Histoplasma capsulatum/Blastomyces dermatitidis, Molecular Detection, PCR, Blood
NY State Approved Indicates the status of NY State approval and if the test is orderable for NY State clients.
Rapid detection of Histoplasma capsulatum and Blastomyces dermatitidis DNA
Real-Time Polymerase Chain Reaction (PCR)
(PCR is utilized pursuant to a license agreement with Roche Molecular Systems, Inc.)
Reporting Name A shorter/abbreviated version of the Published Name for a test; an abbreviated test name
Histoplasma/Blastomyces PCR, B
Specimen Type Describes the specimen type needed for testing
Specimen Required Defines the optimal specimen. This field describes the type of specimen required to perform the test and the preferred volume to complete testing. The volume allows automated processing, fastest throughput and, when indicated, repeat or reflex testing.
Container/Tube: Lavender top (EDTA)
Specimen Volume: 5 mL
Pediatric: 3 mL
Collection Instructions: 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.
Forms: If not ordering electronically, submit a Microbiology Request Form (Supply T244) with the specimen.
Specimen Minimum Volume Defines the amount of specimen required to perform an assay once, including instrument and container dead space. Submitting the minimum specimen volume makes it impossible to repeat the test or perform confirmatory or perform reflex testing. In some situations, a minimum specimen volume may result in a QNS (quantity not sufficient) result, requiring a second specimen to be collected.
Specimen Stability Information Provides a description of the temperatures required to transport a specimen to the laboratory. Alternate acceptable temperature(s) are also included.
|Whole blood||Refrigerated||7 days|
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
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.
Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
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.
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
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.
Clinical Reference Provides recommendations for further in-depth reading of a clinical nature
1. Kauffman CA: Histoplasmosis. Clin Chest Med 2009;30:217-225
2. Wheat LJ, Freifeld AG, Kleiman MB, et al: Clinical practice guidelines for the management of patients with histoplasmosis: 2007 update by the Infectious Diseases Society of America. Clin Infect Dis 2007;45:807-825
3. Chapman SW, Bradsher RW Jr, Campbell GD Jr, et al: Practice guidelines for the management of patients with blastomycosis. Infectious Diseases Society of America. Clin Infect Dis 2000;30:679-683
Method Description Describes how the test is performed and provides a method-specific reference
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 and a 192-bp region of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Histoplasma capsulatum, respectively. The acceptor probe for Blastomyces dermatitidis was labeled with a Red-705 dye, while the acceptor probe for Histoplasma capsulatum was labeled with a Red-640 dye. Labeling the acceptor probes with 2 different dyes allows for simultaneous detection and differentiation of Blastomyces dermatitidis and Histoplasma capsulatum within a single PCR assay.(Unpublished Mayo method)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday through Friday, 3 times per week
Analytic Time Defines the amount of time it takes the laboratory to setup and perform the test. This is defined in number of days. The shortest interval of time expressed is "same day/1 day," which means the results may be available the same day that the sample is received in the testing laboratory. One day means results are available 1 day after the sample is received in the laboratory.
Maximum Laboratory Time Defines the maximum time from specimen receipt at Mayo Medical Laboratories until the release of the test result
Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded
Performing Laboratory Location The location of the laboratory that performs the test
Test Classification Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer's instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR), Investigation Use Only (IUO) product, or a Research Use Only (RUO) product.
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 U.S. Food and Drug Administration.
CPT Code Information Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Medical Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.
87798 x 2
LOINC® Code Information Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the result codes returned for this test or profile.
|Result ID||Reporting Name||LOINC Code|
|32462||Histo/Blasto Result||In Process|