SLIPTA e-Tool improves laboratory audit process in Vietnam and Cambodia.

BACKGROUND: The Stepwise Laboratory Quality Improvement Process Towards Accreditation (SLIPTA) checklist is used worldwide to drive quality improvement in laboratories in developing countries and to assess the effectiveness of interventions such as the Strengthening Laboratory Management Toward Accreditation (SLMTA) programme. However, the paper-based format of the checklist makes administration cumbersome and limits timely analysis and communication of results. DEVELOPMENT OF E-TOOL: In early 2012, the SLMTA team in Vietnam developed an electronic SLIPTA checklist tool. The e-Tool was pilot tested in Vietnam in mid-2012 and revised. It was used during SLMTA implementation in Vietnam and Cambodia in 2012 and 2013 and further revised based on auditors' feedback about usability. OUTCOMES: The SLIPTA e-Tool enabled rapid turn-around of audit results, reduced workload and language barriers and facilitated analysis of national results. Benefits of the e-Tool will be magnified with in-country scale-up of laboratory quality improvement efforts and potential expansion to other countries.

Fate of sputum samples transported in bottles containing cetylpyridinium chloride and sodium chloride: a national reference laboratory study.

The transportation of sputum samples may sometimes take more than one week which results in an increased contamination rate and loss of positive cultures. The current study was planned to analyze the recovery rate of mycobacteria from transported samples with and without Cetylpyridinium chloride (CPC). Addition of CPC is useful for isolation of M. tuberculosis from sputum subjected to long-term storage.

Development and evaluation of a multiplex polymerase chain reaction for the detection of Mycobacterium tuberculosis from pulmonary specimens.

BACKGROUND: The diagnosis of pulmonary tuberculosis is still a major challenge. Using a polymerase chain reaction (PCR), one can detect Mycobacterium tuberculosis in clinical samples within a few hours. However, single gene targets may result in false negativity due to the absence of target DNA in some M. tuberculosis isolates. The objective of this study was to develop and evaluate a multiplex PCR (M-PCR) using IS6110 and devR primers for the detection of M. tuberculosis in sputum samples. METHODS: Sputum samples were collected from: (1) 200 confirmed cases of tuberculosis; (2) 100 suspected cases of tuberculosis diagnosed on the basis of clinical and radiological findings; (3) 200 non-tubercular patients suffering from respiratory diseases other than tuberculosis, in whom tuberculosis had been excluded. All 500 sputum samples were subjected to PCR using IS6110 primers, and M-PCR using IS6110 and devR primers; results were compared with conventional techniques. RESULTS: It was found that M-PCR was 97.5% successful in detecting the presence of tuberculosis in the confirmed tuberculosis group as compared to 84.5% by IS6110-based PCR. In the suspected tuberculosis group, M-PCR could detect 45% of cases as compared to 40% by IS6110-based PCR. Overall, the specificities of both the PCR and M-PCR were found to be 96.5%. CONCLUSIONS: This study demonstrated that the M-PCR assay is more sensitive than the IS6110-based PCR for the detection of M. tuberculosis in sputum specimens and could be applied in situations of highly suspected tuberculosis when all others tests including IS6110 PCR are negative.

Genetic polymorphisms among Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in northern India.

Restriction fragment length polymorphism (RFLP) based on IS6110 is considered the gold standard for Mycobacterium tuberculosis molecular typing. It is useful to discriminate among M. tuberculosis strains, investigate outbreaks and distinguish between reactivation and re-infection. We studied polymorphisms among M. tuberculosis isolates from northern India using RFLP to determine the presence of a correlation between IS6110 based fingerprints and drug resistance and to look for relapse and transmission among patients and their contacts. RFLP patterns of PvuII digested genomic DNA of 100 M. tuberculosis isolates were analyzed using southern blotting with a 245 bp IS6110 probe. Drug sensitivity testing (DST) was conducted for rifampicin (40 microg/ml), isoniazid (1 microg/ml), ethambutol (2 microg/ml) and streptomycin (4 microg/ml) using the proportion method. A high degree of polymorphism was seen among the M. tuberculosis isolates and the number of IS6110 copies varied from 0 to 14, with a predominance of isolates with 11 bands. Seventy-five isolates had a high number of bands, 9 had an intermediate number, 6 isolates had a low number and 10 isolates had no bands. No correlation between IS6110 band numbers and RFLP banding patterns was found with drug resistance or for any particular geographical area, although clustering was seen amongst MDR-TB cases. No cases of relapses or transmissions were seen.

Evaluation of direct method of drug susceptibility testing of Mycobacterium tuberculosis to rifampicin and isoniazid by nitrate reductase assay in a national reference laboratory.

The aim of this study was to evaluate a simple, rapid, and inexpensive colorimetric nitrate reductase assay (NRA) for direct drug susceptibility testing (DST) of Mycobacterium tuberculosis against rifampicin (RIF) and isoniazid (INH). A total of 118 smear-positive specimens were processed from patients on antituberculosis treatment. A comparison was made between the direct NRA of DST with the direct proportion method and with the internationally accepted indirect 1% proportion method as the "gold standard". The sensitivity and specificity of the direct NRA and indirect proportion method were 94% and 98%, and 100% and 98% for RIF and INH, respectively. Excellent agreement was found between the 2 tests with kappa values of 0.92 and 0.98, and P value was less than 0.001 for RIF and INH. The results in most cases were available in 14 days (turnaround time). The direct NRA is a rapid, accurate, simple, and inexpensive method to determine multidrug resistance from sputum. Direct NRA may become an appropriate alternative method, especially for the resource poor settings.

Susceptibility testing of Mycobacterium tuberculosis by broth microdilution method: a rapid alternative method.

A total of 50 clinical isolates of Mycobacterium tuberculosis were tested by the proportion method and the broth microdilution method (BMM) for primary antitubercular drugs. The results were obtained after 14 days of incubation. There was a 100% agreement for all drugs except streptomycin, which showed a 96% agreement. Thus, the BMM was found to be a rapid, reliable, and less labor-intensive method to determine MIC.