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Rapid detection of Histoplasma capsulatum and Blastomyces dermatitidis DNA
Infections with Blastomyces dermatitidis and Histoplasma capsulatum cause a variety of clinical manifestations ranging from self-limited, mild pulmonary illness to potentially life-threatening, disseminated disease. Patients at risk for disseminated disease include neonates and immunosuppressed individuals, particularly those with AIDS, hematologic malignancies, or a recent transplant. Primary infections are acquired through inhalation of microconidia that are present in the environment. In the United States, most cases of blastomycosis and histoplasmosis occur along the Ohio and Mississippi River valleys.
The gold standard for diagnosis of blastomycosis and histoplasmosis remains isolation of the organisms in culture. Although sensitive, recovery in culture and subsequent identification may require days to weeks. The organisms can be identified after growth in culture using traditional macro- and microscopic morphologic techniques or through the use of nucleic acid hybridization probes. Hybridization probe-based procedures are rapid and demonstrate good sensitivity and specificity from culture, although some cross-reactivity with relatively uncommon fungal organisms has been reported. Additional diagnostic tests that can be utilized for these organisms include stains, histopathology, serology, and antigen detection with each of these methods offering advantages and limitations depending on the stage of the illness and the status of the patient. Fungal stains (eg, calcofluor white) offer a rapid diagnostic approach, but demonstrate poor sensitivity and specificity. Serologic tests such as complement fixation and immunodiffusion are noninvasive, but are laborious, subjective, and may show low sensitivity, especially in immunocompromised hosts. Antigen detection also offers a noninvasive approach, but has been demonstrated to show cross-reactivity with antigens from closely related fungal species.
Molecular techniques have been established as sensitive and specific methods for the diagnosis of infectious diseases and have the added advantage of a rapid turnaround time for results. Due to the limitations of conventional diagnostic methods for blastomycosis and histoplasmosis, a single tube, real-time PCR assay was developed and verified for the detection and differentiation of Blastomyces dermatitidis and Histoplasma capsulatum directly from clinical specimens.
A positive result for Histoplasma capsulatum indicates presence of Histoplasma DNA; a positive result for Blastomyces dermatitidis indicates presence of Blastomyces DNA.
A negative result indicates absence of detectable Histoplasma capsulatum and Blastomyces dermatitidis DNA.
For cases of suspected disseminated histoplasmosis, a Histoplasma urine antigen test should be ordered instead of the rapid PCR assay, since the urine antigen test has increased sensitivity.
Blood should only be drawn for suspected cases of disseminated disease where the PCR may be helpful to distinguish between Histoplasma capsulatum and Blastomyces dermatitidis infection. Blood is not an appropriate specimen source for the diagnosis of localized disease (eg, pulmonary histoplasmosis or blastomycosis).
A fungal blood culture must also be performed.
This rapid PCR assay detects Histoplasma capsulatum and Blastomyces dermatitidis nucleic acid and, therefore, does not distinguish between the presence of viable, disease-related organisms and nucleic acid persisting from previous, treated 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 Histoplasma capsulatum or Blastomyces dermatitidis because the organism may be present at levels below the limit of detection for this assay.
Analytical Sensitivity and Specificity:
The analytical sensitivity of the assay was determined to be < or =100 copies/microliter for both Blastomyces dermatitidis and Histoplasma capsulatum. The Blastomyces dermatitidis melt peak is read at 705 nm and has a melting temperature (Tm) of 66 C + or - 0.26 C (mean + or - standard deviation), while the Histoplasma capsulatum read at 640 nm has a Tm of 61 C + or - 0.27 C. A National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) search of the primer, probe, and target sequences for both Blastomyces dermatitidis and Histoplasma capsulatum did not yield any potentially cross-reacting sequences. In addition, testing of nucleic acids from 179 potentially cross-reacting microbes commonly encountered in the clinical laboratory including bacteria, fungi, parasites, and viruses demonstrated no cross-reactivity with other organisms. The list of organisms is available upon request.
Sensitivity and Specificity from Cultured Isolates:
The sensitivity of the assay from isolates grown in culture was 100% (61/61) and 94.5% (51/54) for Blastomyces dermatitidis and Histoplasma capsulatum, respectively. The specificity of the assay was 100% for both organisms as no cross-reactivity was detected in the other non-Blastomyces dermatitidis or non-Histoplasma capsulatum cultures evaluated.
Clinical Sensitivity and Specificity Directly from Specimens:
A total of 797 clinical specimens were tested concurrently by fungal culture and the real-time PCR assay to assess clinical sensitivity and specificity. The sensitivity and specificity of the PCR assay for Blastomyces dermatitidis was 86% (12/14 positive) and 99.4% (778/783 negative), respectively. The overall sensitivity and specificity of the PCR assay for Histoplasma capsulatum was 73.3% (11/15 positive) and 100% (782/782 negative), respectively. Of note, the recovery of Histoplasma capsulatum from bronchoalveolar lavage (BAL) fluid was low (2 PCR positives of 6 culture positives), which accounted for all of the falsely negative specimens. Therefore, a negative result from BAL fluid does not rule out Histoplasma capsulatum infection due to low sensitivity from this specimen source.
Due to the low number of positive specimens obtained clinically despite testing almost 800 specimens over a period of 1 year, spiking studies using a plasmid control for both organisms were also performed using negative specimens representing various specimen types (30 each of pleural fluid, sputum, BAL fluid, cerebrospinal fluid, tissue, bone, sterile body fluids, urine, and blood). The spiking studies demonstrated a sensitivity of the PCR assay of 100% (30/30 positive) for Blastomyces dermatitidis across all specimen types except blood (97% sensitive; 29/30 positive). The PCR assay had 100% (30/30) sensitivity for Histoplasma capsulatum across all spiked specimen types except sputum and blood which had 98% and 97% sensitivity respectively.
The overall extraction and amplification inhibition rate of the assay using both targets was <1% (2/330 inhibited). Extraction inhibition occurred in 1 of 30 blood specimens for Blastomyces dermatitidis and Histoplasma capsulatum, and 1 of 60 sputum specimens for Histoplasma capsulatum.
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