Robustness of the Line Probe Assay for the Rapid Diagnosis and Characterization of Mutations in Extensively Drug-Resistant Tuberculosis

Abstract

Introduction:

Extensively drug-resistant tuberculosis (XDRTB) is a public health concern. We evaluated the diagnostic accuracy of Genotype^® MTBDRsl for detection of resistance to fluoroquinolones (FQs) and second-line injectable drugs (SLIDs) and characterized mutations seen.

Materials and Methods:

MTBDRsl was carried out either directly on sputum samples or indirectly on culture isolates (n = 100) from known multidrug-resistant tuberculosis (MDRTB) patients from July 2015 to September 2017. Diagnostic accuracy for the detection of resistance to FQs and SLIDs was calculated in comparison with conventional culture-based drug susceptibility testing. Mutations at the gyrA and rrs loci, as well as discrepant phenotypic and genotypic results, were studied. A subset of isolates underwent pyrosequencing.

Results:

Out of 100 MDRTB samples/isolates tested, 59% were pre-XDRTB and 7% were XDRTB. The sensitivity and specificity for the detection of resistance to FQs were 96.6% [95% confidence interval (CI): 88.3–99.6] and 80% [95% CI: 64.4–90.9] and those for SLIDs were 70% [95% CI: 34.8–93.3] and 100% [95% CI: 95.9–100]. The most frequent mutations were the absence of wild type 3 with corresponding mutation 3c (20/66) at the gyrA locus, and absence of wild type 1 and corresponding mutation 1 (6/7) at the rrs locus. The absence of a wt2 band with a corresponding mutation at the gyrA locus was seen in four of eight patients with discrepant genotypic and phenotypic results for FQ resistance. All isolates tested by pyrosequencing (n = 5) were concordant with the line probe assay for FQ resistance with identical mutations (D94G) and four of five isolates were concordant with SLIDs with identical mutations (A1401G).

Conclusion:

The MTBDRsl is a useful test for accurate diagnosis of XDRTB and may help to tailor therapy.

Introduction

Drug-resistant tuberculosis (TB) threatens global TB care and prevention, and remains a public health concern. Multidrug-resistant TB (MDRTB) is defined as TB resistant to both rifampicin and isoniazid, the backbone of the first-line regimen. The rollout of Xpert MTB/RIF for simultaneous detection of TB and resistance to rifampicin has ensured that an increasing number of MDRTB cases are being detected and notified.¹ Extensively drug-resistant TB (XDRTB) is defined as MDRTB along with resistance to at least one fluoroquinolone (FQ) and a second-line injectable drug (amikacin, capreomycin or kanamycin) (SLID), the two most important drugs in an MDRTB regimen, and has been reported in 105 countries.² Pre-XDRTB is defined as MDRTB along with resistance to either the FQs or SLIDs.

Appropriate management of these patients is dependent on conventional drug susceptibility testing (DST), often not available in time to tailor an effective regimen.³ Molecular methods have considerable advantages in this setting, offering a rapid and accurate diagnosis. Although several commercial assays are available for the diagnosis of MDRTB, those for XDRTB are limited. The Genotype^®MTBDRsl, Hain Lifescience, Nehren, Germany (henceforth MTBDRsl), a line probe assay, is the only WHO-recommended test currently available for the diagnosis of XDRTB. It incorporates probes to detect mutations at the gyrA and rrs loci for version 1.0 and additional mutations for FQs (gyrB) and kanamycin (eis promoter) in version 2.0.⁴ A WHO Expert Group recommends that the MTBDRsl cannot replace culture-based DST, but may be used as a rule in test for XDRTB.⁵

The mutations detected by the MTBDRsl can predict response to drugs, therefore, allowing modification of therapy early on in the course of treatment.⁶ In addition, recent literature has justified the use of these mutations to predict patient outcomes. Unfavorable outcomes have been identified with gyrA 94 mutants other than 94Ala for FQs. Any mutation at codon 94 can be identified by the MTBDRsl by the absence of the WT3 probe; 94Gly mutations can be detected by MUT3c, which have a poor prognosis. Understanding FQ and SLID resistance mechanisms at a molecular level should facilitate rapid and accurate diagnosis of XDRTB and aid treatment options.⁸ With regard to SLIDs, mutants with the rrs mutation A1401G display low-level resistance to capreomycin and high-level resistance to amikacin and kanamycin, whereas those with the C1402T mutants are susceptible to amikacin and have low-level resistance to kanamycin and high-level resistance to capreomycin. Finally, the rrs G1484T mutants display high-level resistance to all three drugs.⁹ Studies on proteomics have demonstrated that up to a third of isolates can harbor mutations in regions other than the rrs locus for resistance to SLIDs and this is a limitation of this assay.^10–13

There is a paucity of data on mutations causing resistance from our region and, therefore, our objectives were to evaluate the diagnostic accuracy of MTBDRsl v.1 for the rapid diagnosis of XDRTB in a high-burden center in south India, and to characterize common mutations encoding resistance to FQs and SLIDs commonly seen in our patients.

Materials and Methods

Study setting

This study was done at a 2,600-bed tertiary care referral center in Vellore, Tamil Nadu, southern India, with an outpatient turnover of 9,000 cases per day. Patients suspected of TB are seen at the Directly Observed Short Course clinic and the outpatient service of the departments of general medicine, infectious diseases, and pulmonary medicine. The mycobacteriology laboratory is accredited by the Revised National Control Programme for Tuberculosis to perform culture and DST and is part of the programmatic management of drug-resistant tuberculosis.

Study population

Clearance for this study was obtained from the institutional review board (IRB Min No: 11410). One hundred and nineteen consecutive pulmonary MDRTB patients (confirmed by Xpert MTB/Rif or culture-based DST), who were >18 years with a bronchoalveolar lavage (BAL)/sputum sample that had either a smear status of at least 1+ on smear microscopy or growth on culture, were included in this study carried out from July 2015 to September 2017. Of these, 59 had a smear grading of ≥1+ and were processed directly by MTBDRsl, the remaining 60 were graded as smear negative or scanty acid fast bacilli (AFB) and, therefore, culture growth was awaited to carry out testing. Seven samples did not grow on culture and 11 samples did not have DST as a follow on request. One sample was indeterminate by MTBDRsl. Therefore, 100 samples were available for analysis. The patient electronic records were reviewed further for demographic and clinical data.

Sample processing

All samples were processed in a Biosafety Level (BSL) three laboratory in the mycobacteriology laboratory at the department of microbiology. Smear microscopy by auramine staining was done on all unconcentrated sputum samples.

Culture-based DST (1% proportion method on Lowenstein Jensen medium)

Concentration and decontamination were carried out using the Petroffs method preceding solid culture on Lowenstein Jensen (LJ) medium as per standard operating protocol. All contaminated cultures were repeat tested once, and if resolved, they were included for analysis. Confirmation of Mycobacterium tuberculosis complex was performed with an MPT64 protein detection-based immunochomatographic test (SD Bioline Kit; Standard Diagnostics, Inc., Korea). The WHO-recommended 1% proportion method was used for DST by the LJ medium as the reference standard, with recommended concentrations of ofloxacin (4 μg/mL), capreomycin (40 μg/mL), and kanamycin (30 μg/mL).

Genotype MTBDRsl assay

All sputum sediments, or culture isolates (if the sample was smear negative or scanty), underwent DNA extraction using Genolyse A and B as per manufacturer's instructions. The PCR on DNA from culture isolates was performed using the following parameters: 95°C for 15 min, 95°C for 30 sec, 58°C for 2 min (10 cycles), 95°C for 25 sec, 53°C for 40 sec, 70°C for 40 sec (20 cycles), and final extension at 70°C for 8 min (the parameters used for detecting DNA from sputum used 30 cycles of elongation), followed by reverse hybridization according to manufacturer's instructions (MTBDRsl v.1; Hain Lifesciences, Germany). A valid MTBDRsl result was defined by a M. tuberculosis complex-specific control, conjugate controls, and amplification control bands in conjunction with the target gene locus control.

Pyrosequencing

Pyrosequencing was carried out on five representative isolates. In brief, DNA extraction was carried out with Tris–chloroform HCl, amplification of target genes using primer sets as described by Lin et al.¹⁴ followed by sequencing of isolates using the Pyromark Q24 1D system. Sequences were then aligned against the NCBI library containing wild type and mutant sequences.

Statistical analysis

The sensitivity, specificity, and likelihood ratios were calculated for each drug compared with the gold standard of culture-based DST. Statistical analyses were performed using STATA SE (version 12; StataCorp.).

Results

In this study population, by the gold standard culture-based DST, 50% of the MDRTB samples/isolates were resistant to rifampicin, isoniazid, and the FQs; therefore, pre-XDR, 10% were XDRTB, and the remaining 40% were MDRTB. However, by the MTBDRsl assay, 59% of the isolates were pre-XDRTB, whereas 7% of isolates were XDRTB. When compared with the gold standard, the sensitivity and specificity of MTBDRsl (Table 1) were 96.6% [95% confidence interval (CI): 88.3–99.6] and 80% [95% CI: 64.4–90.9] for FQs (+LR ∼4.8 and −LR ∼0.04) and 70% [95% CI: 34.8–93.3] and 100% [95% CI: 95.9–100] for SLIDs, namely capreomycin and kanamycin (−LR ∼0.3), respectively.

Table 1.

Accuracy Indices for MTBDRsl

	Sensitivity (%)	Specificity (%)	+Likelihood ratio	−Likelihood ratio
Ofloxacin (n = 100)	96.6 (88.3–99.6)	80 (64.4–90.9)	4.4 (2.6–9.0)	0.04 (0.01–0.16)
Kanamycin (n = 100)	70 (34.8–93.3)	100 (95.9–100)	NA	0.3 (0.12–.77)
Capreomycin (n = 100)	70 (34.8–93.3)	100 (95.9–100)	NA	0.3 (0.12–.77)

NA, not applicable.

The most frequent mutations (Table 2) were the absence of wild type 3 with corresponding mutation 3c (20/66) at the gyrA locus; a single strain was heteroresistant with both mut 3b and 3c at the locus for gyrA. The absence of wild type 1 and corresponding mutation 1 (six of seven) was most common at the rrs locus.

Table 2.

Frequency of Mutations Detected by MTBDRsl

Locus	Probe	Gene region	Total
gyrA (n = 66)	mut 1	A90V	2
	mut 2	S91P	1
	mut 3b	D94N/Y	3
	mut 3b+mut 3c	D94N/Y, D94G	1
	mut 3c	D94G	4
	Δwt1+ mut 1	85–90, A90V	2
	Δwt2	89–93	2
	Δwt2+ mut 1	89–93, A90V	9
	Δwt2+ mut 2	89–93, S91P	2
	Δwt3	92–97	9
	Δwt3+ mut 2	92–97, S91P	2
	Δwt3+ mut 3a	92–97, D94A	1
	Δwt3+ mut 3b	92–97, D94N/Y	8
	Δwt3+ mut 3c	92–97, D94G	20
rrs (n = 7)	mut 1	A1401G	1
	Δwt1+ mut 1	1401–1402, A1401G	6

Of five isolates that underwent pyrosequencing, all were concordant with the line probe assay for FQ resistance with identical mutations (D94G) and four of five isolates were concordant with SLIDs with identical mutations (A1401G); one isolate failed to bind at the rrs locus (Table 3).

Table 3.

Results of MTBDRsl, Culture-Based Drug Susceptibility Testing, and Pyrosequencing

	MTBDRsl			DST by LJ
ISOLATE	FQs	SLIDs	MTBDRsl mutations	FQs	SLIDs	Pyrosequencing
1	Resistant	Resistant	D94G	Resistant	Resistant	GAC94GGC
1			A1401G			A1401G
2	Susceptible	Susceptible	—	Susceptible	Susceptible	WT
2			—			WT
3	Resistant	Resistant	D94G	Resistant	Resistant	GAC94GGC
3			A1401G			A1401G
4	Resistant	Susceptible	D94G	Resistant	Susceptible	GAC94GGC
4			—			WT
5	Susceptible	Susceptible	—	Susceptible	Susceptible	WT
5			—			Probe fail

DST, drug susceptibility testing; LJ, Lowenstein Jensen; WT, wild type.

Discussion

Molecular-based assays are designed to detect specific drug resistance-encoding mutations in M. tuberculosis and achieve faster turnaround times (within 48 hr) for resistance reporting compared with culture-based DST. In addition, they alert clinicians to the emergence of drug resistance in M. tuberculosis strains from individual patients. Early detection of drug resistance is crucial to preventing the transmission of drug-resistant TB and averting mortality.¹⁵ The MTBDRsl can also play a role in guiding treatment by providing valuable information on mutations of two important classes of drugs used in MDRTB, the FQs and SLIDs. The prevalence of pre-XDRTB and XDRTB in our study was 59% and 7%, respectively. Thus emphasis needs to be given to the identification of pre-XDRTB among MDRTB patients to ensure cure and halt disease progression. Resistance to FQs is associated with its irrational use for other bacterial infections, which has been described in India.¹⁶

The sensitivity of MTBDRsl for the detection of resistance to FQs was 96.6% [95% CI: 88.3–99.6] (Table 1). Mutations in the gyrB gene also cause FQ resistance and could explain the two cases wherein detection of resistance was missed by MTBDRsl and phenotypic resistance was present.¹⁷ This could be overcome by the use of the second version of this assay, which has a locus for the gyrB gene.

However, the assay had a specificity of 80% [95% CI: 64.4–90.9], lower than existing literature both in India and internationally.¹⁸ Eight strains resistant by the MTBDRsl were susceptible by DST, of which four of eight strains had an absence of wt2 with a corresponding mutation. Certain double mutations at gyrA (T80A and A90G substitutions) can lead to false-positive resistance by masking of binding to the wild type probes, commonly wt2.^19,20 Of our false-resistant isolates, however, none had an absence of a wild type band hybridization alone (Table 4). A second explanation, acknowledged by Hain, is that synonymous mutations can result in false-resistant results. For these reasons, Ajileye et al. suggest that FQs may be kept in the regimen but not counted as an effective agent until false positives are systematically excluded by phenotypic DST.²¹

Table 4.

Discrepant Results for Fluoroquinolones Between MTBDRsl and Culture-Based Drug Susceptibility Testing

		MTBDRsl result
S. No.	Diagnosis (phenotypic result)	FQs	SLIDs	Mutation at gyrA	Treatment regimen^a	Treatment completed	Outcome
1	MDRTB	Resistant	Susceptible	Δwt2+ mut 1	Weight-based MDRTB regimen	Defaulter	Expired
2	MDRTB	Resistant	Susceptible	mut 3c	Weight-based MDRTB regimen	No	Smear positive at 3 months of ATT
3	MDRTB	Resistant	Susceptible	Δwt2+ mut 1	Weight-based MDRTB regimen	Yes	Clinically better
3	MDRTB	Resistant	Susceptible	Δwt2+ mut 1	Weight-based MDRTB regimen	Yes	Culture negative
4	MDRTB	Resistant	Susceptible	Δwt3+ mut 3c	Weight-based MDRTB regimen	ND	Clinically better
4	MDRTB	Resistant	Susceptible	Δwt3+ mut 3c	Weight-based MDRTB regimen	ND	Culture negative
5	MDRTB	Resistant	Susceptible	Δwt2+ mut 2	Weight-based MDRTB regimen	Yes	Clinically better
5	MDRTB	Resistant	Susceptible	Δwt2+ mut 2	Weight-based MDRTB regimen	Yes	Culture negative
6	MDRTB	Resistant	Susceptible	mut 3b	Weight-based MDRTB regimen	Yes	Clinically better
6	MDRTB	Resistant	Susceptible	mut 3b	Weight-based MDRTB regimen	Yes	Cultures not received
7	MDRTB	Resistant	Susceptible	Δwt2+ mut 1	Weight-based MDRTB regimen	Yes	Clinically better
7	MDRTB	Resistant	Susceptible	Δwt2+ mut 1	Weight-based MDRTB regimen	Yes	Culture negative
8	MDRTB	Resistant	Susceptible	Δwt3+ mut 3b	Weight-based MDRTB regimen	ND	Clinically better
8	MDRTB	Resistant	Susceptible	Δwt3+ mut 3b	Weight-based MDRTB regimen	ND	Culture negative

With fluoroquinolones.

FQs, fluoroquinolones; SLIDs, second-line injectable drugs; MDRTB, multidrug-resistant tuberculosis.

Table 4 describes the treatment regimens and the outcome of the patients harboring these discrepant strains. In this group, all patients were on a weight-based MDR regimen that included FQs, six of eight patients were clinically better and culture negative at the end of treatment, one of eight patients expired, and one of eight patients had not completed treatment and was smear positive. The most common mutation in this group was the absence of wt2 along with a corresponding mutation, seen in four patients (Table 4), of whom all but one patient, a defaulter, were clinically better and culture negative on follow-up.

The most common mutation (Table 4) was the absence of wild type 3 with corresponding mutation 3c (D94G) at the gyrA locus (n = 20). Our results for detection of resistance to FQs contrast significantly with the results of two studies conducted in Congo and the Democratic Republic of Congo, where >60% of FQ resistance was attributable to the sole absence of wt probe hybridization,^19,20 but are similar to data from a recent multicentric study, including centers from India, where the majority of resistant strains were due to hybridization of mutation probes.^22,23 Recent data from Bangladesh suggest that gyrA mutations at position 94, other than Ala, predict high-level resistance to moxifloxacin, as well as poor treatment outcome in MDRTB patients in whom an injectable agent is still effective, whereas a failure of hybridization of wt3 and mutation 3a (corresponding to 94Ala) has good outcome—we report only one such mutation in this cohort.⁷

Out of the 100 samples tested, MTBDRsl missed the detection of resistance to SLIDs in three isolates when compared with conventional DST, and had a sensitivity of 70% [95% CI: 34.8–93.3]. Specificity was 100% [95% CI: 95.9–100] for capreomycin and kanamycin (Table 1). Discordance of capreomycin resistance between molecular and phenotypic methods is common due to unreliable critical concentrations and lack of consistent molecular markers, which may explain the three discrepant results. Reeves et al. suggest that the use of higher critical concentrations may resolve this issue.²⁴ The Beijing strain, commonly seen in India, has high rates of kanamycin resistance, which this study fails to describe.²⁵ The use of the second version of this assay, which bears a locus for the eis promoter region, may detect more capreomycin-sensitive but low level kanamycin-resistant strains.²⁶ Our data closely mirror that from India and other high-burden centers across the globe for specificity, but are marginally lower than previous studies for sensitivity.^27–29 The mutation most commonly seen at the rrs locus in this study (Table 2) was the absence of wt1 and hybridization of the mut 1 probe (A1401G) (n = 6). This is commonly associated with cross-resistance between kanamycin, amikacin, and capreomycin, and indicates that none of these agents can be used in the treatment regimen.³⁰ The same pattern of mutations was demonstrated in a multicentric study that included India, Moldova, and South Africa.³¹

All isolates that were tested by pyrosequencing (n = 5) confirmed the mutations for FQs and SLIDs, but for one at the rrs locus that was a “probe fail.” The results of a study on pyrosequencing (unpublished data) from our center also described the most common mutations, D94G in 55% of the FQ-resistant clinical isolates and A1401G in 50% of the clinical isolates for SLIDs, that is, resistant to amikacin, kanamycin, and capreomycin.

Limitations

MTBDRsl v.1 appears to have utility predominantly on smear-positive samples—this remains a limitation of the assay.³² With superior sensitivity on smear-negative samples, MTBDRsl v.2 is an improvement over its predecessor.³³ At the onset of this study, the second version was not available commercially, however, is currently being evaluated. The low number of XDRTB isolates was a limitation of this study. Sequencing of all strains was not carried out and this is a limitation that future study must focus on.

Conclusion

The role of molecular testing for the diagnosis is a cornerstone in accurate and rapid diagnosis of drug-resistant TB. The MTBDRsl is a part of the battery of tests available for the diagnosis of drug-resistant TB, and from our study, proves to be a good rule-out test for the diagnosis of FQ resistance and an excellent rule-in test for the diagnosis of resistance to SLIDs. The most common mutations detected correlated well with other studies from India. A large number of pre-XDR cases, for which clear management guidelines are not available, were present from this cohort of patients. Pyrosequencing confirmed the mutations detected by the MTBDRsl in the subset tested. Of interest were the mutations seen in the discrepant results between molecular and phenotypic testing for FQs, which showed that 50% were due to absence of wild type 2 with a corresponding mutation, and of which three of four patients had improved while on an FQ-based regimen, indicating that mutations in this region may promise a good prognosis.

Footnotes

Disclosure Statement

M.M.N., P.R., P.J., and J.S.M. have no competing financial interests/conflicts of interest.

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