First Evaluation of Drug-Resistant Mycobacterium tuberculosis Clinical Isolates from Congo Revealed Misdetection of Fluoroquinolone Resistance by Line Probe Assay Due to a Double Substitution T80A-A90G in GyrA

Abstract

BackgroundTuberculosis (TB) is one of the major public health problems in Congo. However, data concerning Mycobacterium tuberculosis drug resistance are lacking because of the insufficient processing capacity. So, the aim of this study was to investigate for the first time the resistance patterns and the strain lineages of a sample of M. tuberculosis complex (MTBC) isolates collected in the two main cities of Congo.MethodsOver a 9-day period, 114 smear-positive sputa isolated from 114 patients attending centers for the diagnosis and treatment of TB in Brazzaville and Pointe Noire were collected for culture and drug susceptibility testing (DST). Detection of mutations conferring drug resistance was performed by using line probe assays (GenoType MTBDRplus and MTBDRsl) and DNA sequencing. Strain lineages were determined by MIRU-VNTR genotyping.ResultsOf the 114 sputa, 46 were culture positive for MTBC. Twenty-one (46%) were resistant to one or more first-line antiTB drugs. Of these, 15 (71%) were multidrug resistant (MDR). The most prevalent mutations involved in rifampin and isoniazid resistance, D516V (60%) in rpoB and S315T (87%) in katG respectively, were well detected by MTBDRplus assay. All the 15 MDR strains were susceptible to fluoroquinolone and injectable second-line drug. No mutation was detected in the rrs locus involved in resistance to amikacin and capreomycin by both the MTBDRsl assay and DNA sequencing. By contrast, 9 MDR strains belonging to the same cluster related to T-family were identified as being falsely resistant to fluoroquinolone by the MTBDRsl assay due to the presence of a double substitution T80A-A90G in GyrA.ConclusionsTaken together, these data revealed a possible spread of a particular MDR clone in Congo, misidentified as fluoroquinolone resistant by MTBDRsl assay. Thus, this test cannot replace gold-standard culture method and should be interpreted carefully in view of the patient's native land.

MTBDR plus and MTBDR sl Assays: Absence of Wild-Type Probe Hybridization and Implications for Detection of Drug-Resistant Tuberculosis

“…Six percent of FQ resistance, as classified by MTBDRsl, was due to the sole absence of WT probe hybridization. Our results contrast significantly with the results of two studies conducted in the Congo and the Democratic Republic of Congo, where over 60% of FQ resistance was attributable to the sole absence of WT probe hybridization (17,18). Our results more closely resemble those reported by Brossier et al (33) and Huang et al (34), in which less than 20% of FQ resistance was attributable to the sole absence of WT probe hybridization.…”

“…(ii) If no MUT probes hybridize on the test strip and one or more WT probes also do not hybridize, the presence of an unknown mutation in that region is inferred, and the specimen is classified as resistant. Two recent studies assessing the performance of the MTBDRsl assay detected an unusually high proportion of FQresistant specimens that were eventually determined to be falsely positive after comparison to sequencing and/or conventional culture results (17,18). False-positive (FP) FQ resistance results from the two studies were determined to be the result of absent WT probe hybridization (interpreted to mean the presence of a nonwild-type or unknown mutation sequence) rather than direct detection of a known mutation (17,18).…”

“…Two recent studies assessing the performance of the MTBDRsl assay detected an unusually high proportion of FQresistant specimens that were eventually determined to be falsely positive after comparison to sequencing and/or conventional culture results (17,18). False-positive (FP) FQ resistance results from the two studies were determined to be the result of absent WT probe hybridization (interpreted to mean the presence of a nonwild-type or unknown mutation sequence) rather than direct detection of a known mutation (17,18). Given the significant impact of FQ resistance on MDR-TB treatment and the potential for the sole absence of WT probe hybridization to increase the number of false-positive test results, we analyzed individual probe performance to further understand the underlying subtleties of the MTBDRplus v2 and MTBDRsl assay results (19).…”

Performance of the New Version (v2.0) of the GenoType MTBDR sl Test for Detection of Resistance to Second-Line Drugs in Multidrug-Resistant Mycobacterium tuberculosis Complex Strains

“…Previous studies have demonstrated that these substitutions, commonly identified in African countries such as Republic of the Congo, do not confer resistance and are thus responsible for misclassification of FQ resistance by line probe assay (23,24). This problem, already encountered with MTBDRsl v1, could be avoided by inclusion of a comment in the recommendations for interpretation of MTBDRsl v2.0 results in case of the absence of the WT2 probe.…”

Evaluation of GeneXpert MTB/RIF and line probe assay for rapid diagnosis of Mycobacterium tuberculosis in Sudanese pulmonary TB patients

“…Rapid identification is essential to initiate early treatment, improve patient's outcomes, and more effective for public health interventions [5]. Therefore, molecular assays have been used to predict drug resistance in clinical specimens within one working day and are potentially the most rapid methods [6][7][8][9][10]. The GeneXpert MTB/RIF assay is a novel integrated diagnostic device that performs sample processing and semi-nested real-time PCR analysis in a single hands-free step for the diagnosis of tuberculosis and rapid detection of RIF resistance in clinical specimens [4,10].…”