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Open Access Research article

The manual mycobacteria growth indicator tube and the nitrate reductase assay for the rapid detection of rifampicin resistance of M. Tuberculosis in low resource settings

Chamila P Adikaram1*, Jennifer Perera1 and Sandya S Wijesundera2

Author Affiliations

1 Department of Microbiology, Faculty of Medicine, University of Colombo, Kynsey Road, Colombo, 08, Sri Lanka

2 Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Colombo, Kynsey Road, Colombo, 08, Sri Lanka

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BMC Infectious Diseases 2012, 12:326  doi:10.1186/1471-2334-12-326

Published: 27 November 2012

Abstract

Background

Tuberculosis (TB) is a disease of poverty that contributes significantly to ill-health in developing countries. Drug resistant TB is a major challenge to disease control. Early diagnosis and rapid determination of drug sensitivity is of paramount importance in eradication of TB. Although automated liquid culture based methods are available for rapid detection of drug resistance, the high cost of these tests prevent them from being used routinely in low resource settings. This study compares two phenotypic methods, the manual Mycobacteria Growth Indicator Tube (MGIT) and the Nitrate Reductase Assay (NRA) in liquid medium, with the agar proportion method (APM), the gold standard for susceptibility testing of Mycobacterium tuberculosis.

Methodology

Fourteen day old M. tuberculosis strains (n=373) grown on solid media were used for drug susceptibility testing by APM, NRA and the manual MGIT method. Rifampicin free and rifampicin incorporated (final concentration, 1 μg/ml) media were inoculated with the recommended concentrations of mycobacterial suspensions and incubated at 37°C in 5% CO2. In the APM, the proportion of colonies in the drug containing medium was determined. In the NRA, the colour change in the medium was compared with a standard colour series after day 6 and day 12 of incubation. Growth in the MGIT was detected using the manual MGIT reader from day 2 onwards. The 2 methods were compared with the gold standard, APM to determine sensitivity and specificity and agreement between the methods was calculated using kappa statistics.

Results

Thirty one (31) rifampicin resistant isolates were identified. When compared with the APM, the sensitivity of detection of rifampicin resistance was 85% for the NRA and 93% for the manual MGIT and the specificity was 99% and 100% respectively. Both assays, NRA (κ=0.86) and manual MGIT method (κ= 0.94) were in excellent agreement with the APM. The mean turnaround time for manual MGIT method and NRA were 08 days and 10 days respectively.

Conclusion

The NRA in liquid medium and manual MGIT are useful alternatives to APM for drug susceptibility testing of M. tuberculosis in low resource settings.

Keywords:
Drug resistant tuberculosis; Determination of drug sensitivity; Manual MGIT; NRA in liquid medium; Agar proportion method