RESUMO
Objective/background: guidelines for the manipulation of Mycobacterium tuberculosis [MTB] cultures require a Biosafety Level 3 [BSL-3] infrastructure and accompanying code of conduct. In this study, we aimed to validate and apply detection methods for viable mycobacteria from surfaces in a BSL-3 MTB laboratory
Methods: we evaluated phenotypic [Replicate Organism Detection and Counting [RODAC] plates] and molecular [propidium monoazide [PMA]-based polymerase chain reaction [PCR]] approaches for the detection of viable mycobacteria, as well as the effect of 70% ethanol applied for 5 min for disinfection against mycobacteria. For validation of the method, recovery of serial dilutions of Mycobacterium bovis bacillus Calmette-Guerin from glass slides was measured. Subsequently, we stamped surfaces in and around the biosafety cabinet [BSC] after different technicians had manipulated high bacterial load suspensions for routine drug-susceptibility testing in a Class II BSC
Results: RODAC stamping could detect as few as three bacteria on slides stamped either 5 min or 60 min after inoculation. PMA-based PCR, tested in parallel, did not pass validation. Mycobacteria were still detected after 5-min disinfection with ethanol 70%. In the BSL-3, from 201 RODAC-stamped surfaces, MTB was detected in four: three inside a BSC- on a tube cap and on an operator's gloves-and one outside, on an operator's gown
Conclusion: RODAC plates detect mycobacteria at low numbers of microorganisms. In addition, this method allowed us to show that 70% ethanol does not reliably kill mycobacteria when applied for 5 min to a dried surface, and that MTB bacilli may arrive outside a Class II BSC during routine practice, although the route could not be documented
RESUMO
Objective/background: the GeneXpert MTB/RIF assay [Xpert] was endorsed as the initial diagnostic tool in people suspected of human immunodeficiency virus-associated or drug-resistant tuberculosis [TB]. However, information regarding the performance of Xpert for diagnosing smear-negative TB in high burden settings remains limited. We evaluated the diagnostic accuracy of Xpert and the impact of bleach concentration on the performance of Xpert using smear-negative sputum samples from human immunodeficiency virus-negative patients
Methods: one spot and one morning smear-negative sputum samples per patient were examined using Xpert and culture at the Mycobacteriology Research Center of Jimma University, Ethiopia. The sputum culture on both Lowenstein-Jensen and/or Mycobacteria Growth Indicator Tube was the gold-standard
Results: of 185 smear-negative presumptive pulmonary TB cases, 19 [10.3%] had cultureproven TB. The sensitivity of Xpert on spot and morning sputum was similar [63.2%]. Testing two specimens per patient insignificantly increased the sensitivity of Xpert. Bleach concentration and pelleting improved the sensitivity of Xpert over unprocessed sputum in paired samples [73.8% vs. 63.2%] without affecting the specificity [95%]. Bleach concentration and pelleting allowed an additional seven cases of TB [missed on the first and second direct Xperts] to be detected, five of which were from culture-negative cases
Conclusion: testing of a single sputum sample by Xpert can reach reasonable sensitivity and results would be available on the same day, avoiding loss of patients and treatment delay. The sensitivity of Xpert was improved after bleach concentration and pelleting, although its added value needs further study on a larger scale