Introduction

Tuberculosis (TB) presents as a serious public health problem, globally with an estimated 10.4 million new TB cases in 2015 [1]. It is a leading cause of deaths from infectious diseases (with worldwide estimated 1.8 million deaths in 2015), large numbers of deaths mainly occurring in low and middle income countries [1]. In 2015, 480,000 new MDR-TB cases were estimated to occur worldwide [1].

Worldwide, large percentage of cases of MDR-TB remain undiagnosed. Further, there is an increasing trend of prevalence of MDR-TB in Nepal. According to the recent drug resistance surveillance in Nepal, the prevalence of MDR-TB was 2.6% in case of newly diagnosed tuberculosis cases and17.6% among previously treated cases [2].

The conventional Mycobacterium tuberculosis (MTB) drug susceptibility testing is a gold standard technique but requires long period of time (8 weeks) to give definitive report [3]. Further, it requires more sophisticated and higher bio-safety level laboratory along with the well trained staffs [3]. Therefore, the diagnosis of MDR-TB by using the conventional drug susceptibility testing is not possible in resource limited countries like Nepal [3]. In addition, due to the very slow turnaround time of conventional technique the chances for early diagnosis of MDR-TB and proper patient management is very low even in the settings where there is availability of resources for performing the gold standard technique [3]. To address these issues, Xpert MTB/RIF assay was developed and it was endorsed by world health organization (WHO) in December 2010 as an accurate, feasible, rapid, affordable, and near-point-of-care TB diagnostic test for use in resource-limited settings [4]. Many studies on DNA sequencing have reported that more than 95% of rifampicin (RIF) resistant MTB isolates have mutation within the 81-bp hot spot region of the rpoB gene [5, 6, 7]. The Xpert assay works by detecting MTB and RIF resistance by polymerase chain reaction (PCR) based amplification of the 81-bp rpoB gene segment and probing for the mutations that are related to RIF resistance. The assay is automated and completes within 2 hrs [8, 9]. After the redesign of probe B in December 2011 [10], studies have assessed the performance of Xpert in detection of MTB and MDR-TB. We also aimed to evaluate the diagnostic accuracy of Xpert MTB/RIF assay in comparison to conventional DST for diagnosis of MDR-TB in our setting.

Materials and Methods

Results

Summary of anti-tubercular drug susceptibility test

Out of 88 culture positive clinical specimens, 55 were from male, 83 were from pulmonary TB cases and 37 were from newly diagnosed TB cases. The age of culture positive TB patients ranged from 13–82 years with the highest percentage in productive age group i.e. 15–45 years (71.6%) (p<0.05). Out of 87 Mycobacterium spp. isolated from culture positive clinical specimens (excluding the invalid sample), only 16% (n = 14) were sensitive to all 4 drugs (isoniazid, rifampicin, streptomycin, ethambutol) tested and 64.4% (n = 56) were found to be resistant to all 4 drugs. Almost, 82.8% (72/87) of the Mycobacterium spp. isolated were found to be resistant to isoniazid followed by rifampicin (81.6%) (Table 1).

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Table 1. Summary of anti-tubercular drug susceptibility test.

https://doi.org/10.1371/journal.pone.0169798.t001

Performance of GeneXpert as compared to gold standard conventional drug susceptibility test

Diagnostic efficacy of GeneXpert was assessed in 85 MTB culture positive cases with the overall sensitivity, specificity, PPV and NPV at 95% confidence interval of 98.6% (92.3–99.8), 100% (78–100), 100% (94.7–100) and 93.8% (69.7–98.9) respectively. Only 1/85 and 2/85 cases reported as RIF sensitive by GeneXpert assay were found to be resistant to RIF and isoniazid (INH) respectively by conventional DST. Statistically, there was no significant difference between Xpert MTB/RIF assay and conventional DST method for detection of MDR-TB, (p>0.05) (Table 2).

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Table 2. Performance of GeneXpert as compared to gold standard conventional drug susceptibility test.

https://doi.org/10.1371/journal.pone.0169798.t002

Discussion

In a Cochrane review [12], Xpert MTB/RIF pooled sensitivity for rifampicin resistance detection was 95% (95% CI, 90% to 97%; 17 studies) which was found to be lower than in our study i.e. 98.6% (95% CI, 92.3–99.8) and pooled specificity was 98% (95% CI, 97% to 99%; 24 studies) which was again lower than our study i.e. 100% (95% CI, 78–100). One case found to be positive for resistance to RIF phenotypically (detected by conventional DST method), was found to be negative for resistance to RIF genotypically by Xpert. The reason for this discrepancy might be due to mutation in other sites rather than hot spots, which needed to be solved by further sequencing the rpoB gene. Further, in our study no false positive cases were detected. This is the strength in favor of using Xpert for the detection of RIF resistance.

Furthermore, Xpert has very short turnaround time (<2 hrs) with low biohazard risk [13]. Rifampicin resistance can be detected in less than one day with Xpert MTB/RIF assay while it takes an average of 75 days for diagnosis of MDR-TB with the help of conventional DST [14]. And of much concern about in this study is 86.5% (32/37) of the new and 79.2% (38/48) of the retreatment TB patients had MDR-TB, which indicates the high level of resistance even when the patients were first infected and this high rate of MDR-TB in new cases might be due to sample selection bias, sample being taken from the TB reference center where culture positive cases from various geographical regions are referred. Further, the other reason for this may be due to patients being reluctant in providing information about their previous exposure and treatment failure.

Conclusions

The sensitivity and specificity of GeneXpert MTB/RIF assay for the diagnosis of MDR-TB was found to be comparable to gold standard, conventional DST. Further, due to short turnaround time, it is highly significant to use GeneXpert MTB/RIF assay for diagnosis of MDR-TB in country with high prevalence of MDR-TB. It will be helpful to TB control program by helping in early case finding in country like Nepal, where the prevalence of MDR-TB is increasing.

Acknowledgments

We kindly acknowledge German Nepal Tuberculosis Project (GENETUP)- National Reference Laboratory for providing the proper platform and resources for the study and Nepal Health Research Council for financial support for the study. The authors would also like to thank all the patients and technical staffs for their help during the study.