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Rapid detection of Coccidioides DNA
An aid in diagnosing coccidioidomycosis
Coccidioidomycosis is caused by the dimorphic fungi, Coccidioides immitis and Coccidioides posadasii. These organisms are endemic to the southwestern regions of the United States, northern Mexico, and areas of Central and South America. The illness commonly manifests as a self-limited upper respiratory tract infection, but can also result in disseminated disease that may be refractory to treatment.(1) Clinical onset generally occurs 10 to 16 days following inhalation of coccidioidal spores (arthroconidia).(2) Disease progression may be rapid in previously healthy or immunosuppressed individuals.
At present, the gold standard for the diagnosis of coccidioidomycosis is culture of the organism from clinical specimens. Culture is highly sensitive and, with the implementation of DNA probe assays for confirmatory testing of culture isolates, yields excellent specificity.(3) However, growth in culture may take up to several weeks. This often delays the diagnosis and treatment of infected individuals. In addition, the propagation of Coccidioides species in the clinical laboratory is a significant safety hazard to laboratory personnel, serving as an important cause of laboratory-acquired infections if the organism is not quickly identified and handled appropriately (ie, in a Biosafety Level 3 facility). Serological tests including immunodiffusion and complement fixation are widely used for the detection of antibody against Coccidioides. Serology for Coccidioides can be limited by delays in antibody development or nonspecificity due to cross-reactions with other fungi. In addition, immunodiffusion and complement fixation tests are highly labor intensive and are generally limited to reference laboratories.
Molecular methods can identify Coccidioides species directly from clinical specimens, avoiding the need for culture and allowing for a more rapid and safer diagnosis.
A positive result indicates presence of Coccidioides DNA.
A negative result indicates absence of detectable Coccidioides DNA.
This test should always be performed in conjunction with fungal culture.
This rapid PCR assay detects Coccidioides nucleic acid and, therefore, does not distinguish between viable, disease-related organisms and transient colonizing organisms or nucleic acid persisting from old disease. Test results should be correlated with patient symptoms and clinical presentation before a definitive diagnosis is made.
A negative result does not rule out the presence of Coccidioides or active disease because the organism may be present at levels below the limit of detection for this assay.
This test does not distinguish between the Coccidioides immitis and Coccidioides posadasii.
Formalin-fixed, paraffin-embedded tissue is not an optimal source for PCR analysis as formalin fixation has been demonstrated to decrease the sensitivity of PCR.(4) Therefore, a negative PCR result from fixed tissue should be interpreted with caution if the clinical presentation is suggestive of coccidioidomycosis.
Analysis of 266 respiratory specimens (bronchoalveolar lavage, sputum, induced sputum, bronchial wash, pleural fluid) by LightCycler PCR for Coccidioides species detection demonstrated 100% sensitivity and 98.4% specificity compared with culture. Analysis of 66 fresh tissue specimens yielded 92.9% sensitivity and 98.1% specificity compared with culture. The sensitivity and specificity of the assay testing 148 paraffin-embedded tissue specimens was 73.4% and 100% respectively.
The limit of detection of the assay was determined to be between 1 and 10 copies of target/microliters (<50 copies of target/reaction). The analytic specificity of the assay was determined by performing a Basic Local Alignment Search Tool (BLAST) search of the primer and probe sequences on the National Center for Biotechnology Information website (http://www.ncbi.nml.nig.gov). In addition, an extensive panel of nucleic acid extracted from 114 potentially cross-reacting organisms including fungi, bacteria, mycobacteria, viruses, and human DNA was tested. The assay did not demonstrate cross-reactivity with any of the organisms included in the specificity panel.
1. Chiller TM, Galgiani JN, Stevens DA: Coccidioidomycosis. Infect Dis Clin North Am 2003;17:41-57
2. Feldman BS, Snyder LS: Primary pulmonary coccidioidomycosis. Semin Respir Infect 2001;16:231-237
3. Padhye AA, Smith G, Standard PG, et al: Comparative evaluation of chemiluminescent DNA probe assays and exoantigen tests for rapid identification of Blastomyces dermatitis and Coccidioides immitis. J Clin Microbiol 1994;32:867-870
4. Inoue T, Nabeshima K, Kataoka H, Koono M: Feasibility of archival non-buffered formalin-fixed and paraffin-embedded tissues for PCR amplification: an analysis of resected gastric carcinoma. Pathol Int 1996;46:997-1004