The evaluation of the diagnostic utility and sensitivity of the Xpert® MTB/RIF in the detection of Mycobacterium tuberculosis and rifampicin resistance on bone marrow aspirate samples
Date
2017
Authors
Subramony, Nishanti Nadhiya
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Abstract
In South Africa, the World Health Organisation estimated 454 000 new cases of Mycobacterium Tuberculosis (M.tb) infection (MTB) in 2015. Disseminated tuberculosis arises from haematogenous spread of the bacilli and seeding of the bacilli in extrapulmonary sites. The current gold standard for the detection of MTB of the bone marrow is TB culture which has an average turnaround time of 6 weeks. Although shorter, histological examinations of trephine biopsy cores to diagnose MTB also have a time delay owing mainly to the 5-7 day processing period prior to microscopic examination. Adding to the diagnostic delay is the non-specific nature of granulomatous inflammation which is the hallmark of MTB involvement of the bone marrow. A Ziehl-Neelson stain (which highlights acid-fast bacilli) is therefore mandatory to confirm the diagnosis but can take up to 3 days for processing and evaluation. Owing to this delay in diagnosis, many patients are lost to follow up or remain untreated for up to six weeks while the results are awaited, thus encouraging the spread of undiagnosed TB.
The Xpert MTB/RIF (Cepheid, Sunnyvale, CA) is the molecular test used in the South African national TB program as the initial diagnostic test for pulmonary TB in adults and children. In 2013 the Xpert MTB/RIF was applied to diagnose extrapulmonary TB and despite being available in 207 testing sites nationwide, it was never investigated for its potential application in diagnosing TB in bone marrow. Therefore, this study investigates the optimisation and performance of Xpert MTB/RIF on bone marrow aspirate specimens.
BMA specimens received for routine immunophenotypic analysis as part of the investigation into disseminated MTB or in the evaluation of cytopenias in immunocompromised patients were used in this study. Processing BMA on the Xpert® MTB/RIF was optimised to ensure bone marrow in EDTA and heparin did not inhibit the PCR reaction. Inactivated M.tb from an Xpert MTB/RIF external quality assessment program was spiked into the clinical bone marrow specimen and distilled water (as a control). A volume of 500μl and an incubation time of 15 minutes with sample reagent were investigated as the processing protocol.
A total of 135 BMA specimens had sufficient residual volume for Xpert® MTB/RIF testing however 22 specimens (16.3%) were not included in the final statistical analysis as an adequate trephine biopsy and/or TB culture was not available. Xpert MTB/RIF testing was possible in
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the presence of heparin or EDTA, but the overall detection of MTB in BMA was low compared to histology and culture.
The sensitivity of the Xpert® MTB/RIF when compared to both histological and culture findings was 8.7% with a 95% confidence interval (CI) of 1.07-28.04%. The sensitivity of the Xpert® MTB/RIF compared to histological findings only was 11.1% with a 95% CI of 1.38-34.7%. The specificity of the Xpert® MTB/RIF was 98.9% (95% CI: 93.9-99.7%).
Although the Xpert® MTB/RIF has a shorter turnaround time than histology and TB culture and is less expensive than culture and drug susceptibility testing, the low sensitivity of the Xpert® MTB/RIF in this study limits its current use for the diagnosis of MTB in bone marrow aspirate specimens until the diagnostic algorithm or the assay is further defined.
Description
A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Medicine in the Branch of Pathology (Haematology). Johannesburg, 20 November 2017.
Keywords
Xpert® MTB/RIF