Elsevier

Tuberculosis

Volume 110, May 2018, Pages 59-67
Tuberculosis

Accuracy of whole genome sequencing versus phenotypic (MGIT) and commercial molecular tests for detection of drug-resistant Mycobacterium tuberculosis isolated from patients in Brazil and Mozambique

https://doi.org/10.1016/j.tube.2018.04.003Get rights and content

Abstract

Background

The fast and accurate diagnosis of drug-resistant tuberculosis (DR-TB) is critical to reducing the spread of disease. Although commercial genotypic drug-susceptibility tests (DST) are close to the goal, they are still not able to detect all relevant DR-TB related mutations. Whole genome sequencing (WGS) allows better comprehension of DR-TB with a great discriminatory power. We aimed to evaluate WGS in M. tuberculosis isolates compared with phenotypic and genotypic DST.

Methods

This cross-sectional study evaluated 30 isolates from patients with detected DR-TB in Brazil and Mozambique. They were evaluated with phenotypic (MGIT-SIRE™) and genotypic (Xpert-MTB/RIF™, Genotype-MTBDRplus™, and MTBDRsl™) DST. Isolates with resistance to at least one first- or second-line drug were submitted to WGS and analyzed with TB profiler database.

Results

WGS had the best performance among the genotypic DST, compared to the phenotypic test. There was a very good concordance with phenotypic DST for rifampicin and streptomycin (89.6%), isoniazid (96.5%) and ethambutol (82.7%). WGS sensitivity and specificity for detection resistance were respectively 87.5 and 92.3% for rifampicin; 95.6 and 100% for isoniazid; 85.7 and 93.3% for streptomycin while 100 and 77.2% for ethambutol. Two isolates from Mozambique showed a Val170Phe rpoB mutation which was neither detected by Xpert-MTB/RIF nor Genotype-MTBDRplus.

Conclusion

WGS was able to provide all the relevant information about M. tuberculosis drug susceptibility in a single test and also detected a mutation in rpoB which is not covered by commercial genotypic DST.

Introduction

In 2016, globally there were an estimated 10.4 million incident cases of tuberculosis (TB), 1.3 million TB-related deaths among HIV-negative and 374.000 deaths among HIV-positive people [1]. Drug-resistant tuberculosis (DR-TB) is increasing and may undermine efforts to eradicate TB in many countries [2]. Global data from 2016 estimated 490.000 new MDR-TB cases and around 50% related deaths. But even so, information about DR-TB in Brazil and Mozambique is scarce, mainly due to the lack of laboratory facilities for drug susceptibility tests (DST) [1]. From 1998 to 2015, the concept of a high-burden country (HBC) became familiar and widely used and widely used in the context of TB, TB-HIV, and multi-drug resistant TB (MDR-TB). In 2015, Mozambique was among the three lists and Brazil in the former two [1]. New accurate and rapid diagnostic strategies for drug-resistance are urgently required to ensure that patients are diagnosed early and initiated onto appropriate therapy to improve outcomes and prevent the spread of DR bacilli [[3], [4], [5], [6]]. Currently, phenotypic DST is the gold standard for diagnosis of drug resistance. However they are time-consuming, require expensive laboratory facilities, are not available in many HBC and are not standardized for all anti-TB drugs [[7], [8], [9]].

Recently World Health Organization (WHO) endorsed three rapid genetic-based DST: Xpert-MTB/RIF, MTBDRplus, and MTBDRsl line-probe assays [1]. Although these tests are fast and easy to perform, they are not able to detect all the mutations associated with DR-TB [[10], [11], [12]]. Whole genome sequencing (WGS) allows the analysis of Mycobacterium tuberculosis genome enabling the identification of mutations which confer resistance, mutations compensating for fitness cost and has an extremely high discriminatory power that to measure the transmission of M. tuberculosis [[13], [14], [15]]. The development of next-generation sequencing technologies has reduced costs and time required to sequence the M. tuberculosis genome, making it progressively more affordable to study the epidemiology of disease as well as to describe the mechanisms of drug resistance [16]. However, the development and standardization of robust platforms that enable reliable analysis and interpretation of WGS data remain to be achieved [8,17,18].

We used WGS to characterize the mutations conferring resistance in clinical isolates of M. tuberculosis from Brazil and Mozambique and to compare these results to those obtained by phenotypic and commercial genotypic DST.

Section snippets

Study design and population

This is a cross-sectional study evaluating DR M. tuberculosis isolated from different patients in Southeastern Brazil (São Paulo state) and Central Mozambique (Sofala province). All the isolates were tested with phenotypic, commercial genotypic DST and were submitted to WGS if resistance was detected to at least one first- or second-line drug by one of these tests.

Drug-susceptibility tests

Phenotypic DST was conducted using MGIT-960 SIRE kit (MGIT-960; Becton Dickinson Diagnostic Systems, Sparks, MD). The critical

Patients' demographic characteristics

Among the 17 patients from Brazil, 15 (88.2%) were male, 16 (94.1%) had a pulmonary disease, and none were co-infected with HIV. One female patient (case #2370) was born in Angola (Africa) and had been living in Brazil for five years when she was diagnosed with extensively drug-resistant TB (XDR-TB). Among the 13 patients from Mozambique, seven (53.8%) were male, all presented pulmonary disease and six (46.1%) were co-infected with HIV.

Phylogenetic results

Among Mozambican isolates, 9 (69.2%) had a lineage of

Discussion

One of the most important clinical applications of WGS of M. tuberculosis isolates is the prediction of phenotypic drug resistance. However, the confidence at which these predictions are made is dependent on our knowledge of the association between phenotype and genotype [8]. Several studies have investigated the utility of WGS as a tool for DST [13,33,35]. The accuracy of predicting resistance varies among different classes of drugs as well as different drugs from the same class. M.

Conclusion

To investigate DR-TB in clinical practice we start with phenotypic DST for first-line drugs and only if resistance is detected, injectable second-line drugs (iSLD) and fluoroquinolones are tested. The same flow is followed for DR-TB investigation with genotypic DST. If rifampicin resistance is detected with Xpert or Genotype MTBDRplus, another genotypic DST for fluoroquinolones and iSLD susceptibility evaluation is required. WGS offers all the information about drug resistance in a single test.

Conflict of interest statement

We, the authors (CSF; EN; JRP; KP; AD; RMW; WASJ and VRB) state there is no conflict of interest for the authorship of this paper.

Funding information

This work was mainly supported by a grant from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) - Processo 15/13333-3, and partially supported by Fogarty International Center HIV Research Training Program grant, National Institutes of Health, to the University of Pittsburgh (D43TW009753) and Fundaçao de Apoio ao Ensino, Pesquisa e Assistencia do HCFMRP-USP (2015-2017).

These data were not presented before at any place or time.

Acknowledgments

We want to thank Professor Lee Harrison (University of Pittsburgh) for research support; Margarida Passeri Nascimento (Mycobacteriology Lab at HC FMRP-USP) for technical support; Center for Medical Genomics (CMG) at the Clinics Hospital of Ribeirão Preto (FMRP-USP) for the technical and research support; and Brazilian National Program for Tuberculosis Control (Denise Arakaki-Sanchez) for the support to perform Xpert-MTB/RIF in these isolates.

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