Evaluation of the GenoType MTBDRsl Version 2.0 Assay for Second-Line Drug Resistance Detection of Mycobacterium tuberculosis Isolates in South Africa

DISCUSSION

To our knowledge, the evaluation of GenoType MTBDRsl VER 2.0 in conjunction with a reproducibility assessment is the first such study conducted in a high TB-HIV setting in Africa. GenoType MTBDRsl VER 2.0 has shown an improvement in sensitivity and specificity for the determination of molecular resistance to both FLQ (100% [95% CI, 95.8 to 100%] and 98.9% [95% CI, 96.1 to 99.9%]) and SLID (89.2% [95% CI 79.1 to 95.6%] and 98.5% [95% CI 95.7–99.7%]) compared with the pooled sensitivity and specificity of GenoType MTBDRsl VER 1.0 reported by the WHO Expert Group (12) and a meta-analysis by Theron and colleagues (5). Agreement between the phenotypic gold standard and GenoType MTBDRsl VER 2.0 for FLQ (OFX) and SLID was 99.3% and 96.3%, respectively, across a wide distribution of mutations. The assay was found to be highly reproducible in terms of interlaboratory (93 to 96%) and interoperator (100%) assessment.

FLQ resistance in M. tuberculosis is ascribed mainly to gyrA mutations, with 57.5% of mutations detected at codon 94 and 31.5% at codon 90 (15). Consistent with published data, the highest frequency of mutations conferring FLQ resistance was observed in gyrA codon 94, followed by codon 90 (Table 3). Furthermore, gyrA mutations D94G and D94N were observed in the majority of the isolates tested. Previous studies have indicated that isolates harboring these mutations exhibit high levels of resistance to FLQ (15, 16). The gyrA MUT3D/D94H mutation, reported as a rare in silico mutant (13, 17), was observed in 9/85 (10.6%) of mutations identified from these clinical isolates. Heteroresistance involving either FLQ or SLID was detected in 18/85 (21.2%) of isolates, with multiple mutation bands observed in some isolates (Table 3), and were confirmed as phenotypically resistant. Cohen et al. reported on mixed-strain infections in a hospital setting in KwaZulu-Natal, South Africa (18). Further investigation is required to elucidate the frequency and clinical relevance of mixed infections across a more widespread South African setting. Based on our findings, interpretation of these strains as resistant was consistent with the phenotypic DST results.

Undefined mutations with the absence of gyrA wild-type bands were confirmed as phenotypically resistant. WGS results available for four isolates confirmed the presence of gyrA mutations that the assay missed. The GenoType MTBDRsl VER 2.0 guideline for interpretation of results states that “only bands with intensities as strong as or stronger than the amplification control zone (AC) are to be considered” (13). Three out of four of these discordant isolates had gyrA MUT2/S91P bands with intensities less than that of the AC zone and therefore were interpreted as negative and reported as resistant due to the absence of gyrA wild-type bands. No weak-intensity mutation bands were detected in the fourth isolate. An isolate phenotypically susceptible to OFX with gyrA MUT 3C/D94G bands and a confirmed Asp94Gly mutation on WGS had gyrA WT bands with less intensity than the AC control band. The discrepant OFX phenotypic DST is possibly due to a mixed population. The inclusion of selected gyrB probes offered limited enhancement to the identification of FLQ mutations, as only the absence of gyrB WT was observed in 3/268 (1.1%) isolates. GenoType MTBDRsl VER 2.0 is limited to detecting mutations in selected areas of the quinolone resistance-determining regions (QRDR) of gyrA and gyrB only; therefore, FLQ resistance mechanisms outside these regions are likely to be missed but do not appear to be a major concern in our setting presently.

Consistent with published data (17), rrs MUT1 A1401G (translating to high-level SLID resistance) was the most frequently observed mutation (77.5%) among tested isolates. The overall SLID sensitivity has improved in the new assay to 89.2% compared with previously published data (5, 12). The inclusion of eis promoter region probes improved the detection of SLID resistance, as observed in 8/62 (12.9%) low-level KAN-resistant isolates, all phenotypically KAN resistant and not associated with rrs mutations. Two isolates, with rrs WT1 bands absent, no corresponding mutation band observed, wild type by WGS, and interpreted as resistant to SLID by the assay, were phenotypically susceptible to SLID. The omission of WT bands in the line probe assay is not a reliable indication of phenotypic resistance and would require confirmatory phenotypic DST (19). In 6 phenotypically KAN-resistant isolates that were AMK susceptible, no mutations were detected by the assay or WGS. Although other factors related to phenotypic resistance cannot be excluded, the discrepant results may be due to the inherent challenges of in-house (nonstandardized) second-line drug preparations available for testing (19). Five isolates that were phenotypically susceptible to CAP, interpreted as SLID resistant by the assay and wild type by WGS, displayed resistance only to KAN and AMK phenotypically. As indicated by Georghiou et al. (20), rrs MUT1 A1401G mutation is a moderate predictor of CAP resistance. Therefore, CAP resistance should be confirmed phenotypically prior to exclusion of the drug from a treatment regimen (21).

There were 31 isolates identified as phenotypically XDR, and the GenoType MTBDRsl VER 2.0 assay achieved 97.0% agreement between the index and gold standard tests. Of the four XDR cases misclassified by the assay, all showed FLQ resistance but missed SLID resistance. All were phenotypically KAN resistant. One isolate was classified as XDR by LPA with gyrA MUT1 (A90V) and a missing eis WT2 only (C-12T/G-10A) but was phenotypically susceptible to KAN. We used strict criteria, and resistance to any one SLID was accepted as a criterion to classify a strain as XDR. Due to limited availability of standardized drug preparations for second-line DST, in routine practice, laboratories would normally only test one drug out of this class for resistance determination. This is a limitation of the data available for this sample set, as mutations in the assay are known to correlate well with OFX resistance and is evident in our observations. As reported, correlation of these mutations with higher FLQs is uncertain and may still show phenotypic susceptibility despite the presence of these mutations; thus, phenotypic testing remains important for these drugs (22, 23).

The reproducibility assessment of selected routine laboratories showed excellent performance consistent with other data on molecular diagnostics (Table 6) (24). GenoType MTBDRsl VER 2.0 LPA performed well with excellent reproducibility and high sensitivity and specificity for FLQ and SLID resistance determination. The average reporting turnaround times for GenoType MTBDRsl VER 2.0 LPA varied from 2 to 4 days subsequent to the identification of a positive TB culture, whereas second-line phenotypic DST results were only available in 14 to 21 days. The longer TAT for DST results is largely dependent on the viability/fitness of the isolate, especially for XDR isolates. It also highlights the importance of direct testing in providing clinically relevant and reliable results.

Furthermore, adoption of the assay in laboratories already performing the GenoType assays was relatively easy and quick to implement. GenoType MTBDRsl VER 2.0 LPA is therefore suitable for testing clinical isolates as a rapid screening tool for detection of second-line drug resistance in countries with a high burden of MDR-TB.

A limitation of the study was that only culture isolates were used and not clinical specimens, as this study was performed to compare directly the GenoType MTBDRsl VER 2.0 assay with the gold standard, phenotypic DST. However, there is a need for further studies evaluating the GenoType MTBDRsl VER 2.0 assay on clinical specimens from patients with MDR-TB, thereby enhancing the turnaround time for resistance reporting and pre-XDR/XDR case detection in high-risk settings. Another limitation was that of the FLQs, only OFX was tested and none of the other later-generation FLQs (isolates collected between 2012 and 2014). This probably explains the higher sensitivity found in our study compared with other studies (25). Phenotypic DST for later-generation FLQs (moxifloxacin) was introduced in 2015 per WHO recommendations (22, 23). A general limitation of second-line drug testing is the limited availability of standardized drug preparations (19). A further limitation was that samples from the Braamfontein TB Referral Laboratory could not be included for WGS resolution testing, as the DNA was not available.

In a press release by the WHO in May 2016, the use of the GenoType MTBDRsl VER 2.0 assay as “an initial test, instead of phenotypic culture-based DST” to detect FLQ and SLID resistance in confirmed RIF-R and MDR patients was recommended (26, 27). Appropriately trained laboratory staff, quality assurance, and availability of laboratory infrastructure are requisite recommendations of the WHO to implement use of this assay (26, 27). Our study provides support and evidence for these recommendations and the implementation of the assay in South Africa.