|Year : 2018 | Volume
| Issue : 1 | Page : 74-81
Genetic polymorphism of rare mutations in Mycobacterium tuberculosis-infected patients in Delhi
Himanshu Vashistha1, Mahmud Hanif1, Kamal Kishore Chopra1, Divya Shrivastava2, Ashwani Khanna3
1 New Delhi Tuberculosis Centre, New Delhi, India
2 School of Life Sciences, Jaipur National University, Jaipur, Rajasthan, India
3 State Tuberculosis Office, Gulabi Bagh Chest Clinic, Delhi State, Delhi, India
|Date of Web Publication||5-Mar-2018|
Dr. Mahmud Hanif
New Delhi Tuberculosis Centre, Jawaharlal Nehru Marg, Delhi Gate, New Delhi - 110 002
Source of Support: None, Conflict of Interest: None
Background: There is a wide variation in existing Mycobacterium tuberculosis strains across the globe, and false results in line probe assay (LPA) can occur due to the presence of unique genetic mutations in different settings. Objectives: An attempt was made to observe uncommon mutations in multidrug-resistant tuberculosis (MDR-TB) strains and determination of genetic diversity by spoligotyping and to study the treatment outcome in patients with uncommon mutations. Materials and Methods: Band pattern analysis of LPA strips was performed as per manufacturer's instructions. DNA sequencing was performed to confirm the presence of uncommon mutations in Intermediate Reference Laboratory in Delhi state. Results: Four uncommon mutations were recognized along with 12 unique spoligotype patterns which serve as an update to worldwide databases. The noteworthy presence of a spoligotype previously rarely seen in India was the SIT53/T1 pattern. Central Asian (CAS) spoligotype was highly associated with MDR followed by Beijing type. During follow-up, the treatment outcomes of cases showing uncommon mutations were considered as cured, after completion of their treatment. Conclusion: The rifampicin resistance appears to be an effective marker of MDR-TB. The presence of uncommon mutations confirms genetic polymorphism that may require treatment targeted at both drug-resistant and drug-susceptible phenotypes for the better management of patients with MDR-TB.
Keywords: Multidrug resistant, sequencing, spoligotyping, treatment outcome, uncommon mutations
|How to cite this article:|
Vashistha H, Hanif M, Chopra KK, Shrivastava D, Khanna A. Genetic polymorphism of rare mutations in Mycobacterium tuberculosis-infected patients in Delhi. Biomed Biotechnol Res J 2018;2:74-81
|How to cite this URL:|
Vashistha H, Hanif M, Chopra KK, Shrivastava D, Khanna A. Genetic polymorphism of rare mutations in Mycobacterium tuberculosis-infected patients in Delhi. Biomed Biotechnol Res J [serial online] 2018 [cited 2019 May 19];2:74-81. Available from: http://www.bmbtrj.org/text.asp?2018/2/1/74/226576
| Introduction|| |
The emergence of drug-resistant M. tuberculosis strains is posing a significant challenge to tuberculosis control programs. Multidrug-resistant tuberculosis (MDR-TB), which is caused by M. tuberculosis isolates that are resistant to, at least, rifampin (RIF) and isoniazid (INH), is a serious public health hazard., As a result, patients with MDR-TB who do not respond to treatment are a constant source of transmission of MDR-TB.,,,
Genotype MTBDRplus assay (Hain Lifesciences, GmbH, Germany) detects mutations associated with both rifampicin and INH resistance simultaneously. Line probe assay (LPA) allows detection of low levels of resistant bacteria amid a predominantly susceptible population, providing a more accurate representation of the susceptibility of the infecting bacteria., The recent WHO 2015 guidelines for surveillance of drug-resistant tuberculosis recommend incorporation of molecular technologies into surveys, either alone or as a screening tool before culture-based methods.
Drug resistance in M. tuberculosis develops by sequential acquisition of mutations in target genes. Different mutations lead to varying degrees of resistance and influence bacterial ability to multiply. Therefore, the validation of LPA has usually begun by testing for RIF resistance because more than 95% of RIF-resistant isolates have mutations in the 81-bp core region of the rpoB gene. Most studies showed mutations in codons 531 and 526 in 40%–65% and 10%–40% of RIF-resistant isolates, respectively.,,
As per recent reports, since the relative frequency of alleles associated with resistance varies geographically, therefore use of sophisticated techniques such as DNA sequencing to detect drug resistance mutations can serve as epidemiological markers., Spacer oligonucleotide typing (spoligotyping) is a rapid and convenient genotyping method that is well suited for the identification of different family of M. tuberculosis strains. The response of the patients and treatment outcome depends, in addition to diagnosis, appropriate and timely treatment, and host factors, on the virulence of M. tuberculosis and genetic polymorphism prevalent in clinical isolates of the bacterium.
An attempt has been made to catalog the observed variations and to update the TBDreamDB database  that has been developed for cataloging, storing, and dissemination of genetic polymorphism information. Hence, the present study focuses on the prevalence of MDR-TB and pattern of drug resistance among pulmonary TB patients from a tertiary care center in Delhi. Moreover, the determination of their frequency distribution of uncommon mutations and genomic diversity and their treatment outcome may be useful for better management of MDR-TB patient.
| Materials and Methods|| |
The present study was carried out at New Delhi Tuberculosis Centre, IRL-STDC, New Delhi, from February 2015 to January 2017. Ethical clearance for the study was obtained from the Institutional Ethics Committee and Institutional Scientific Committee.
A total of 641 rifampicin-resistant cases were included in the study. Patients within the 14–70 years' age group were considered. Sputum samples were collected in 50 ml sterile Falcon tube from MDR-TB suspects registered with the directly observed therapy short-course centers of the Revised National Tuberculosis Control Program (RNTCP) in 17 associated chest clinics. A unique laboratory accession number was provided to each sample.
Direct smears were prepared, stained by auramine O fluorescent staining, and microscopically examined. Specimens were decontaminated by N-acetyl-L-cysteine and sodium hydroxide method. Two 500 μl aliquot of the processed sputum deposits were made, one was used for the LPA test and the other was used for MGIT 960 liquid culture.
LPA was conducted and interpreted as per the manufacturer's instruction. For batch quality control, a known pan-sensitive H37Rv strain of M. tuberculosis was used. All decontaminated specimens (0.5 ml) were inoculated on MGIT 960 liquid culture.
Amplification of rpoB gene including the 81 bp hotspot region was carried out using customized primers. These primers were designed using the SG Primer program [Figure 1]., Twenty-five DNA isolates were randomly selected (which were showing uncommon mutations in genotypic LPA and drug susceptibility testing [DST]) for sequencing by dideoxy terminator cycle sequencing kit and analyzed by ABI Prism automated instrument.
|Figure 1: The NI, NII, RRDR, CII, and CIII fragments that were amplified to scan for mutations in rpoB are shown|
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Spoligotyping assay was outsourced to external agency, and results were analyzed and documented. Only 25 DNA isolates which were suspected of having uncommon mutations were processed for spoligotyping.
Patients were followed up for 2 years to determine the treatment outcome as per standard outcome definition of RNTCP guidelines.
| Results|| |
Line probe assay
A total of 3610 (Dx = 3610) smear-positive diagnosis specimen results were obtained from February 2014 to December 2014. Among the 3610 specimens with interpretable LPA results, rifampicin drug resistance was observed in 641 (17.75%) cases. Five hundred (13.85%) were MDR-TB and 141 (3.9%) had RIF monoresistance. Four hundred and fifty-one (12.5%) specimen had INH monoresistance, 2504 (69.35%) were sensitive to both drugs, and 14 (0.4%) were negative for M. tuberculosis complex.
Mutation analysis in line probe assay
The most common mutation detected in 186 samples by LPA in the rpoB gene was Ser516 Leu (29.0%), diagnosed by the absence of MUT3 band in LPA. Of the overall 641 samples in whom R-resistance was detected, 330 (51.48%) were on the basis of missing wild-type probes along with the absence of any mutation probe (uncommon mutations). Samples showing absence of wild type probes along with the presence of corresponding mutant probe were 311 (48.52%) [Table 1]. Heteroresistant mutations were suspected in 249 (38.8%) cases out of 641, with the presence of both wild-type probes and their corresponding mutation probe [Table 1].
|Table 1: Frequency of mutations detected in the rpoB gene by line probe assay (n=641)|
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All of the resistant samples, 500 MDR-TB, 141 RIF monoresistant, and 451 INH monoresistant specimens were inoculated in liquid culture (1092) on MGIT 960, from the 1 ml processed sputum deposited earlier.
Of the 1092 resistant samples, 966 (88.5%) were culture positive, 59 (5.4%) were contaminated, and 67 (6.1%) were culture negative. Genomic DNA was extracted from positive cultures using phenol-chloroform method.
Confirmation of uncommon mutations
DNA sequencing was performed for 100 culture isolates showing that uncommon mutations in LPA were selected randomly. An initial polymerase chain reaction (PCR) primer set was designed to detect mutations in the N1 region, rifampin resistance-determining region (RRDR), and C2 and C3 region of rpoB [Figure 1].
Uncommon mutations confirmed and reported in this study include mutations from CAG (Gln) to CAT (His) at codon 517, AGC (Ser) to AGG (Arg) at codon 512, ACA (His) to GCA (Ala) at codon 526, and TTG (Leu) to CTG (Leu) s at codon 524 [Table 2] and [Figure 2]. Heteroresistant mutations were confirmed in 64 (64%) cases among the selected 100 cases [Table 2]. The sequences with uncommon mutations found in this study were deposited for gene bank with submission ID (NCBI-SUB 1766772) for obtaining nucleotide accession number.
|Table 2: Mutations in Mycobacterium tuberculosis rpoB gene detected by DNA sequencing|
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|Figure 2: DNA sequence for rifampicin resistance due to uncommon mutations|
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Spoligotyping and database comparison
Comparison of our spoligotyping results was done in binary format with the SITVIT2 database, SpolDB4.0. Spoligotype international type (SIT) designates spoligotypes shared by two or more patient isolates, as opposed to “orphan,” which designates patterns reported for a single isolate.
Spoligotyping results show that out of 100 DNA isolates, the most predominant was CAS type 1 family exhibited in 56 (56%) isolates. Twenty (20%) isolates belonged to Beijing family with East-Asian Beijing lineage and eight (8%) belonged to T1 type family with Euro-American lineage. Four (4%) isolates each belonged to T2 type family with Euro-American lineage, LAM9 type family with Euro-American lineage, X3 type family with Euro-American lineage, and EAI5 type family with Indo-Oceanic lineage [Table 3].
Out of these 56 isolates belonging to CAS type 1 family, 8 isolates belonged to Indo-Oceanic lineage while rest of the 36 (48%) isolates belonged to a novel type with unknown lineages as per the worldwide acceptable databases, i.e. SPOLDB3 and SPOLDB4 [Table 3]. Spoligotyping resulted in 18 distinct patterns. Nearly 52.0% isolates were grouped in 7 clusters while the remaining isolates (48%) were unique. Among the three MDR DNA isolates which were showing uncommon mutations, 2 isolates belonged to CAS type 1 family with unknown lineages while 1 belonged to LAM9 type family with Euro-American lineage.
Another interesting case concerns, the noteworthy presence of a spoligotype previously rarely seen in India – the SIT53/T1 pattern – found in 2/25 (8%) TB cases [Table 3]. In an investigation between M. tuberculosis spoligotypes and genotypic drug resistance, we found that CAS spoligotype was highly associated with MDR followed by Orphan, Beijing, T, X, LAM, EAI types.
A minimum spanning tree (MST) was constructed using BioNumerics software, version 3.5 (Applied Maths, Sint-Martens-Latem, Belgium) based on spoligotyping. An MST is a unidirectional network in which all the isolates are linked together with the fewest possible linkages between nearest neighbors. A radial tree was also drawn as a hierarchical layout using the SpolTools software to visualize parent-to-descendant spoligotypes (http://www.emi.unsw.edu.au/spolTools).
A spoligotype-based dendrogram was generated by the unweighted pair group method with arithmetic average calculation [Figure 3]. A cluster was defined as two or more isolates from different patients with identical spoligotype patterns. Clusters were made according to different spoligotype patterns obtained [Figure 4]. Unique (nonclustered) spoligotypes did not cluster with any other sample. Orphan was defined as a unique spoligotype pattern not described in the SITVIT2 database.
|Figure 3: Cluster formation through unweighted pair group method with arithmetic averages dendrogram|
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|Figure 4: Evolutionary relationships among Mycobacterium tuberculosis spoligotypes from tuberculosis patients in Delhi|
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DNA isolates showing uncommon mutations (4) were studied for treatment outcome also. All 4 patients were followed up for 24 months for studying parameters as listed in RNTCP guidelines.
All four patients have completed their treatment and were found as “cured” as the smear results were negative for AFB and after inoculation on liquid culture; all of these specimens were turned out as culture negative after 42 days of incubation. We also found that 1 isolate (25%) out of 4 cured patients did not show any mutations and 3 (75%) out of 4 cured patients were MDR as they showed rpoB gene mutations. Out of four isolates with uncommon mutations, one that was resistant to rifampicin was a female, and out of the three males, one was showing sensitive results to rifampicin in phenotypic DST. Impact of uncommon mutations in context of treatment outcome needs to be extensively studied further to serve the MDR-TB patients.
| Discussion|| |
Techniques which detect MDR mutations in new cases at onset or during therapy would enable rapid identification of MDR and facilitate the modification of regimens with improvement to program practices.
In this study, among the smear-positive diagnosis cases, an overall increase in the culture positivity (88.5%) shows a good sign of recovery of M. tuberculosis in these patient samples. Furthermore, similar recovery rates were observed in some studies like Bicmen et al. (63.2% MGIT culture positive).
In our study, the value of RIF as a surrogate MDR marker has been documented also and further corroborated like Yadav et al., 2013. Despite their advantages, genotypic methods do not always identify phenotypically resistant strains, highlighting the limitations of molecular testing and need for supplementation with culture or additional probes.
Our LPA results show that 500 (13.85%) were MDR-TB and141 (3.9%) had rifampicin monoresistance. Similarly, 28% of MDR-TB cases were evidenced in a study from Yadav et al. In addition, resistance can be inferred from the absence of a wild-type signal alone, without confirmation by a mutant probe signal (in 51.48% of our isolates) and may be due to a mutation in a region not associated with resistance. Such susceptible isolates would be called resistant leading to the unnecessary removal of RIF and/or INH from therapy.
And, samples showing absence of wild type probes along with the presence of corresponding mutant probe were 311 (48.52%). This highlights the need for the interpretation of genotypic data in conjunction with patient clinical status and the determination of mutations specific to certain geographical locales.
The frequency of mutations in the rpoB gene of RIF-resistant M. tuberculosis isolates varies between different geographical regions of India. A previous study from Asia also indicated that 47.51% of isolates carried the most common mutation Ser531 Leu while 8.45%, 7.70%, and 4.23% of isolates had His526Tyr, His526Asp, and Asp516Val mutation. However, Herrera et al. and other authors have detected mutations, associated with RIF resistance, outside the 81 bp core region, such as at the codons 481, 490, 498, 505, 534, 535, 553, 561, 571, 572, 633, and 672, although less frequently.,,,
In this study, the most commonly observed mutations in the region of rpoB was Ser516 Leu (29.0%) diagnosed by the absence of MUT3 band in LPA strip. This is similar to the findings of a South African study. Overall, 18 distinct DNA alterations within the RRDR were detected in these isolates, and a total of 13 distinct mutations were detected in the N1 and C2 region in rpoB gene. Uncommon mutations confirmed and reported in this study include mutation at codons 517, 512, 526, and 524.
Heteroresistance, reflecting the slow evolution of bacteria from a sensitive to resistant profile, is not uncommon in M. tuberculosis. However, our study reports relatively significant levels of heteroresistance in rpoB gene in an endemic setting. The detection of heteroresistance seems to support our finding of an association between a clean resistant profile for the rpoB, katG, and inhA and a poor outcome since the presence of extensively resistant strains is more likely to result in nonresponsiveness to treatment.
Genetic polymorphism by spoligotyping shows interesting outcomes as the presence of a spoligotype previously rarely seen in India – SIT53/T1 pattern – found in 2/25 (8%) TB cases. Although considered a ubiquitous pattern, this spoligotype was rarely reported from Indian subcontinent countries, including India. This Mycobacterium tuberculosis complex pattern has apparently reached India recently through travelers from one of these countries or through Indian migrants returning home from such host countries.
Our findings show the potential use of sequencing for complementing Drug Resistance Surveys (DRSs) or surveillance in this setting, with good specificity compared to genotypic LPA DST. The reported uncommon mutations can be included in molecular assays, and population-based phylogenetic studies can track transmission of MDR-TB including the CAS1 and Beijing family strains in the country.
Despite the large number of mutations already reported in other studies, the evidence of uncommon mutations in this study indicates that mutations continue to arise, probably due to the ability of M. tuberculosis to adapt to drug exposure. For the control of MDR-TB, elimination of incomplete treatment is critical. This possibility of uncompleted treatments results in escalating rise in the prevalence and strength of MDR-TB. Hence, early detection of the MDR-TB may well be critical to durable control of tuberculosis.
Our study has limitations, to find any association between a particular mutation and the occurrence of monoresistance or MDR. Furthermore, due to financial limitations and budget constraints, DNA sequencing and spoligotyping were done for only 25 samples. Moreover, follow-up was done only for patients confirmed with uncommon mutations. Moreover, future studies are also encouraged to investigate in this context.
| Conclusion|| |
The rifampicin resistance (RIFr) appears to be an effective marker of MDR-TB. Confirmation with PCR-DNA sequencing can detect the presence of RIFr M. tuberculosis within 2 days with a clear differentiation between RIFr and RIF strains. New and improved diagnostic and drug-susceptibility testing methods that can detect heterogeneous subpopulations need to be developed to improve treatment of MDR-TB. The presence of uncommon mutations confirms genetic polymorphism that may require treatment targeted at both drug-resistant and drug-susceptible phenotypes for the better management of patients with MDR-TB.
Consent for publication
All authors have given consent after reading and approving the final manuscript for publication.
Availability of data and material
All study data and study materials are available on request.
We are sincerely thankful for the FIND India for supporting this study. We are indebted to Dr. Dheeraj and Ms. Vartika Sharma from ICGEB, India, for their support in this study.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for images and other clinical information tobe reported in the journal. The patients understand that name and initials will not be published and due efforts will be made to conceal identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
This work was partially supported by the Tuberculosis Association of India through a Collaborative Research Grant to the New Delhi Tuberculosis Centre.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]