Can inhA mutation predict ethionamide resistance?

SETTING: A tertiary care centre in Mumbai, India. OBJECTIVE: To estimate the extent of association between inhA mutant isoniazid (INH) resistant strains and ethionamide (ETH) resistance. DESIGN: A total of 140 clinical isolates processed for INH and ETH phenotypic drug susceptibility testing, and molecularly processed with the line-probe assay (LPA) Genotype® MTBDRplus, were considered. RESULTS: Among the 112 phenotypically determined INH-resistant strains, 69 (61.6%) strains were ETH-resistant. An inhA promoter mutation was identified in 24 (21.4%) INH-resistant isolates, 21 (87.5%) of which were ETH-resistant (P < 0.0001). CONCLUSION: An inhA promoter mutation could be considered as a marker for the determination of ETH resistance in India, where the use of LPA is being expanded.

Using likelihood ratios to estimate diagnostic accuracy of a novel multiplex nested PCR in extra-pulmonary tuberculosis.

SETTING: A tertiary care centre in Mumbai with a referral bias towards treatment failures. OBJECTIVE: To standardise and evaluate a novel single tube multiplex nested polymerase chain reaction (PCR) targeting insertion sequence (IS) 6110, mpb64, rrs and rpoB genes for rapid diagnosis of extra-pulmonary tuberculosis (EPTB). METHODS: The PCR assay was evaluated among 489 consecutive consenting patients, and results were compared against a composite reference standard comprising smear microscopy, culture, clinical symptoms, radiological scan and histology. RESULTS: PCR assay reported a pooled sensitivity of 94.5% (242/256, 95%CI 91-97): 91.9% (125/136, 95%CI 86-96) for smear-negative composite reference standard (CRS) positive cases and 97.5% (117/120, 95%CI 93-99) for smear-positive CRS-positive cases. The PCR positivity rate increased from 91.7% (235/256, 95%CI 88-95) when presence of IS6110 was considered alone for reporting a test as positive to 94.5% (242/256, 95%CI 91-97) when used in combination with other three gene targets, with a specificity of 96.4% (212/220, 95%CI 93-98). A positive likelihood ratio of 26 (95%CI 13-51) and a negative likelihood ratio of 0.06 (95%CI 0.03-0.09) makes the test useful for ruling out and ruling in the disease. CONCLUSION: Culture should not be replaced by PCR as a gold standard; however, PCR can be used as a rapid, accurate tool in the diagnosis of EPTB.

Evaluation of light emitting diode-based fluorescence microscopy for the detection of mycobacteria in a tuberculosis-endemic region.

OBJECTIVES: To evaluate fluorescence microscopy (FM) using light emitting diode (LED) technology for the detection of acid-fast bacilli at a tertiary referral centre in Mumbai, India, a tuberculosis-endemic country. DESIGN: LED FM was introduced into a laboratory experienced with Ziehl-Neelsen (ZN) microscopy but unfamiliar with FM. It was evaluated in parallel with routine ZN microscopy services and compared with mycobacterial culture as a reference standard. RESULTS: A total of 1357 pulmonary and 917 extra-pulmonary specimens were examined during the study. LED FM had 78.3% sensitivity and 92.0% specificity against mycobacterial culture when using pulmonary specimens, and 34.0% sensitivity and 88.8% specificity for extra-pulmonary specimens. The mean time per smear examination was 2.48 min for ZN vs. 1.41 min for LED FM. Several biases in study design and operation identified during analysis, which are likely to lead to underestimates of LED FM accuracy, are discussed in the context of future LED FM evaluations. CONCLUSIONS: Although LED FM has significant benefits over both ZN microscopy and conventional FM, its implementation and validation may be prone to difficulties which could hamper evaluation of its performance. Adequate training and detailed standard operating procedures are important to maximise accuracy.