Conventional methods for mycobacteriological culture and DST are slow and labour intensive, requiring sequential procedures for isolation of mycobacteria from clinical specimens, identification of MTB complex, and in vitro testing of strain susceptibility to anti-TB drugs. During this time patients may be prescribed inadequate treatment, thus fuelling the development and/or spread of drug resistance. Novel technologies for rapid detection of anti-TB drug resistance have therefore become a priority in TB research and development, and molecular line probe assays focused on rapid detection of RIF resistance (alone or in combination with INH) are now commercially available.
In June 2008, concomitantly with the first published meta-analysis on MTBDR line probe assays , WHO has made public the policy statement on molecular line probe assays for rapid screening of patients at risk of MDR-TB . However, adoption of line probe assays does not eliminate the need for conventional culture and DST capability, as culture remains necessary for definitive diagnosis of TB in sputum smear-negative patients, while conventional DST of second line drugs is required to diagnose XDR-TB. Moreover, culture and DST are still essential for patient follow-up during treatment.
The aim of our study was to evaluate the molecular assay GenoType® MTBDRplus for detecting DR-TB directly in sputum specimens as a means to provide a more accurate management of chronic TB patients in Burkina Faso. Our study demonstrates that the GenoType® MTBDRplus assay can be used to identify MDR-TB cases correctly in the absence of culture facilities in TB patients classified as "chronic" according to internationally accepted criteria. Moreover, this study strongly suggests that sputum smear-microscopy as the only criteria to identify chronic TB patients is inadequate because of the risk of false positives.
The use of the GenoType® MTBDRplus molecular test, with further confirmation from culture and DST, allowed readjustment of patients' treatment in over half of the cases studied: 1) patients who were classified and treated as MDR-TB cases harbouring RIF- and INH-S strains (n 24); 2) patients negative for MTB complex DNA among those enrolled at M0 or remaining smear-positive during follow-up (n 24); 3) patients with a NTM infection (n 13). In addition, the correct classification of patients allowed to reconsider their need for hospitalization in MDR-TB wards limiting nosocomial exposure to MDR-TB.
The GenoType® MTBDR plus assay performed directly on clinical specimens is less expensive than culture-based DST. We recommend that cost-effectiveness in applying the test should be carefully evaluated in each setting taking into account specific situations (e.g. TB and HIV prevalence), technical skills available on site, and frequency of mutations among resistant samples to evaluate the sensitivity of the molecular assay.
More data is needed to validate the use of molecular assays for rapid detection of drug resistance to key anti-TB drugs such as RIF and INH under routine conditions.
Our study found that the GenoType® MTBDR plus yielded interpretable results in more than 99% of the cases, among both smear-positive and smear-negative sputum samples.
Reported sensitivities for RIF- and INH- resistance for the GenoType® MTBDR plus are ≥ 97% and ≥ 90%, respectively . Also specificities are accounted to be ≥ 99% for both RIF- and INH- resistances. In our study, 4 samples showed INH susceptibility by molecular line probe assay, while these were identified as INH-resistant by culture based DST. A possible explanation is the lower sensitivity of the test on INH resistance since the GenoType® MTBDR plus targets only 2 of the several genomic regions involved in determining INH-resistance .
Three samples designated as MDR-TB by GenoType® MTBDR plus assay, were not confirmed to be RIF resistant on culture-based DST. Further investigation on these 3 cases identified mutations in the rpoB gene (M515I+H526N, L533P, and H526N respectively). Discrepancies between culture and molecular DST can be interpreted by studying the MICs of the corresponding strains. In fact, strains carrying the substitution L533P in rpoB gene have been demonstrated to have different MIC values (from 0.5–1.0 μg/mL up to 32 μg/mL) [27, 28]. Further evaluation on the three strains carrying substitutions in rpoB gene allowed to identify increased MIC values.
Our study had several limitations. Discrepant results in sputum smear microscopy in Burkina Faso and Italy could be due to differences in the sampling time and in recruitment of patients by peripheral centers with suboptimal capacity in smear microscopy.
Discrepant GenoType® MTBDRplus-positive and culture-negative samples may be justified in two ways. First, some samples were collected and transported to Italy under sub-optimal conditions and this may have affected their culture results . Second, most of the culture-negative samples harboured DNA from MTB susceptible strains by GenoType® MTBDRplus. For these cases we speculate that standard TB treatment was effective and prescription of WHO Category IV regimen unnecessary.
One case with negative culture and with a positive MDR profile on molecular assay may be due to the fact that this case was sampled six months after the beginning of the therapy with category IV regimen. In fact, we observed during treatment follow-up that the molecular assay became negative between month 3 and month 6 in patients under effective treatment (data not shown).
Two sputum smear-negative samples harbouring NTM (M. intracellulare, M. avium) were identified as MTB complex positive by the GenoType® MTBDRplus. This may be due to successful treatment that contributed to select microorganisms not belonging to the MTB complex.
Results obtained in our sample suggest that in Burkina Faso the frequency of mutations involved in RIF resistance differs from that of other settings. The commonest mutation is the D516V substitution in the hotspot region of rpoB gene, detected entirely in 43.8% (14/32) of RIF resistant cases. The S531L mutation is responsible of resistant phenotype in only 9.4% (3/32) in our study, whereas this substitution affects the majority of RIF resistant strains in most of other countries worldwide . In this study we observed that mutations in the promoter region of inhA gene had a minor role in conferring resistance to INH. As these data were obtained from chronic patients undergoing two cycles of standard first-line drugs regimen, we believe that low-level resistance mutations have favoured secondary selections for high-level resistance mutations such as S315T substitution in the katG gene , and we recommend further studies be conducted.
The molecular test correctly identifies as MTB-complex negative all the patients in which the smear positivity was probably due to the presence of NTMs as suggested by culture results. The addition of a Mycobacteria genus specific probe could address this problem improving the clinical usefulness of the test, especially in high HIV prevalence settings.
As shown in other studies the molecular assay has been successfully performed on contaminated cultures to obtain drug susceptibility data . This provides important drug resistance data in settings where culture and culture DST testing are not conducted correctly as confirmed by quality control systems.