Elsevier

Journal of Infection

Volume 77, Issue 4, October 2018, Pages 328-334
Journal of Infection

Multi-center evaluation of GenoType MTBDRsl line probe assay for rapid detection of pre-XDR and XDR Mycobacterium tuberculosis in China

https://doi.org/10.1016/j.jinf.2018.06.014Get rights and content

Highlights

  • Compared to phenotypic DST, the second version of MTBDRsl assay proven its satisfactory performance in detecting resistance to fluoroquinolone and 2nd-line injectable agents both in TB high and low burden settings of China.

  • The molecular diagnosis assay significantly reduced the turnaround time between sample collection and result reporting.

  • Specific genetic mutations identified by molecular diagnosis assay, were strongly associated with different levels of resistance to second-line drugs.

  • The assay can be used routinely in high MDR-TB burden settings to shorten the drug susceptibilities testing and guide the treatment.

Summary

Objectives

The implementation of rapid and reliable drug susceptibilities diagnosis is fundamental for effective treatment of multidrug-resistant tuberculosis(MDR-TB). The present study aimed to assess the diagnostic performance of the 2nd-version GenoType MTBDRsl kit as well as the impact of its implementation on the turnaround time in a multi-center Chinese study.

Methods

Totally 353 MDR-TB patient specimens were consecutively tested. The 2nd-version GenoType MTBDRsl assay, drug susceptibility testing with the MGIT 960 system, and sequencing were performed and compared.

Results

MTBDRsl testing identified the major genotypes associated with fluoroquinolones resistance, predominated by gyrA MUT3B (Asp94Asn and Asp94Tyr, 26.5%) and MUT3C (Asp94Gly, 19.5%). The genotypes associated with resistance to 2nd-line injectable drugs(SLIDs) were rrsMUT1(A1401G, 64.9%) and absence of WT1(C1402T, 10.5%). The sensitivities for detection of resistance to fluoroquinolones, SLIDs, and their combination (extensively drug resistance, XDR) were 80.5%, 80.7% and 73.5% and specificities were 100.0%, 99.3% and 99.1%, respectively. Implementation of this test significantly reduced the turnaround time between sample collection and result reporting from 45 to 3 days, a reduction by 93.3% (p, 0.001).

Conclusion

With a favorable diagnostic performance and short turnaround time, the 2nd-version GenoType MTBDRsl assay proves its value for early diagnosis of resistance to 2nd-line drugs as well as of XDR-TB in China.

Introduction

Drug-resistant tuberculosis (TB) remains to be an important public health concern worldwide.1 The efficacy of standard chemotherapy is undermined by the emergence of multidrug-resistant (MDR) TB (i.e. simultaneous resistance to at least isoniazid and rifampicin) and treatment has become even more troublesome due to the emergence of extensively drug-resistant (XDR) tuberculosis (i.e. MDR-TB with additional resistance to fluoroquinolones (FQs) and at least one of the 2nd-line injectable drugs (SLIDs)).2, 3 According to the latest global tuberculosis report published by the World Health Organization (WHO), there were 490, 000 cases of MDR-TB worldwide in 2016, and 6.2% of them were XDR-TB.1 But the actual global incidence of XDR-TB could be greatly underestimated, since in many high burden countries, second-line drug susceptibility testing (DST) is not performed.

The WHO has developed guidelines for phenotypic DST of first-/second-line anti-tuberculosis drugs on Löwenstein–Jensen medium or Middlebrook agar using the proportion method.4 However, solid culture-based DST of M. tuberculosis (Mtb) is demanding, expensive, and time-consuming. After primary isolation, the test results take at least 4 to 6 weeks to become available.5, 6 This delay could lead to continued transmission of MDR-TB in the community, amplified drug resistance and a less good treatment outcome.7 Therefore, there is a major interest in implementing a cost-effective, reliable, and rapid molecular method for detection of drug resistant TB. Rapid detection of MDR-TB, pre-XDR and XDR-TB is crucially important for early referral of patients to specialist centers, to ensure that effective treatment can be initiated in a timely way and under proper infection control measures.8

Currently, direct identification of the drug resistance related gene mutations is the only rapid method for predicting drug resistance in Mtb. The GenoType MTBDRsl assay (Hain Lifesciences, Germany) was developed for fast identification of Mtb resistance against 2nd-line drugs.9 The 1st version of the GenoType MTBDRsl test had been extensively evaluated in different settings albeit with limited sample sizes, and the performance was unsatisfactory due to limitations in the detected genes and mutations.10 The 2nd version of the test has been improved by including two new target regions: the gyrB and eis promoter region which confer resistance to FQs and SLIDs, while the target region for Ethambutol has been removed due to its low sensitivity and limited use in MDR-TB treatment.9 Although the 2nd version of the MTBDRsl assay has been recommend by WHO, its evaluation has so far been limited in areas with a high TB burden like China.11, 12 Therefore, more data are required in order to better understand the clinical applicability of this assay in various high burden settings.

The MDR-TB and XDR-TB rates have been on a stable increase in China.1 The availability of fast, accurate and reliable methods to detect the 2nd line drug resistance is important for optimizing the treatment.13 Here we describe a multicenter study across four sites with different social-demographic conditions and TB burden aimed at evaluating the performance of Genotype MTBDRsl assay version 2.0. We investigated its diagnostic accuracy of detecting resistance to commonly used FQs (Ofloxacin, Levofloxacin and Moxifloxacin) and SLIDs (Kanamycin, Amikacin and Capreomycin) with phenotypic DST as a reference. We also studied the drug resistance-associated genes and loci in comparison to direct Sanger sequencing and evaluated the implementation of this assay in terms of turnaround time and agreement.

Section snippets

Study setting

The Jiangsu, Yunnan, Liaoning, and Sichuan provinces are located respectively in eastern, southern, northern, and mid-western China. The population in Jiangsu, Yunnan, Liaoning, and Sichuan provinces were 80.0 million, 46.0 million, 43.8 million, and 82.6 million respectively, and the annual per capital gross domestic product (GDP) ranged from 32,326 China Yuan (CNY) (≈ 5,077 US dollar) in Yunnan province to 95,259 CNY (≈ 14,960 US dollar) in Jiangsu province. The estimated TB incidence rate in

MTBDRsl LPA Procedures

A 500 µl portion of the decontaminated sample (the remaining portion was used for culturing) was used to perform the 2nd version of the MTBDRsl assay according to manufacturer's instructions.9 A portion of extracted DNA was kept refrigerated (+ 4°C) until receiving the MTBDRsl assay results. The saved DNA pellet was centrifuged at 13,000 G for 5 min and 5 µl of DNA was added to 45 µl amplification mix and amplified using 42 PCR cycles based on the manufacturer's recommendation for clinical

Time difference in diagnosis

Among the isolates collected between 2016 and 2017, by implementing Genotype MTBDRsl assay and other microbiological testing in real time, turnaround time (TAT) was defined as the time between the dates of sputum sampling and the readout dates of the phenotypic DST and MTBDRsl and was calculated from the Chinese national TB reporting system.

Subjects inclusion

Within the study period, totally 391 were smear-positive and diagnosed as MDR-TB. Among the 368 culture positive MDR-TB isolates, 5 failed in the culture and/or phenotype DST due to the lost viability in the solid media or contamination, while the 10 non-interpretable results arose from failure in the PCR amplification. Thus totally 353 (95.9%) had both phenotypic DST and MTBDRsl assay results for 2nd-line drugs and were available for the accuracy analysis. The assay gave interpretable results

Discussion

The present multicenter diagnostic accuracy study aimed to assess the performance of the assay by including a large and diverse collection of clinical TB isolates, which have been collected to represent a varied scenario of drug resistance profiles in different Chinese settings.

Detected by GenoType MTBDRsl, the proportion of gyrA mutations conferring FQ resistance was comparable to that of previously published studies (71% to 92%).15, 16, 17 However, the frequency of Asp94Gly (24.2%) was a

Conclusion

The GenoType MTBDRsl assay was shown to be useful for early diagnosis of resistance to FQs, SLIDs and XDR-TB. The test is able to detect mutations in clinical samples without any culture which ensures a short turnaround time and a reduced biosafety hazard to laboratory staff. Apart from allowing a short turnaround time, the use of this test also offers an increased level of standardization when determining resistance in M tuberculosis.

Conflict of interest

None.

Acknowledgements

We thank all the collaborative staffs in four study sites for their assistance. This work was supported by the grants from the National Natural Science Foundation of China (NSFC) (PI, Yi Hu, No. 81373063), Sweden-China (VR-grant no 540-2013-8797 and NSFC No. 81361138019) project (PIs, Biao Xu and Sven Hoffner) as well as the Fourth Round of Three-Year Public Health Action Plan of Shanghai, China (Grant No. 15GWZK0101). The content of the paper is solely the responsibility of the authors and

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