Abstract
Fluoroquinolones are the core drugs for the management of multidrug-resistant tuberculosis (MDR-TB). Molecular drug susceptibility testing methods provide considerable advantages for scaling up programmatic management and surveillance of drug-resistant TB. We describe here the misidentification of fluoroquinolone resistance by the GenoType MTBDRsl (MTBDRsl) (Hain Lifescience GmbH, Nehren, Germany) line probe assay (LPA) encountered during a feasibility and validation study for the introduction of this rapid drug susceptibility test in Kinshasa, Democratic Republic of Congo. The double gyrA mutation 80Ala and 90Gly represented 57% of all fluoroquinolone mutations identified from MDR-TB patient sputum samples, as confirmed by DNA sequencing. This double mutation was previously found to be associated with susceptibility to fluoroquinolones, yet it leads to absent hybridization of a wild-type band in the MTBDRsl and is thus falsely scored as resistance. Our findings suggest that MTBDRsl results must be interpreted with caution when the interpretation is based solely on the absence of a wild-type band without confirmation by visualization of a mutant band. Performance of the MTBDRsl LPA might be improved by replacing the gyrA wild-type probes by additional probes specific for well-documented gyrA mutations that confer clinically relevant resistance.
INTRODUCTION
Fluoroquinolones (FQs) are essential drugs for the management of multidrug-resistant tuberculosis (MDR-TB) (1). Resistance to FQs is associated with poor treatment outcome in MDR-TB and is also one of the defining conditions of extensively drug-resistant tuberculosis (XDR-TB). XDR-TB is defined as MDR-TB with additional resistance to any FQ and a second-line injectable drug such as kanamycin, amikacin, or capreomycin, and pre-XDR-TB is defined as MDR-TB associated with resistance to FQ or a second-line injectable, but not both (2–5). In areas with high rates of TB and MDR-TB, it is extremely important to monitor resistance to these drugs, especially where FQs are widely used for treatment of other bacterial infections. Molecular drug susceptibility testing (DST) methods have considerable advantages for scaling up programmatic management and surveillance of drug-resistant TB. Currently, compared to conventional DST, these methods offer improvements in speed of diagnosis, standardized testing, potential for high-burden settings, and lower levels for laboratory biosafety (6). In 2008, the World Health Organization (WHO) endorsed molecular line probe assay (LPA) technology for rapid detection of MDR-TB that brings results within 2 days—even from clinical specimens (7). The GenoType MTBDRplus assay (MTBDRplus) (Hain Lifescience GmbH, Nehren, Germany) was one of the first commercially available LPAs. Because of its accuracy and rapidity, genotypic detection of rifampin (RMP) and isoniazid resistance with the MTBDRplus has emerged as an essential tool for the diagnosis of MDR-TB (8, 9). It has also been suggested as an alternative approach for conducting drug resistance surveys (DRS) in settings with limited capacity to perform phenotypic DST (10). In 2009, the company Hain Lifescience introduced a new format of the LPA, the GenoType MTBDRsl test (MTBDRsl), for the rapid determination of genetic mutations associated with resistance to FQs, second-line injectable drugs, and ethambutol. The main mechanism of resistance to FQs in Mycobacterium tuberculosis is caused by mutations affecting DNA gyrase, which consists of the GyrA and GyrB subunits, encoded by the gyrA and gyrB genes, respectively (11). Most mutations conferring bacterial resistance to FQs occur in a short segment termed the quinolone resistance-determining region (QRDR) in the gyrA gene (12, 13). Analysis of the QRDR alone by genotypic tests has been suggested as sufficient for rapid identification of the vast majority of FQ-resistant M. tuberculosis strains, with an estimated sensitivity around 85% for FQ resistance (14, 15). The identification of resistance to FQs by the MTBDRsl is based on this principle. The format of the MTBDRsl is similar to that of the MTBDRplus and it also has a turnaround time of 48 h. Compared to phenotypic DST, the sensitivity of the MTBDRplus for detection of FQ resistance by identification of the best known gyrA mutations (but, for instance, not the gyrB mutations) has been generally evaluated as suboptimal. For this reason, the MTBDRplus has not yet been endorsed by the WHO, but its specificity is considered to be very high (15, 16). Here we report that, in addition, the specificity of the MTBDRsl LPA was inadequate for the detection of FQ resistance during a validation study of the assay in Kinshasa.
MATERIALS AND METHODS
Patients and specimens.
From March 2011 to June 2013, a feasibility and validation study with both the MTBDRplus and MTBDRsl LPAs was carried out at the National Public Health Laboratory of the Democratic Republic of Congo (DRC)—the Institut National de Recherche Biomédicale (INRB)—in Kinshasa. Kinshasa is the capital city of DRC, with an estimated 10 million inhabitants. With a total of 137 TB clinics, the city of Kinshasa is notifying more than 80% of all MDR-TB suspects in the country (DRC National TB Program, unpublished data). We prospectively collected sputum specimens of 587 MDR-TB suspects in 50 out of the 137 TB clinics which were purposefully selected as sentinel sites in Kinshasa for rapid detection of drug-resistant TB. Consenting MDR-TB suspects defined according to the WHO categories of treatment (17) have been consecutively included in the study, in addition to smear-positive contacts of known MDR-TB cases.
MTBDRsl.
Sputum specimens without any additives were transported from the TB clinics. Patient information related to their demographic data and clinical TB history was collected. Sputum specimens were processed according to standard methods previously described, which included decontamination and processing with NaOH according to a modified Petroff technique, with a final concentration of 2% (18). The sediment obtained was tested by the MTBDRplus according to the manufacturer's instructions (19). All specimens showing resistance to RMP and isoniazid or to RMP alone by the MTBDRplus were concurrently tested by the MTBDRsl from the same DNA extract. Strips were interpreted according to the manufacturer's instructions (20). For each gene, the test evaluates the presence of wild-type (WT) and/or mutant (MUT) sequences, thus covering all high-confidence resistance mutations. For gyrA these are 90Val, 91Pro, and the codon 94 mutations Ala, Asn, Gly, His, and Tyr. When all the WT probes of a specific gene appear as bands on the strip, there is no detectable mutation within the region examined and the strain is considered sensitive to the corresponding drugs. In case of a mutation, the amplicon cannot bind to the corresponding WT probe, but it may bind to one of the MUT probes provided this specific mutation is represented on the strip. The absence of a WT band or appearance of a MUT band at least as strong as the amplification control band must be interpreted as resistance to the respective drugs.
The INRB laboratory follows a strict unidirectional workflow for all molecular testing. For quality control, each test batch included a known pan-susceptible TB strain (H37Rv). Negative controls (water) were included during all steps of the procedure. The strips were interpreted on a regular schedule by two different readers (M.K.K. and M.A.) who were blinded to the results of genetic sequencing. Discrepancies between both readers were uncommon (<10%) and were resolved by consensus.
Genetic sequencing.
Sequencing of the gyrA was performed at the Supra National Reference Laboratory (SRL) in Antwerp, Belgium, on all available DNA extracts from Kinshasa harboring FQ-resistant patterns on LPA. The methodology for PCR amplification and sequencing of genes encoding gyrase A and B has been published elsewhere (21).
Ethical considerations.
The study was approved by the Ethics Committee of the University of Antwerp, Belgium, and the National Tuberculosis Program DRC. All data and sputum specimens were collected in the context of routine care and no additional data collection or contact with patients occurred for this study. The data were completely delinked from any personal identifiers before analysis.
RESULTS
MTBDRsl test.
Of 587 consecutive individual MDR-TB suspects who submitted their sputum specimens at TB clinics during the study period, a total of 211 MDR-TB and 28 RMP monoresistant results were obtained from DNA extracts tested with MTBDRplus. All 239 extracts with RMP resistance were also tested by MTBDRsl. Of those, 87% (209/239) yielded an interpretable test for FQ gyrA, with the remainder invalid due to absence of the gyrA control band. As shown in Table 1, of 209 samples with an interpretable result, 177 (85%) were identified as FQ susceptible and 32 (15%) as FQ resistant. Out of 32 FQ-resistant samples, 20 (63%) were identified as resistant based only on the lack of hybridization with WT probe number 2 (WT2), while for the other 12 samples, one or two specific mutation bands appeared.
TABLE 1.
Genotypic pattern obtained by MTBDRsl line probe assay on 209 sputum DNA extracts from documented multidrug-resistant tuberculosis in Kinshasa
| MTBDRsl result | gyrA patterna | No. (%) of strains |
|---|---|---|
| Resistant (n = 32) | MUT3A | 4 (12.5) |
| MUT3C | 3 (9.3) | |
| MUT1 | 2 (6.2) | |
| MUT3A plus MUT3C | 2 (6.2) | |
| MUT2 | 1 (3.1) | |
| ΔWT2 | 20 (62.5) | |
| Sensitive (n = 177) | WT1 plus WT2 plus WT3 | 177 (84.7) |
MUT1, MUT2, MUT3A, and MUT3C, mutation bands appearing; WT1, WT2, and WT3, wild-type DNA bands appearing; ΔWT2, wild-type 2 band disappearing.
The distribution of the 14 gene mutations found in the 12 FQ-resistant samples with a MUT band on the MTBDRsl strips is shown in Table 1. The predominant mutations identified as conferring FQ resistance were gyrA MUT3A (94Ala) (6/14 [43%]), followed by MUT3C (94Gly) (5/14 [36%]), MUT1 (90Val) (2/14 [14%]), and MUT2 (91Pro) (1/14 [7%]), with Arg94Ala and Arg94Gly found twice as a double mutation. No MUT3B (94Asn or Tyr) or MUT3D (94His) mutations were found in our study.
DNA sequencing results.
gyrA sequencing was performed on 25 (25/32 [78%]) DNA extracts identified by MTBDRsl as FQ resistant. DNA sequencing confirmed mutations in the gyrA QRDR for 23/25 (92%), but two contained only WT DNA. Table 2 shows the types of mutations detected by DNA sequencing and their frequencies, stratified by WHO patient category and compared to MTBDRsl results. There were 4 patients in WHO category 4 (recurrence after second-line TB treatment) versus 12 in category 2 (recurrence after re-treatment with first-line drugs) and 9 in category 1 (recurrence after primary treatment with first-line drugs). The 94Gly substitution was detected in three of the four DNA extracts from category 4 patients, once as a triple mutation (80Ala, 90Gly, and 94Gly), while the fourth showed a 94Ala mutation. Mutations in this group were detected directly with MTBDRsl by hybridization with the gyrA probes in MUT3C or MUT3A and were missing corresponding WT bands, except for the 94Ala, which showed as MUT3A and MUT3C bands without loss of WT3. Eleven samples from WHO category 2 patients were confirmed to contain multiple mutations, but one showed only wild-type DNA on sequencing. The two multiple mutations (94Tyr and 94Ala and 80Ala, 90Gly, and 94Ala) showed only a gyrA MUT3A band on the MTBDRsl strip. The expected MUT3B was missing for the first, and for both all of the wild-type bands were still present. The nine remaining, with an 80Ala and 90Gly mutation on sequencing, showed only as an absent WT2 band in the MTBDRsl. From WHO category 1 patient samples, two single and six double mutations were identified by DNA sequencing, but only wild-type DNA was found in one sample. Both single mutations (90Val and 91Pro) and one of the six double mutations (80Ala and 90Val) were detected with the MTBDRsl by hybridization with the gyrA probe in MUT1 or MUT2 and absence of the WT2 band. The five remaining double mutations (80Ala and 90Gly and 80Ala and 90Arg) were all associated with lack of hybridization with WT2 in MTBDRsl without a MUT band appearing. Overall, results by sequencing showed that the double mutations 80Ala and 90Gly represent 57% of all 23 confirmed gyrA mutations among MDR-TB patients in Kinshasa, DRC.
TABLE 2.
MTBDRsl patterns versus gyrA mutations detected by DNA sequencing among 25 of the 32 DNA extracts, by WHO patient category
| Type of patienta | gyrA MTBDRsl patternb | gyrA sequencing data, codons, and amino acid (nucleotide) substitutions | No. (%) detected |
|---|---|---|---|
| Cat. 4 | ΔWT3 plus MUT3C | 94Gly (GGC) | 2 (8) |
| ΔWT2 plus ΔWT3 plus MUT3C | 80Ala (GCC), 90Gly (GGG), 94Gly (GGC) | 1 (4) | |
| MUT3A plus MUT3C | 94Ala (GCC) | 1 (4) | |
| Cat. 2 | MUT3A | 94Tyr (TAC), 94Ala (GCC) | 1 (4) |
| MUT3A | 80Ala (GCC), 90Gly (GGG), 94Ala (GCC) | 1 (4) | |
| ΔWT2 | 80Ala (GCC), 90Gly (GGG) | 9 (36) | |
| ΔWT2 | WTc | 1 (4) | |
| Cat. 1 | ΔWT2 plus MUT1 | 90Val (GTG) | 1 (4) |
| ΔWT2 plus MUT1 | 80Ala (GCC), 90Val (GTG) | 1 (4) | |
| ΔWT2 plus MUT2 | 91Pro (CCG) | 1 (4) | |
| ΔWT2 | 80Ala (GCC), 90Gly (GGG) | 4 (16) | |
| ΔWT2 | 80Ala (GCC), 90Arg (AGG) | 1 (4) | |
| ΔWT2 | WT | 1 (4) |
Cat. 1, 2, and 4, WHO patient categories of recurrences after first-line primary treatment, first-line re-treatment, and second-line treatment, respectively.
ΔWT, omission of the respective wild-type band.
WT, only wild-type DNA detected.
DISCUSSION
This is the first assessment of the performance of LPA technology under routine diagnostic conditions in the capital city of DRC, one of the 22 TB high-burden countries. According to the MTBDRsl results, the proportion of FQ resistance among MDR-TB (i.e., RMP-resistant) samples was alarming, at 15%. Of these gyrA mutants, 63% were indirectly detected by MTBDRsl through lack of hybridization with the WT2 or with any mutant gyrA probes. However, besides a few cases without any mutation detected, DNA sequencing showed for more than half of those a combination of the mutations 80Ala and 90Gly, which has previously been demonstrated to confer FQ hypersusceptibility (22). Studies by the same group suggest that this hypersusceptibility may be caused by a stronger covalent bond and resulting enzyme blockage if the WT 90Ala is replaced by 90Gly because of its smaller side chain, while a bulkier side chain, as with the 90Val mutation, has the opposite effect and causes resistance (23). 80Ala is not detected by MTBDRsl LPA, since codon 80 is not covered by the test. The gyrA codons analyzed range from 89 to 93, including codon 90. Our sequence results revealed a mutation in 90Gly (GGG), while the only mutation probe included on the strip for position 90 is 90Val (GTG), explaining why only the absence of WT2 was found in these samples but no confirmation by appearance of a mutant probe. The MTBDRsl, which contains mutant DNA probes only for the most frequent gyrA QRDR mutations observed (12, 24) at codons 90, 91, and 94, has previously been assessed as highly specific in several countries worldwide (25–31). In our series in Kinshasa, the majority of the QRDR mutations observed were not associated with true FQ resistance. Mutation prevalence might differ by geographical areas and by preselection of patients (26). Negative controls, included in DNA extraction, PCR amplification, and hybridization, never showed evidence of contamination. Moreover, 14/20 profiles with a lacking WT2 were single occurrences found in as many runs, and the remaining six were found per two in three runs. For these reasons we believe that contamination or cross-contamination of the tests is highly unlikely to explain the high frequency of this unusual pattern.
Fundamentally, the problem is that interpretation of the MTBDRsl is based on absence of WT bands, which is equated with resistance (20). Errors are known to occur because of silent mutations, with a change of nucleotide resulting in a different code but for the same amino acid (26). The systematic error we report here was hitherto hardly known, i.e., a polymorphism not associated with resistance that appears to be more common in Kinshasa, DRC. In contrast to what was reported elsewhere (27–30), the predictive value for demonstration of FQ resistance of the assay in Kinshasa is therefore low, since the proportion of test results falsely indicating resistance to FQs was high. We erroneously alarmed the DRC National TB Program by reporting that FQ resistance had taken a big leap, based on these LPA results, before finding out through sequencing that the combinations of 80Ala (GCC) and 90Gly (GGG) mutations were in fact not associated with true FQ resistance. After this correction, the proportion of FQ resistance did drop to 5%. Considering sequencing as the gold standard, two samples had been wrongly classified as resistant because of a missing WT2 band. This may have been due to poor amplification compared to the control, leading to erroneous interpretation. Aberrant results were also seen for two samples with multiple mutations on DNA sequencing, but only a MUT3A band appearing on the LPA, and no wild-type band disappearing. Another sample with only a 94Ala mutation detected by sequencing showed two mutation bands on LPA, MUT3A, and MUT3C. These differences may have been caused by different proportions of the alleles present in the aliquots used for the different tests, possibly together with some remaining wild-type DNA. At a too low proportion, the alleles would not be reliably detected by either test. DNA sequencing performed in our study showed gyrA 80Ala (GCC) and 90Arg (AGG) in a double mutation, and to the best of our knowledge, this was never described previously.
In low-income countries with high TB burdens, resistance to FQ is not routinely tested because of the very limited laboratory infrastructure. New molecular techniques that do not have the same biosafety requirements as conventional techniques have the potential to overcome this problem and are an alternative to periodic or continuous surveillance of resistance against this important class of drugs. Local validation of a novel molecular assay will require assessing its accuracy compared to a reference, ideally composed of standard pheno- and genotypic techniques. For MDR-TB management, knowledge of FQ susceptibility is crucial, as FQs represent the core drugs in all the second-line drug regimens. Failure to detect mutations conferring resistance (i.e., poor sensitivity) or overdetection of false resistance (i.e., poor specificity) results in poor programmatic management of MDR-TB cases. FQ resistance in M. tuberculosis has a major impact on MDR-TB patient outcome, and removal of FQ seriously jeopardizes the strength of the second-line regimen, so false-positive results should be avoided. Molecular differentiation of the gyrA mutations 80Ala and 90Gly has important clinical consequences, since these mutations are not associated with FQ resistance. In Kinshasa, managing MDR-TB cases based on results of the MTBDRsl LPA only would thus have been detrimental for patient prognosis.
In this study, a significant proportion of DNA extracts from MDR patients had mutations observed in the QRDR, suggesting a rapid increase or even an outbreak of pre-XDR-TB in Kinshasa. Although not confirmed as pre-XDR, an MDR-TB strain with clonal expansion in Kinshasa is suggested by the high frequency of the unusual combination of 80Ala and 90Gly mutations. These mutations do not confer any advantage due to FQ resistance and were also not observed among category 4 recurrences. Their high prevalence may thus point to continued MDR transmission due to delayed or absent detection and treatment of MDR-TB. Further studies using genotyping techniques with higher resolution should clarify the proportion of MDR-TB due to recent transmission.
This study has some limitations. First, we could not correlate the mutations with FQ resistance (level) in our population, since only a few strains were still available and local phenotypic DST appeared unreliable. In turn, this was caused by extremely high culture contamination rates in addition to numerous operational problems, illustrating the higher feasibility of genotypic than conventional DST, even at the capital. Second, DNA sequencing was not performed on susceptible RMP and on susceptible FQ to verify if this polymorphism is also frequent in Kinshasa in the absence of MDR-TB. Moreover, fingerprinting of these isolates was not done to identify whether the 80Ala (GCC) and 90Gly (GGG) mutations are characteristic of a single MTBc lineage that is common in Kinshasa. Further studies are necessary to characterize the biological significance and potential selective advantage of these mutations not conferring resistance.
Our results warrant caution in the interpretation of the MTBDRsl when the only sign of resistance is the absence of WT2 band hybridization, without the presence of confirmatory mutation bands. Such instances may lead to the false interpretation of the result as FQ resistance in settings with high prevalence of the 80Ala and 90Gly polymorphisms that do not confer resistance. Performance of the MTBDRsl LPA could thus be improved by omitting all gyrA WT probes and adding the few missing mutant probes well documented to confer FQ resistance, so that all clinically relevant mutations are confirmed by a mutant band.
ACKNOWLEDGMENTS
This work was funded by a grant from the Directorate General of Development Cooperation of the Belgian Government through the institutional collaboration program between the ITM and the INRB and a grant by the Vlaamse Inter Universitaire Raad (VLIR) (Belgium).
Footnotes
Published ahead of print 28 May 2014
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