies from Histoplasma capsulatum and other fungi are possible. Similarly, falsely negative reactions are also possible.

Complement fixation detects IgG class antibodies and titers of ≥1:2 may suggest active disease. However, it is important to remember that titers may persist for months after infection has resolved. Increasing titers in serial specimens collected several weeks apart are diagnostic of active disease. Complement fixation is more sensitive than immunodiffusion but less specific.

In this slide, I discuss the growth of Coccidioides species in culture. The organism grows relatively quickly appearing first moist and glaborous on the culture plate much like a yeast but it becomes mold-like over time with a white or grayish-white appearance on a culture plate in about 3 to 10 days on routine mycologic media such as inhibitory mold agar. It can also grow on bacteriologic medium such as blood agar so bacteriologists should be cautious in handling plates with a mold-like colony on them, especially if they know the patient has been to an area endemic for Coccidioides. Since this often isn’t known by the lab, referral of plates containing a mold to the mycology laboratory where they can be safely processed is very important. Culture of Coccidioides species on mycologic medium has good sensitivity with approximately 90% of clinical cases producing an isolate. Because the arthroconidia from a mature culture plate can be easily aerosolized, Coccidioides poses a significant risk to lab staff for a laboratory-acquired infection. Therefore, all work with cultures of Coccidioides species must be done in a certified class II biological safety cabinet by highly trained personnel. Further, at this time, Coccidioides species remain on the CDC and APHIS Select Agents lists as potential agents of bioterrorism and for this reason, laboratories must make certain isolates are secured and destroyed within 7 days of identification. Destruction must be promptly reported to the CDC’s Select Agent Program.

This slide shows the fluffy white colonial morphology of Coccidiodies species on the left and the microscopic morphology with the alternating arthroconidida on the right. Note the empty disjunctor cells between the barrel-shaped arthroconidida.

Next I will discuss the methods that are available for the identification of Coccidioides species after growth on a culture plate. As described earlier, macroscopic and microscopic morphology can be used for identification from a culture plate but this is not recommended due to the subjectivity sometimes associated with morphologic identification and due to the safety hazard that mature cultures of this organism present to laboratory personnel. Instead, molecular methods such as nucleic acid hybridization probes, DNA sequencing, or PCR testing are recommended.

This slide provides a schematic of the nucleic acid hybridization probe method which can be utilized to identify Coccidioides species from a pure culture. Use of the nucleic acid hybridization probes first requires lysis of the organism to release the ribosomal RNA from the cells. This RNA is present in multiple copies which enhances sensitivity of the method. After the RNA is released, single-stranded, complementary, acridinium-ester-labeled DNA probes are added which bind to the target RNA from Coccidioides if present. After a short hybridization period, a selection reagent is added to solution which cleaves the ester label off of any nonhybridized probes. Then any RNA-DNA hybrids formed by the specific binding of the probe to Coccidioides target RNA are detected using a chemiluminscent reaction. The probe is FDA-cleared and has a reported sensitivity of 98.8% with a specificity of 100% according to package insert data. The probe does not distinguish between Coccidioides immitis and Coccidioides posadasii but as discussed earlier, there is no clinical need to do so.

The next identification method that I will discuss is DNA sequencing. This slide depicts the typical workflow used for sequencing of Coccidioides species 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.

The DNA sequence obtained from the patient-derived isolate is compared to fungal sequence libraries containing well-characterized sequences.

The DNA sequence can be obtained in as little as 8 hours after an organism has grown in culture although isolates are typically batched by laboratories providing this service as so a more typical turnaround time is identification in about 24 hours after growth in culture. This is still a very rapid turnaround time as compared with phenotypic methods for the identification of Coccidioidies.

Now that we have discussed the use of nucleic acid hybridization probes and DNA sequencing for the identification of Coccidioides species growing in a pure culture, I will focus the remainder of the presentation on the use of rapid cycle, real-time PCR for the detection and identification of Coccidioides species both from a culture isolate but also directly from selected specimen types without the need to wait for growth in culture. The Coccidioides species real-time PCR assay can detect both Coccidioides immitis and Coccidioides posadasii in respiratory specimens, sterile body fluids including CSF and both fresh and formalin-fixed, paraffin-embeded tissue. This assay was developed to decrease the time required for detection of 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 CDC 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. Rapid identification allows the physician to make an accurate diagnosis and administer the appropriate therapeutic agent as soon as possible, which is critical to reducing the morbidity and mortality associated with this agent.

A full description of the Coccidioides PCR assay has been published in the Journal of Clinical Microbiology and I have provided the reference in this slide if you are interested in reading more of the details. Briefly, the workflow for all of our real-time PCR assays is the similar and is depicted in this slide. We begin with the patient specimen or a pure culture of the organism, 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.

This slide provides an example of the FRET probe melt curve analysis used for the detection of Coccidioides species following the PCR reaction. 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. The assay does not distinguish between Coccidioides immitis and Coccidioides posadasi but as I mentioned earlier, there is no clinical reason to do so at this time. We have utilized the Coccidoidies PCR assay for approximately 5 years and in the next few slides, I will briefly highlight a couple of examples of how the PCR assay has been useful in our practice.

Since we now have several years of experience utilizing the Coccidioides real-time PCR assay, we have been able to review the clinical utility of this approach and the study has been published in Mycopathologia. I have provided the reference information if you would like to learn more. In this study, we reviewed the medical charts of 145 patients with suspected coccidiomycosis and found that the real-time PCR assay was similar to fungal culture in terms of sensitivity for the diagnosis of pulmonary disease but I should caution that the number of positive cases in this study was small. The PCR assay was also found to be highly useful in rapidly ruling out coccidiomycosis with a greater than 99% negative predictive value.

In a second publication also in Mycopathologia, we described 2 cases of Coccidioides meningitis that were rapidly diagnosed using the real-time PCR assay. In the first case, the PCR result was available 3 days before positive serologic findings and 22 days prior to the culture result which actually was negative for this case. So without the PCR result, physicians would have had to rely solely on serology and clinical findings to make the diagnosis. The rapid availability of the PCR result provided them with a rapid, definitive diagnosis. In the second case, the positive PCR result was also available prior to a positive culture result. Laboratory studies on cerebrospinal fluid 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 rapid and accurate diagnosis of meningeal coccidiomycosis.

In summary, I would like to conclude with a couple of take home points for this Hot Topic presentation. For patients who reside in or who have traveled to an endemic area such as Arizona or the San Joaquin Valley and who have suspected coccidioidomycosis, the Coccidioides PCR test and routine fungal culture should be ordered. The Coccidioides PCR test has demonstrated nearly equivalent sensitivity when compared to routine fungal culture. In contrast to culture however, the results of the PCR assay are available in hours compared to the days to weeks required for culture adding a valuable rapid diagnostic tool to our mycology toolkit for Coccidioides species. It is important to remember that no method is perfect and although the PCR assay is extremely useful for the rapid detection of many cases of coccidioidomycosis, the performance of culture in addition to PCR remains important to find those cases which may be PCR negative. Thank you.