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 Table of Contents  
Year : 2022  |  Volume : 11  |  Issue : 2  |  Page : 183-189

Diagnostic utility of GenoType MTBDRsl assay for the detection of moxifloxacin-resistant mycobacterium tuberculosis, as compared to phenotypic method and whole-genome sequencing

1 Department of Microbiology, National Reference Laboratory and Canter of Excellence (TB) WHO, National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India
2 Department of Thoracic Surgery and Surgical Anatomy, National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India

Date of Submission18-Feb-2022
Date of Decision10-May-2022
Date of Acceptance28-May-2022
Date of Web Publication14-Jun-2022
Date of Print Publicaton14-Jun-2022

Correspondence Address:
Ritu Singhal
Department of Microbiology, National Reference Laboratory and Center of Excellence (TB) WHO, National Institute of Tuberculosis and Respiratory Diseases, New Delhi - 110 030
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmy.ijmy_70_22

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Background: Recently, moxifloxacin (MFX)-resistant results of Mycobacterium tuberculosis (Mtb) obtained by GenoType MTBDRsl (second-line line probe assay [SL-LPA]) have been stratified to determine their resistance level; however, its accuracy has not been well studied. Therefore, the study aimed to evaluate the diagnostic accuracy of SL-LPA, with phenotypic drug susceptibility testing (pDST) and whole-genome sequencing (WGS) for the detection of MFX-resistant Mtb and their resistance level. Methods: A total of 111 sputum samples were subjected to SL-LPA according to the diagnostic algorithm of the National Tuberculosis Elimination Program. Results were compared with pDST of MFX (at critical concentration [CC, 0.25 μg/ml] and clinical breakpoint [CB, 1.0 μg/ml] using BACTEC mycobacterial growth indicator tube-960), and WGS. Results: At CC, SL-LPA and pDST yielded concordant results of MFX for 104 of 111 (94%). However, at CB, 23 of 30 (77%) isolates carrying gyrA mutation known to confer low-level resistance to MFX were scored as susceptible by pDST. Among 46 Mtb isolates carrying gyrA mutations known to confer high-level resistance to MFX, 36 (78%) isolates yielded concordant results, while 10 (22%) isolates were scored as susceptible at CB by pDST. WGS identified gyrA mutations in all isolates suggested by SL-LPA. Conclusion: It is concluded that the stratification of MFX-resistant results by SL-LPA/genotypic method is not very well correlated with pDST (at CB), and hence, pDST may not be completely replaced by SL-LPA. gyrA D94G and gyrAA90V are the most prevalent mutations in MFX-resistant Mtb.

Keywords: Clinical breakpoint, critical concentration, drug-resistant tuberculosis, moxifloxacin, whole-genome sequencing

How to cite this article:
Yadav RN, Bhalla M, Kumar G, Sah GC, Dewan RK, Singhal R. Diagnostic utility of GenoType MTBDRsl assay for the detection of moxifloxacin-resistant mycobacterium tuberculosis, as compared to phenotypic method and whole-genome sequencing. Int J Mycobacteriol 2022;11:183-9

How to cite this URL:
Yadav RN, Bhalla M, Kumar G, Sah GC, Dewan RK, Singhal R. Diagnostic utility of GenoType MTBDRsl assay for the detection of moxifloxacin-resistant mycobacterium tuberculosis, as compared to phenotypic method and whole-genome sequencing. Int J Mycobacteriol [serial online] 2022 [cited 2023 Mar 31];11:183-9. Available from: https://www.ijmyco.org/text.asp?2022/11/2/183/347526

  Introduction Top

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a leading cause of mortality worldwide. Reduced access to early diagnosis may increase the number of people dying from TB. Only 71% (2.1/3.0 million) of persons with bacteriologically proved pulmonary TB were tested for rifampicin (RIF) resistance in 2020, and testing of fluoroquinolone (FLQ) resistance was also extremely low, a little over 50%.[1] The rise of drug resistance TB is a severe impediment to TB eradication. Moxifloxacin (MFX) is an important FLQ drug recommended by the World Health Organization (WHO) for the treatment of multidrug-resistant TB (MDR-TB; defined as Mtb strains resistant to first-line anti-TB drugs RIF and isoniazid).[2],[3],[4],[5],[6] Inappropriately use of FLQ has resulted in the establishment of FLQ-resistant TB, contributed to the rise of extensively drug-resistant TB (XDR-TB; defined as Mtb resistant to RIF, plus any FLQ, plus at least one of the drugs bedaquiline [BDQ] and linezolid [LNZ]).[1]

Early identification of drug-resistant profile is very crucial for appropriate administration of effective treatment. Commercial genotypic methods GenoType MTBDRplus assay (first-line line probe assay, FL-LPA) version 2.0 and GenoType MTBDRsl assay (second-line line probe assay [SL-LPA]) version 2.0 are front-line tests available for early detection of DR-TB from acid-fast bacillus (AFB)-positive pulmonary samples and Mtb isolates. The FL-LPA is used to detect Mtb complex resistant to first-line anti-TB drugs and is therefore suited to diagnose MDR-TB.[7] The SL-LPA is being used as follow on test, for detecting additional resistance to second-line anti-TB drugs (FLQ, aminoglycosides [AG] and cyclic peptides).[8] However, these rapid tests are only capable of detecting preset drug resistance mutations in small segments of gene, while there are already over 1000 drug resistance mutations in online databases.[9] Whole-genome sequencing (WGS), however, offers a full genetic profile for all known drug resistance mutations.

Phenotypic drug susceptibility testing (pDST) depends on the determination of a single critical concentration (CC) of drug, which is used to distinguish resistant from susceptible Mtb strains. However, knowing the DST at the clinical breakpoint (CB, minimum inhibitory concentration (MIC) above CC) is also very important in the case of certain drugs, as it distinguishes Mtb strains that will likely respond to treatment from those that will not respond.[10] Therefore, mutations conferring resistance to MFX obtained by SL-LPA test has recently been stratified as follows: mutation associated with high-level increase in MIC detected (high-level MFX resistance detected) which is associated with resistance at CB; mutation associated with at least low-level increase in MIC detected (low-level MFX resistance detected) which is associated with MIC lies between CC and CB; and low-level resistance inferred (precise mutation unknown).[10] However, there is scarcity of the study to know the correlation and implication of stratified SL-LPA results with pDST (traditional gold standard) and WGS (molecular gold standard).

The aim of the present study is to know the accuracy of SL-LPA to identify the MFX-resistant Mtb as compared to gold standard pDST (by using mycobacterial growth indicator tube [MGIT]) and WGS, and its stratified MFX DST results correlation with pDST.

  Methods Top

Study settings and design

The study was conducted at the National Reference Laboratory and WHO Center of Excellence for TB, Department of Microbiology, National Institute of TB and Respiratory Diseases, New Delhi, India, from September 2020 to February 2022. The sputum samples were subjected to AFB microscopy by Ziehl–Neelsen (ZN) staining method and results were graded according to standard protocol.[11] All sputum samples were digested, decontaminated, and concentrated by N–Acetyl L-cysteine– sodium hydroxide (NALC-NaOH) method.[12] DNA was extracted by Genolyse kit (Hainlife science, Nehren, Germany) for LPA, directly from those sputum samples which had AFB smear-positive results.[13] The FL-LPA (Version 2.0) was carried out with extracted DNA according to the manufacturer's instruction.[14] Results were considered valid only when both AC and CC probes were present. Probe was only considered as present when its intensity of color was similar or more than that of AC probe. As per the National Tuberculosis Elimination Program diagnostic algorithm,[15] if samples were found as resistant to RIF and/or isoniazid, were further subjected to SL-LPA (version 2.0). H37Rv was included as a positive control and molecular grade water as DNA extraction negative control. Master mixture negative control was used during polymerase chain reaction (PCR) step. Mycobacterial culture was performed with MGIT-960 system among all selected samples where SL-LPA showed resistance to FLQ class drug and/or (SLID). Indirect FL-LPA was performed with AFB smear-negative sputum samples[16] and those with indeterminate/invalid/TUB negative FL-LPA results, through mycobacterial culture (with Mtb identification in case of positive culture results), followed by DNA extraction with same (GenoLyse) kit according to the manufacturer's instructions. The pDST and WGS was performed on Mtb-positive culture isolates.

Second-line line probe assay test

The GenoTypeMTBDRsl assay (SL-LPA) Version 2.0 was performed with manufacturer instruction.[17] Briefly protocol includes DNA extraction, PCR amplification, hybridization, and finally result interpretation. DNA was extracted using Genolysekit (Hain Lifescience, Nehren, Germany) provided with manufacturer. Master mixer for PCR was prepared by using amplification mixes A and B (AM-A and AM-B), provided in kit, with final volume 50 μl that included 35 μl of AM-B, 10 μl of AM-A, and 5 μl extracted DNA. Amplification was performed with preset program on thermal cycler. Hybridization was performed using an automated GT blot (Hain Lifescience, Nehren, Germany) as per the manufacturer's instruction. Each SL-LPA strip contains 27 reaction zones with impregnated probes for all specific mutations to be detected. It includes seven probes for gyrA [A90V (gyrAMut1), S91P (gyrA MUT2), D94A (gyrA MUT3A), D94N/Y (gyrA MUT3B), D94G (gyrA MUT3C), and D94H (gyrA MUT3D)] and two probes for gyrB [N538D (gyrB MUT1), E540V (gyrB MUT2)] to detect resistance to FLQ [Figure 1]. For the detection of SLID resistance, it includes rrs probes (A1401G, C1402T, G1484T) and eis probes.
Figure 1: Representative hybridized strips of GenoTypeMTBDRsl ver. 2 (SL-LPA): Lane 1: Resistance not detected. Lane 2, 3: Mutation associated with at least low-level increase in MIC for MFX (low level MFX resistance) detected, and resistance not detected for SLIDs. Lane 4: Mutation associated with high-level increase in MIC for MFX (high-level MFX resistance) detected high-level resistance to MFX and resistance not detected for SLIDs. Lane 5: Master mixture negative control. Lane 6: DNA-negative control. Lane 7: Positive control (H37RV). MFX: Moxifloxacin, SLID: Second-line injectable drug, SL-LPA: Second-line line probe assay

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Missing (Δ) of WT probe and/or presence of MUT probes was considered as FLQ resistance, as per the manufacturer's instruction. Further, FLQ-resistant results for MFX was stratified as follows: Mutation associated with high-level increase in MIC for MFX detected (high-level MFX-resistant detected), which was represented by the presence of any gyrA MUT3B, MUT3C, MUT3D with or without the presence of other probes); mutation associated with at least low-level increase in MIC for MFX detected (low-level MFX resistance detected) which was represented by the presence of any probe among MUT1, MUT2, MUT3A of gyrA; and/or MUT1, MUT2 probes develops in gyrB region and the absence of other MUT probe); mutation associated with at least low-level increase in MIC inferred (resistance inferred), which was represented by the absence of any WT probe without the presence of any MUT probe, according to recent guidelines;[10] to compare the pDST of MFX.

Mycobacterial culture and phenotypic drug susceptibility testing

Mycobacterial culture was performed with MGIT-960 system consisting of Middle brook 7H9 media. After sample processing (digested, decontaminated and concentrated), sputum sediment was mixed with 1–2 ml phosphate buffer (pH 6.8). A 500 μl of resuspended sputum sediment was inoculated with MGIT tube which had previously been inoculated with growth supplement and reconstituted antimicrobial PANTA (supplied by manufacturer), as per standard protocol.[12] Each inoculated 7 ml MGIT tubes were scanned and entered in BACTEC MGIT-960 instrument. All culture tubes flagged positive by instruments underwent ZN AFB microscopy and immunochromatic assay test (SD bioloine, TB Ag, MPT64 kit) to confirm the Mtb positive.[18] DST for MFX was performed only with Mtb culture positive isolates by MGIT instrument having Middle brook 7H9 liquid medium, following to standard procedure of the WHO with two different final drug concentrations: 0.25 μg/ml and 1.0 μg/ml.[19]

Whole-genome sequencing

DNA extraction for whole-genome sequencing

DNA extraction was performed by bead beating technology using FastPrep-24TM instrument (MP Biolmedicals). Briefly, around 3.6 ml of mycobacterial liquid culture growth (1.8 ml in 2 tubes per sample) was taken, followed by heat killing at 100°C for 30 min. Molecular grade water was taken as a negative control and H37RV strain of Mtb for positive control. It was centrifuged at 13,200 rpm for 15 min to pellet the culture. Supernatant was discarded and pellet was then washed with 1 ml saline solution using centrifugation at the 13,200 rpm for 15 min. Finally pellet was resuspended with 140 μl molecular grade water. 280 μl (140 μl × 2 tubes per sample) of resuspended solution was added to lysis matrix-B tubes (MP Biomedicals). Now, sample tubes was loaded on FastPrep24 instruments (MP Biolmedicals) and bead beating was performed three times with speed of 6.0 m/s for 40 s at 5 min intervals. Once beating was completed sample tubes was centrifuged at 13,200 rpm for 10 min to settle down the beads. Around 50 μl supernatant DNA was transferred to 96-wells plate for further cleaning. For cleanup, this 50 μl of DNA was mixed properly with 90 μl of AMPure XP beads (Agencourt) and kept for room temperature (RT) for 10 min. Plate was then kept on magnetic stand (Thermo Fisher) for 3 min and then supernatant was removed. Samples was then washed two times with 200 μl of 80% chilled ethanol without disturbing the plate, while it was remain kept on magnetic stand. Beads were allowed to air dry for 10 min and plate was removed from magnetic stand. Now, 26 μl elution buffer (EB, Qiagen) was added and resuspended properly and kept on RT. It was again kept on magnetic stands for around 3–4 min then 25 μl of supernatant/DNA (without beads) was transferred to new labeled tubes and kept at –20°C. DNA concentration was measured using the Qubit2.0 flurometer. If any reading was <0.2 ng/μl, it was excluded for further process; whereas, in case of reading more than 60 ng/μl, it was diluted 10 times with EB for further use. All DNA were normalize with EB for final concentration to 0.2 ng/μl for further use.

Library preparation and illumina MiSeq sequencing

Libraries were prepared with normalized DNA for the Illumina MiSeq Sequencing using Nextera XT DNA Library Preparation Kit (Catalog # FC-131-1096). Nextra XT DNA Library Prep protocol was used for library preparation with some minor modification.[20] Original Nextra protocol mentioned 12 cycles for thermal cycler during index PCR amplification, but it was extended to 14 cycles in the present study.[21] Normalization of library (to 1.6 ng/μl equivalent to 4nM) was performed by manually with 0.1% Tween-20 (20 μl of 10% Tween-20 + 1980 μl of Qiagen buffer EB = Buffer A). Library pooling was done by transfer of 5 μl of each normalized library to a single tube and vortexed. The 4 nM pooled library was denatured with equal amount of freshly prepared 0.2N NaOH, and then, it was diluted to 20 pM with HT1 buffer as detailed in protocol.[22] Finally, 12.5 pM library pool was obtained by diluting 375 μl of 20 pM library DNA with 225 μl HT1 buffer and vortexed. A PhiX control was prepared by diluting 10 nM PhiX to 4nM PhiX with buffer A. Again, this PhiX was denatured and diluted to final concentration 12.5 pM, with given protocol (MiSeq system, denature and dilute libraries guide, p. 10-11). Now, 1% PhiX was added to the pool by combining 6 μl of 12.5 pM PhiX to 594 μl of denatured and diluted library pool. It was vortexed and spined and ultimately added into MiSeq cartridge that was further loaded to Illumina MiSeq instrument for sequencing.

Data analysis

FASTQ files were obtained from IlluminaMiSeq sequencer. The raw sequence data were analyzed using command prompt program to perform analysis using mykrobe software to generate SNPs and lineage. The results of all tests were gathered in common excel sheet for the analysis.

  Results Top

A total of 111 samples were included having availability of direct or indirect SL-LPA result. All the sputum samples were further subjected to mycobacterial culture. All the corresponding culture isolates were subjected to WGS; and MFX pDST (at two different concentrations, 0.25 μg/ml [CC] and 1.0 μg/ml [CB]). Any SL-LPA invalid/indeterminate, TUB probe negative, culture contamination and unsuccessful WGS output were excluded from the study. Among these 111 SL-LPA results, 88 were declared as MFX resistance according to the manufacturer's instructions,[10] whereas, in 23, MFX resistance were not detected. Out of 88 MFX-resistant strains, 86 (98%) were confirmed by pDST at CC, while remaining two were found as sensitive. However, all these SL-LPA detected MFX resistance strains showed mutation in gyrA gene by WGS and no mutation was found in gyrB gene. SL-LPA declared resistance not detected in 23 samples [out of which similar results were observed by pDST (at CC) and WGS in 19; whereas, remaining four demonstrated as phenotypic resistant (of which WGS identified mutation in 2: GyrA D94G in 1 and D94N in another one], The concordance for MFX DST results of SL-LPA were 94% (104/111) and 98% (109/111) with pDST at CC, and WGS, respectively.

Furthermore, these 88 FLQ-resistance results were subsequently categorized as high level resistance detected (46, 52.2%), low-level resistance detected (30, 34%) and low-level resistance inferred (12, 13.6%); according to standard guidelines[10] and compared again these categorical results, with WGS, and PDST (at CC and CB). Out of 46 high level MFX-resistant isolates, MFX pDST showed resistant in all at 0.25 μg/ml (CC), however, at higher drug concentration of 1.0 μg/ml (CB), the pDST results were same in 36 (78%) cases with remaining 10 (22%) observed as sensitive, as detailed in [Table 1].
Table 1: Correlation of second-line line probe assay for fluoroquinolone susceptibility results of Mycobacterium tuberculosis with phenotypic drug susceptibility testing and whole-genome sequencing

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Among 30 low-level MFX resistance detected by SL-LPA, MFX pDST at CC also showed resistance in 29 (97%) cases and one (3%) isolate was sensitive, while at CB, most (23/30, 77%) were sensitive, but 7 (23%) showed resistant. All the high level and low level detected results showed corresponding mutation with WGS, as shown in [Table 2]. Similarly, among 12 SL-LPA detected low-level resistance inferred cases, 11 (91%) were resistant in CC according to pDST and remaining one (9%) was sensitive. However, pDST at CB showed resistant in 3 (25%) and 9 (75%) were found as sensitive.
Table 2: Mutation patterns of fluoroquinolone resistant Mycobacterium tuberculosis detected by Second-line line probe assay Version 2.0 and whole-genome sequencing in 88 fluroquinolone-resistant Mycobacterium tuberculosis

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Gene mutation patterns detected by second-line line probe assay and whole-genome sequencing

gyrA Δ WT3/MUT3C was the most prevalent (35/88, 40%) mutations/probe patterns observed by SL-LPA, followed by gyrA Δ WT2/MUT1 (24/88, 27%), gyrA Δ WT3/MUT3B (7/88, 8%), gyrA Δ WT2/MUT2 (5/88, 6%); and the corresponding mutations identified by WGS were gyrA D94G, gyrA A90V, gyrA D94N, and gyrA S91P respectively.

The following mutations/probe patterns were found among high-level MFX resistance detected by SL-LPA: GyrA Δ WT3/MUT3C (35/46, 76%), gyrA Δ WT3/MUT3B (7/46, 15.3%), gyrA MUT3C (4/46, 8.7%). All the SL-LPA identified gyrAWT3/MUT3C and gyrA WT3/MUT3B mutations were confirmed by WGS, which revealed matching mutations as gyrA D94G, and gyrA D94N, respectively. However, WGS revealed dual gyrA (D94G + D94Y) mutations in one isolate, and identical corresponding mutations (gyrAD94G) in the remaining isolates among the gyrA MUT3C (without missing of WT probe, hetero-resistance) mutations detected by SL-LPA.

The most common mutation in low-level MFX resistance detected was gyrA Δ WT2/MUT1 (24/30, 80%), which was identified by WGS as gyrA A90V mutations. gyrA Δ WT2/MUT2 (5/30, 16.7%) was another mutation found by SL-LPA, while the matching mutations identified by WGS was gyrA S91P. In one (3.3%) sample, SL-LPA revealed MUT1 probe without missing of any WT probe (hetero-resistance), whereas, WGS revealed gyrA A90V mutation.

SL-LPA showed low-level resistance inferred in 12 isolates with following mutation/probe patterns; gyrA Δ WT1 (3/12, 25%), gyrA Δ WT2 (5/12, 41.7%) and gyrA Δ WT3 (4/12, 33.3%) probes. Among 3 gyrA Δ WT1 probes, WGS identified gyrA G88C mutation in 2 (66.7%) and gyrA G88A (33.3%) mutation in remaining one. All gyrAWT2 missing results detected by SL-LPA were also identified by WGS as gyrA A90V mutation. Out of four samples showing missing of WT3 probes, in three cases WGS identified mutation D94A in the same gene region, however, gyrAA90V mutation was found in one. A total 20 types of lineage and sub lineage were identified in these 88 FLQ-resistant Mtb by WGS through Mykrobe software in the present study, among which most prevalent were: Lineage 3 (28, 32%), lineage 2.2 (13, 15%), lineage 2.2.3 (11,12%), lineage 2.2.7 (9, 10%), lineage 4.1 (5, 6%).

  Discussion Top

Over the last decades, tremendous progress has been achieved in the early diagnosis of DR-TB, which helps in prompt treatment of TB patients and hence curtailing spread of DR-TB. In the present study, SL-LPA results of MFX DST were correlated with standard MFX pDST at CC (0.25 μg/ml) and CB (1.0 μg/ml) using MGIT-960 and WGS. The concordance for MFX DST results of SL-LPA were 94% (104/111) and 97% (108/111) with pDST at CC, and WGS, respectively. All the 88 samples (showing resistant on SL-LPA (based on alteration in gyrA gene sequence/probe), had also showed mutation (100%) with WGS. Therefore, the results of the present study showing the good performance of SL-LPA for detecting MFX-resistant Mtb. These findings are in agreement with previous study where authors found 94% sensitivity of SL-LPA for detection of MFX-resistant Mtb with pDST at this CC.[23]

The relation between genomic mutations and phenotypic resistance is particularly significant for therapeutic decision as different mutations might induce distinct resistant profiles.[24] In the present study, 23% (7/30) of low-level resistance detected and 42% (5/12) of low-level resistance inferred SL-LPA findings were found to be resistant at CB of pDST. Showing that not all low-level resistance detected/inferred by SL-LPA would be sensitive at CB with pDST and hence, these finding supports the recommendation that SL-LPA detected low-level resistance/inferred should be subjected to pDST at CB for re-evaluation of treatment regimen, as these patients may responds treatment at high dose of MFX.[10],[19] In addition, 22% (10/46) of SL-LPA high-level MFX resistant detected results, found as sensitive at pDSTs CB. Hence, the present findings showing that stratified genotypic MFX DST results may not be accurately standardized with pDST at higher drug concentration (CB). In our knowledge, it is the first kind of study to describe the frequency of correlation of stratified MFX DST results of SL-LPA (version 2.0) with pDST at CB.

Spontaneous chromosomal mutations occur at a frequency of 10-6–10-8 mycobacterial replications, resulting in the genetic resistance to anti-TB drug.[25] Mtb resistance to FLQ is mostly caused by mutations in the quinolone resistance determining region of DNA subunits A (gyrA) and B (gyrB), which encode type II DNA topoisomerases.[26] The previous studies substantiating that FLQ resistance of Mtb is predominantly (60%–100%) arises through gyrA gene (codons 74–113, particularly at codons 88, 90, 91, 94); whereas, mutation in gyrB gene (codons 500–538) is occasional and often co-occur with canonical gyrA mutations.[27],[28],[29],[30],[31] In a systematic review conducted by Avalos et al.,[30] 87% of MXF-resistant Mtb isolates had mutations in their gyrA gene. In their study, the cumulative frequency of individual mutation for FLQ resistance was highest for gyrA D94G (ranging from 21% to 32%), followed by gyrA A90V (ranged 13%–20%), whereas, the gyrA G88C and D94V were found as least frequent (1%–2%). The development of such resistance can lead to the transition of MDR-TB patients to pre-XDR-TB, and then, they can become XDR-TB with further resistance to at least BDQ or LNZ.

In the present study, all the mutated Mtb strains showed mutations in the gyrA gene only (100%). Among these, the most prevalent mutation detected by SL-LPA was gyrA ΔW3 /MUT3C (35/88, 40%) and gyrA ΔWT2/MUT1 (24/88, 27%) and the corresponding mutations revealed by WGS was gyrA D94G and gyrA A90V, which is in agreement with previous study from India.[32]

The present study also found discordance in mutation profile obtained between LPA and WGS. One strain demonstrated gyrA ΔWT3 results with SL-LPA but WGS gyrA A90V mutation. Both SL-LPA and WGS could not find mutation for FLQ resistance from two samples which were resistant with pDST at CC. It may be due to the presence of mutations in gene region which was not incorporated in LPA[33] and could not available in catalogue of Mykrobe software. The average time for WGS test from DNA extraction to results interpretation was 1 week. LPA has several advantages including comparatively simple protocol (as compared the WGS) to carried out the test and able to provide results within 2-3 days. However, it is not applicable to all important anti-TB drugs and not relevant directly to extrapulmonary samples which is paucibacillary in nature.

These days, a lot of attention has been given to mycobacterial WGS. By referencing frequently updated resistance database, mycobacterial WGS can offer complete genetic resistance profile for all known drug resistance mutations in one test. It has the ability to monitor emergence of new resistance mechanism as well as high-resolution epidemic surveillance. However, there are limitations with WGS as: It relies on having excellent quality DNA, which necessitates an initial culture stage to cover significant coverage depth.[9] Furthermore, it has comparatively complex protocol and requirement of enough skill sets to perform the test and therefore, it might be challenging for using frequently as a routine test in high burden and resource constraint setting.

  Conclusion Top

Based on this study, it is concluded that, stratification of MFX-resistant results by genotypic method/SL-LPA is not well correlated with pDST (at CB). gyrA D94G and A90V are the most prevalent mutations in MFX-resistant Mtb demonstrated by LPA and WGS. Further studies with large number of samples required to enhance our understanding about clinical correlation of MFX stratified results of genotypic DST and pDST.

Limitation of the study

The study has some limitations. The study has been conducted with conveniently selected samples with small sample size. Second, we could use freely downloadable Mykrobe software for mutation analysis with WGS results. The software may capture only defined mutations and therefore, it could overlooked other mutations that were beyond to its catalogue. This software is providing drug resistance mutations for 14 anti-TB drugs at a time along with lineage information; however, presently, it is unable to providing mutations conferring resistance to newer anti-TB drugs such as BDQ and LNZ.


We are thankful to all staffs of Department of Microbiology in completing this work. Authors acknowledge the support of Foundation for Innovative New Diagnostics and Central TB Division, Ministry of Health and Family Welfare.

Ethical clearance

The study was conducted in samples collected in national programme without patient interview, thus ethical clearance not required.

Financial support and sponsorship

Logistic support from Foundation for Innovative New Diagnostics.

Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Table 1], [Table 2]


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