Pneumocystis jiroveci, Molecular Detection, PCR
NY State Approved Indicates the status of NY State approval and if the test is orderable for NY State clients.
Preferred test for detection of Pneumocystis
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
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.
The high sensitivity of amplification by PCR requires the specimen to be processed in an environment in which contamination of the specimen by Pneumocystis species DNA is not likely.
Forms: If not ordering electronically, submit a Microbiology Request Form (Supply T244) with the specimen.
Preferred Specimens: Cerebrospinal fluid (CSF), respiratory specimens (eg, bronchoalveolar lavage fluid, bronchial washing, sputum), pleural fluid, respiratory tissue (fresh)
Acceptable Specimens: If no fresh specimen is available, digested respiratory specimens treated with N-acetyl-L-cysteine (NALC)/NaOH are acceptable (eg, bronchoalveolar lavage [BAL] fluid, bronchial washing, respiratory fluid, sputum, or tracheal secretion), as are NALC/NaOH-treated gastric washes.
Unacceptable Specimens: Body fluid other than CSF or pleural fluid, blood, bone, nonrespiratory tissue, bone marrow, organ tissues other than lung, paraffin-embedded tissue, specimen in anaerobe vial or viral transport medium (including but not limited to M4, M5, BD viral transport media, thioglycolate broth), stool, swab, tissue in formalin fluid, urine, specimen >7 days old
Specimen must arrive within 7 days of collection; specimen >7 days will be rejected.
Specimen source is required.
Submit only 1 of the following specimens:
Specimen Type: Body fluid
Sources: CSF or pleural
Container/Tube: Sterile container
Specimen Volume: 1 mL
Specimen Type: Respiratory
Sources: BAL, bronchial washing, or sputum
Container/Tube: Sterile container
Specimen Volume: 1 mL
Specimen Type: Tissue
Container/Tube: Sterile container
Specimen Volume: 5-10 mm
Collection Instructions: Submit fresh tissue.
NALC/NaOH-digested specimen must arrive within 7 days of digestion.
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
1. Submit digested specimen treated with NALC/NaOH.
2. Clearly indicate on container and order form that specimen is a digested specimen.
Specimen Stability Information: Refrigerated (preferred) 7 days/Ambient 7 days/Frozen 7 days
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.
Body fluid other than CSF or pleural fluid, blood, bone, non-respiratory tissue, bone marrow, organ tissues other than lung, paraffin-embedded tissue, specimen in anaerobe vial or viral transport medium, stool, swab, tissue in formalin fluid, urine, specimen >7 days old
Specimen Stability Information Provides a description of the temperatures required to transport a specimen to the laboratory. Alternate acceptable temperature(s) are also included.
|Varies||Refrigerated (preferred)||7 days|
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Pneumocystis pneumonia is an important cause of opportunistic infection in immunocompromised patients, particularly those with HIV. The causative agent, Pneumocystis jiroveci, cannot be cultured in vitro and, therefore, laboratory detection has historically relied upon microscopic identification directly from patient specimens using fluorescent stains or antibodies. Unfortunately, stains often lack sensitivity and require expertise on the part of the reader in order to differentiate Pneumocystis jiroveci from staining artifacts and other fungi. This real-time PCR assay provides sensitive (21% more sensitive than direct detection using fluorescent calcofluor white stain), specific, and objective detection of Pneumocystis from bronchoalveolar lavage fluid and other 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 indicates the presence of Pneumocystis DNA.
A negative result indicates the absence of detectable Pneumocystis DNA.
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
Test results should be used as an aid in diagnosis and should not be considered diagnostic in themselves. The literature indicates that Pneumocystis can cause asymptomatic colonization of healthy and immunocompromised individuals. Therefore, test results should be correlated with patient symptoms and clinical presentation.
A negative result does not rule out the presence of Pneumocystis or active disease because the organism may be present at undetectable levels.
A total of 221 bronchoalveolar lavage (BAL) fluid samples were evaluated for the presence of Pneumocystis DNA by the LightCycler and compared to fluorescent microscopy using calcofluor white staining. Of the 221, 24 were positive and 190 were negative by both detection methods. The remaining 7 were positive by PCR and negative by microscopy. The 7 specimens that were positive using LightCycler PCR alone were tested using another PCR assay targeting a second Pneumocystis gene. All 7 specimens were positive using the second target suggesting that they were true positives that were undetected using the microscopic method. The sensitivity, specificity, positive and negative predictive values of this real-time PCR assay is 100%, 96%, 77%, and 100%, respectively. The analytical sensitivity of the method is 5.6 copies/mcL of positive plasmid control or approximately 28 copies/reaction. The analytical sensitivity in spiked, pooled BAL specimens was found to be 56 targets/mcL using the positive control plasmid. PCR inhibition was tested by spiking 50 extracted negative respiratory specimens (including 10 BAL fluid specimens) with 100 copies of target/mcL using a positive control plasmid. No PCR inhibition was detected. The specificity of the PCR assay was determined by evaluating DNA extracted from pure cultures of a variety of bacteria and fungi. Extracted human DNA was analyzed as well. None of the microbial or human DNA was amplified by the Pneumocystis LightCycler assay indicating that the assay is specific for Pneumocystis species.
Clinical Reference Provides recommendations for further in-depth reading of a clinical nature
1. Cushion MT: Pneumocystis. In Manual of Clinical Microbiology. Eighth edition. Edited by PR Murray, EJ Baron, JH Jorgensen, et al: Washington, DC, ASM Press, 2003, pp 1712-1725
2. Maskell NA, Waine DJ, Lindley A, et al: Asymptomatic carriage of Pneumocystis jiroveci in subjects undergoing bronchoscopy: a prospective study. Thorax 2003;58(7):594-597
3. Miller RF, Ambrose HE, Wakefield AE: Pneumocystis carinii f. sp. hominis DNA in immunocompetent health care workers in contact with patients with P. carinii pneumonia. J Clin Microbiol 2001;39(11):3877-3882
4. Takahashi T, Goto M, Endo T, et al: Pneumocystis carinii carriage in immunocompromised patients with and without human immunodeficiency virus infection. J Med Microbiol 2002;51(7):611-614
5. Vargas SL, Hughes WT, Santolaya ME, et al: Search for primary infection by Pneumocystis carinii in a cohort of normal, healthy infants. Clin Infect Dis 2001;32(6):855-861
6. Wakefield AE, Lindley AR, Ambrose HE, et al: Limited asymptomatic carriage of Pneumocystis jiroveci in human immunodeficiency virus-infected patients. J Infect Dis 2003;187(6):901-908
Method Description Describes how the test is performed and provides a method-specific reference
Bronchoalveolar lavage fluid and sputum are liquified using N-acetyl-L-cysteine. Following liquification, cells are pelleted by centrifugation and resuspended in S.T.A.R. buffer (Roche). Nucleic acids are extracted using the MagNA Pure LC Instrument (Roche). The extract is then transferred to individual self-contained cuvettes for amplification using the LightCycler real-time PCR platform (Roche). 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 on 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.(Arcenas RC, Uhl JR, Buckwalter SP, et al: A real-time PCR assay for detection of Pneumocystis from bronchoalveolar lavage fluid. Diagn Microbiol Infect Dis 2006;54:169-175)
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 Sunday
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
3 to 14 days
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.
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|