Rapid detection of Histoplasma capsulatum and Blastomyces dermatitidis DNA
An aid in the rapid diagnosis of histoplasmosis and blastomycosis
See Meningitis/Encephalitis Panel Algorithm in Special Instructions.
Real-Time Polymerase Chain Reaction (PCR)
See Meningitis/Encephalitis Panel Algorithm in Special Instructions.
Varies
Urine is not an acceptable source for this assay. Studies indicate that Histoplasma DNA is not routinely found in the urine of patients with disseminated histoplasmosis. Therefore, the UHIST / Histoplasma Antigen, Urine is the recommended test for this specimen source.
This test should always be performed in conjunction with fungal culture, order FGEN / Fungal Culture, Routine.
Specimen must arrive within 7 days of collection; specimens received after 7 days will be rejected.
NALC/NaOH-digested specimen must arrive within 7 days of digestion.
Specimen source is required.
Question ID | Description | Answers |
---|---|---|
SRC78 | Histo/Blasto Source |
The high sensitivity of amplification by PCR requires the specimen to be processed in an environment in which contamination of the specimen by Histoplasma or Blastomyces species DNA is not likely.
Submit only 1 of the following specimens:
Specimen Type: Body fluid
Sources: Body, CSF, bone marrow
Container/Tube: Sterile container
Specimen Volume: 1 mL
Specimen Type: Respiratory
Sources: BAL, bronchial washing, sputum
Container/Tube: Sterile container
Specimen Volume: 1 mL
Specimen Type: Tissue or bone
Container/Tube: Sterile container
Specimen Volume: 5-10 mm
Collection Instructions: Collect a fresh tissue or bone specimen.
Acceptable:
Specimen Type: NALC/NaOH-digested respiratory specimens
Sources: Lavage fluid, bronchial washing, gastric washing, respiratory fluid, sputum, or tracheal secretion
Container/Tube: Sterile container
Specimen Volume: 2 mL
Collection Instructions:
1. Submit digested specimen treated with NALC/NaOH.
2. Clearly indicate on container and order form that specimen is a digested specimen.
If not ordering electronically, complete, print, and send a Microbiology Test Request (T244) with the specimen.
Body Fluid or Respiratory Specimen: 0.5 mL
Other | Specimen in anaerobe vial or viral transport medium (including but not limited to M4, M5, BD viral transport media, thioglycolate broth) Feces Swab Tissue in formalin fluid Urine Specimen >7 days old |
Specimen Type | Temperature | Time | Special Container |
---|---|---|---|
Varies | Refrigerated (preferred) | 7 days | |
Ambient | 7 days | ||
Frozen | 7 days |
Rapid detection of Histoplasma capsulatum and Blastomyces dermatitidis DNA
An aid in the rapid diagnosis of histoplasmosis and blastomycosis
See Meningitis/Encephalitis Panel Algorithm in Special Instructions.
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
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 B dermatitidis/gilchristii and H capsulatum directly from clinical specimens.
Not applicable
A positive result for Histoplasma capsulatum indicates presence of Histoplasma DNA; a positive result for Blastomyces dermatitidis/gilchristii indicates presence of Blastomyces DNA.
A negative result indicates absence of detectable H capsulatum and B dermatitidis/gilchristii DNA. Fungal culture has increased sensitivity over this PCR assay and should always be performed when the PCR is negative.
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 H capsulatum or B dermatitidis/gilchristii because the organism may be present at levels below the limit of detection for this assay.
The sensitivity of the PCR assay from bronchoalveolar lavage (BAL) fluid is low and a negative result does not rule out infection.
Analytical Sensitivity and Specificity:
The analytical sensitivity of the assay was determined to be less than or equal to 100 copies/microliter for both Blastomyces dermatitidis/gilchristii and Histoplasma capsulatum. The B dermatitidis/gilchristii melt peak is read at 670 nm and has a melting temperature (Tm) of 66 degrees C + or - 0.26 degrees C (mean + or - standard deviation), while the H capsulatum read at 610 nm has a Tm of 61 degrees C + or - 0.27 degrees C. A National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) search of the primer, probe, and target sequences for both B dermatitidis/gilchristii and H 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 B dermatitidis/gilchristii and H capsulatum, respectively. The specificity of the assay was 100% for both organisms as no cross-reactivity was detected in the other non-B dermatitidis/gilchristii or non-H 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 B dermatitidis/gilchristii was 86% (12/14 positive) and 99.4% (778/783 negative), respectively. The overall sensitivity and specificity of the PCR assay for H capsulatum was 73.3% (11/15 positive) and 100% (782/782 negative), respectively. Of note, the recovery of H 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 H 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 B dermatitidis/gilchristii across all specimen types except blood (97% sensitive; 29/30 positive). The PCR assay had 100% (30/30) sensitivity for H capsulatum across all spiked specimen types except sputum and blood which had 98% and 97% sensitivity respectively.
Inhibition studies:
The overall extraction and amplification inhibition rate of the assay using both targets was less than 1% (2/330 inhibited). Extraction inhibition occurred in 1 of 30 blood specimens for B dermatitidis/gilchristii and H capsulatum, and 1 of 60 sputum specimens for H capsulatum.
1. Kauffman
2. Wheat LJ, Freifeld AG,
3. Chapman SW, Bradsher RW Jr, Campbell GD Jr, et al: Practice guidelines for the management of patients with blastomycosis. Infectious Diseases Society of
Following specimen processing, nucleic acids are extracted using the MagNA Pure Compact (Roche Applied Sciences). The extract is then transferred to a cuvette for amplification using the LightCycler real-time PCR platform (Roche Applied Sciences). The LightCycler is an automated instrument that amplifies and monitors the development of target nucleic acid (amplicon) after each cycle of PCR. The detection of amplicon is based of fluorescence resonance energy transfer (FRET), which utilizes hybridization probes. The presence of the specific organism nucleic acid is confirmed by performing a melting curve analysis of the amplicon. Primers and FRET hybridization probes were designed to target a 174-base pair (bp) region of the histidine kinase (DRK-1) gene of Blastomyces dermatitidis/gilchristii and a 192-bp region of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Histoplasma capsulatum, respectively. The acceptor probe for B dermatitidis/gilchristii was labeled with a Red-705 dye, while the acceptor probe for H capsulatum was labeled with a Red-640 dye. Labeling the acceptor probes with 2 different dyes allows for simultaneous detection and differentiation of B dermatitidis/gilchristii and H capsulatum within a single PCR assay.(Babady NE, Buckwalter SP, Hall L, et al: Detection of Blastomyces dermatitidis and Histoplasma capsulatum from Culture Isolates and Clinical Specimens by Use of Real-Time PCR. J Clin Microbiol 2011;49:3204-3208)
Monday through Friday
This test was developed, and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.
87798
Test Id | Test Order Name | Order LOINC Value |
---|---|---|
HBRP | Histoplasma/Blastomyces PCR | 81653-8 |
Result Id | Test Result Name |
Result LOINC Value
Applies only to results expressed in units of measure originally reported by the performing laboratory. These values do not apply to results that are converted to other units of measure.
|
---|---|---|
SRC78 | Histo/Blasto Source | 31208-2 |
32457 | Histo/Blasto Result | 81653-8 |