|Year : 2018 | Volume
| Issue : 4 | Page : 295-299
Genetic mutations at rifampicin resistance-determining region of RpoB gene in conferring resistance to isolated multidrug-resistant tuberculosis strains
Prasanta Kumar Das, Somtirtha B Ganguly, Bodhisatya Mandal
Intermediate Reference laboratory State TB Demonstration and Training Centre, Kolkata, West Bengal, India
|Date of Submission||17-Sep-2018|
|Date of Decision||03-Oct-2018|
|Date of Acceptance||07-Oct-2018|
|Date of Web Publication||11-Dec-2018|
Dr. Prasanta Kumar Das
State TB Demonstration Cum Training Centre, Kolkata - 700 010, West Bengal
Source of Support: None, Conflict of Interest: None
Introduction: Drug resistant tuberculosis (TB) among the new and retreatment cases was seen to be 5%. Multidrug resistant TB to at least isoniazid and Rifampicin is an alarming threat to the community and the scenario is even worse for retreatment cases showing rising trend over the years. A systematic analysis of the nature of the resistance conferred may contribute in understanding the common as well as rare genetic alterations that ultimately led to the genotypic resistance. The study thus involves a probe into the most common and rare patterns of genetic mutation that have led to the rifampicin resistance among the retreatment cases. The common and rare codons along with their change in amino acid sequences involved in conferring the resistance were looked for. Methods: The detection of the patterns genetic mutation is done by the Genotype MTBDR plus V2 kits (Hains Life Sciences) based on DNA strip technology (line probe assay). Results: The analysis has shown that only around 14.93% cases reflected exclusive mutations at around 531-533 region of the RpoB gene. Presence of both the wild and mutant gene segments simultaneously (WildType [WT] and MUT3) accounted to a striking percentage of 60.3% showing a heterogenous generation of susceptible and resistant strains at the same time. This pattern analysis showing maximum of heterogenous strains may be suggestive of capturing a generation under the process of conversion from WT to mutant type. About 95% reproducibility in liquid culture system further established the fact that most of the strains might be a cause for the rising attrition toward the first line of drugs. Conclusion: This pattern analysis showing maximum of heterogenous strains may be suggestive of capturing a generation under the process of conversion. The retrospective analysis showed how heterogenous population prevailed in the retreatment groups.
Keywords: Rifampicin Resistance determining region: Mutation, RpoB gene, TB Labs, Intermediate Reference laboratory, State TB Demonstration and Training Centre, Mycobacterium tuberculosis Complex, Retreatment
|How to cite this article:|
Das PK, Ganguly SB, Mandal B. Genetic mutations at rifampicin resistance-determining region of RpoB gene in conferring resistance to isolated multidrug-resistant tuberculosis strains. Biomed Biotechnol Res J 2018;2:295-9
|How to cite this URL:|
Das PK, Ganguly SB, Mandal B. Genetic mutations at rifampicin resistance-determining region of RpoB gene in conferring resistance to isolated multidrug-resistant tuberculosis strains. Biomed Biotechnol Res J [serial online] 2018 [cited 2021 Apr 14];2:295-9. Available from: https://www.bmbtrj.org/text.asp?2018/2/4/295/247236
| Introduction|| |
Worldwide, tuberculosis (TB) accounts for the highest morbidity and mortality among the infectious diseases. The drug resistance has further worsened the situation increasing the resistance toward the first line of drugs.
Drug resistant tuberculosis (TB) among the new and retreatment cases was about 5%. This was in accordance to the surveillance done in the year 2000 and 2001.
The overall proportion of multidrug resistant TB (MDR TB), defined as TB resistant to at least isoniazid (INH) and rifampicin (RMP), with or without resistance to other first line drugs, was 5.3%, ranging from 0% to 35% of reported TB cases. The percentage among the retreatment cases showed a steady rise over the years. The retreatment cases contributed about 20% of multidrug resistant TB among the high TB burden nation. In 2009, only 30,000 (7%) of the 440,000 estimated MDR TB cases globally were notified, and of them, only 11,000 (3%) were put on treatment known to be consistent with international guidelines. Majority of the analysis were done by reversed hybridization using oligonucleotide probes.
In 1994, in 35 countries, the first WHO-IUATLD anti-TB drug resistance surveillance was carried out. The study showed primary and acquired multidrug resistance as 1.4% and 13%, respectively. An estimate of 489,139 cases of multidrug-resistant TB (MDR-TB) emerged in 2006.
The percentage among the retreatment cases showed a steady rise over the years. The retreatment cases contributed about 20% among the high Tb burden nations. In the year 2012, percentage was about 20%. About 3% of the MDR cases.
It has been understood that the nature of the resistance plays an important role in conferring resistance and contributes substantially in understanding the epidemiology of transmission of strains among the population.
The study involves a probe into the most common patterns genetic mutation that has led to the rifampicin resistance among the retreatment cases.
This resistance pattern obtained showed maximum of heterogenous strains. This may be suggestive of capturing a generation under the process of conversion. Population of both the mutated strains as well as wildType (WT) are within the range of analytical sensitivity of MTBDR Genotype Version 2 among the majority of retreatment cases tested for.
The study design involved conduction of the line probe assay (LPA) on the sputa collected from retreatment cases that showed recurrent smear positivity even after treatment completion with the first line of anti-TB drugs. The patterns obtained based on the presence or absence of the WT and mutant type of gene segments were recorded.
| Methods|| |
Reversed hybridization by the oligonucleotide probes for the designated gene segments of the RpoB gene was tested for 442 samples. LPA was done using the MTBDR Genotype Version 2. Reproducibility of LPA results was very high with 98.1% concordance of results between the two laboratories. The proposal was reviewed and approved by Research and Ethical committee of Intermediate Reference laboratory State TB Demonstration and Training Centre, Kolkata, West Bengal, India. The Ethics Committee Approval number was, STDC/IRL – DRTB National working formulation by First line LPA- 011/ 2012 dated 13th Feb 2012-2018.
Smear-positive samples were subjected to the LPA directly after subsequent extraction of the genome and multiplex polymerase chain reaction (PCR) of the specific gene segments using biotinylated primers.
The extraction of the bacterial genome was done using Lysis buffer. The multiplex PCR cycle involved 15 min at 95°C (1 cycle) for denaturation, 30 s 95°C, and 2 min 65°C (20 cycles) for annealation. Twenty-five seconds 95°C, 40 s 50°C, and 40 s 70°C (30 cycles) for amplification and 8 min 70°C (1 cycle) for extension.
The targeted genetic segment ranged from codon 505 to codon 533 of the rifampicin resistance determining region (RRDR).
The most common among the absence of a single WT segment were WT2, WT3, and WT 8, which was around 2.71%. The point mutations involved codons 505-509.
The most common among the exclusive presence of mutation bands with corresponding absence of the WT band at codon 530–533 (around 14.93%) [Figure 1].
Among the majority of cases considering all possible combinations, it was found that combination exhibiting the presence of both WT8 and MUT3 contributed to the most of the rifampicin resistance conferred, (about 60.63%) involving the codon 530–533 [Chart 1].
| Results|| |
A total of 442 cases of rifampicin resistance among retreatment cases were studied. Genetic pattern analysis of these isolates was done by reversed hybridization technology using oligonucleotide probes covering codon 505–531 of the rifampicin resistant determining region [Figure 2].
|Figure 2: RMP resistance region of the RpoB gene: rpoB WT 1–8: rpoB wild-type probes. The numbers specify the position of the amino acids (codons listed in the table. The codons for which mutation probes were designed are highlighted|
Click here to view
Among the resistance only due to the absence of WT band(s) with no positive mutation band absence of WT2, WT3, and WT 8 contributed to the majority cases of rifampicin resistance (2.71%). This involves the point mutations, sequencing of the strains would specify the point mutations that have occurred in this regions. Absence of more than one WT bands was shown in 4.07% of cases.
Among the resistance due to development of positive mutation band(s) and absence of corresponding WT band(s). Presence of MUT3 with the absence of WT8 showed 14.93%.
Among the cohort showing resistance due to the presence of mutation band(s) whereas corresponding WT band is also positive MUT3 Pos along with WT 8 pos contributes to the majority of the rifampicin-resistant cases about 60% (with confidence interval ± 2.52 at 95% confidence limit).
As an inference, it can be drawn that heterogenous strains may be suggestive of capturing a generation under the process of conversion. Population of both the mutated strains as well WT are within the range of analytical sensitivity of MTBDR Genotype Version 2 among the majority of retreatment cases.
Among the 442 cases, no mutation was observed at codon 518–521.
The presence of the most common pattern Presence of WT8 and MUT3. The pattern contributes most to the rifampicin resistance isolates among retreatment.
| Discussion|| |
In the study, it was observed that the most common resistance pattern was exhibited by the combination of the presence of Mut3 along with WT8 (about 60.63%).
WHO validated the technology of Line probe assay for rapid screening of Multidrug resistant cases.
Reversed hybridization using oligo nucleotide probes were done for the RRDR region following previous studies.
The probes that targeted the RpoB in the assay, successfully detected >93% of the rifampicin resistant strains (both monoresistant and MDR), national recovery of 95%.
Presence of both WT8 along MUT3 indicates heterogenous strains in majority cases of the individual undergoing the retreatment regime. Further sequencing can give us the idea about the particular point mutations that have occurred.
The majority of the rifampicin resistance in the study occurred due to the mutations in the rifampicin resistance determining Region81 bp region of the Rpo B gene.
This pattern was reported at a lower percentage in an earlier study, that is, 2.6% among newly treated cases. The higher percentage may be a reason of heterogenous population found predominantly in the retreatment group. As the study included MDR suspects within the retreatment group as per the programmatic diagnostic criteria.
Among the 442 cases, 3 were extrapulmonary sample among 10 cases. Earlier studies have shown mono rifampicin resistance detection of 3.90%. The sample is statistically not significant to comment on any definite pattern dominance among the extrapulmonary rifampicin resistant cases.
Here, it is to be noted that the MUT3 involves a mutation at the codon 531 and 533 regions. Within the RRDR of RpoB gene.
The area involves the occurrence of four point mutations out of which the most common is the S531 L. The other rare mutations are S531Q, S531Q, and L533P [Table 1].
|Table 1: The Common mutation on Rifampicin resistance - determining region|
Click here to view
Sequencing would definitely reveal that whether any rare mutations prevail in this demographic zone.
It has been observed that the occurrence of mutations in the RpoB gene of RIF-resistant Mycobacterium tuberculosis isolates varies in different parts of India. A previous study from Asia showed about 47.51% of isolates common mutation Ser531 Leu.
Earlier studies shown S531 L (61.9%) and S315T (71.4%) mutations were the most common mutations in rifampicin-resistant TB isolates among extrapulmonary TB cases.
The analysis has shown that only around 14.93% cases reflected exclusive mutations at around 531 533 region. Presence of both WT and MUT3 accounted to a striking percentage of 60.3%. [Table 2] showing a heterogenous generation of susceptible and resistant strains at the same time.
| Conclusion|| |
This pattern analysis showing maximum of heterogenous strains may be suggestive of capturing a generation under the process of conversion. Population of both the mutated strains as well WT are within the range of analytical sensitivity of MTBDR Genotype Version 2 among the majority of retreatment cases. A machinery to quantify the strains among the group and thereby calculating a standard deviation from the mean would give us a clear picture about proportion of population getting mutated during the first-line retreatment regime. Spoligotyping would have been of considerable help in determining strain-specific mutations.
The retrospective analysis showed how heterogenous population prevailed in the retreatment groups. Treatment failure default rates thus complicated the drug-resistance scenario which occurred due to the lack of diagnostic methodology. The study reiterated the decision of universal DST testing in order to impart early treatment on early diagnosis.
We are thankful to all the patients suffering and staffs contributing for their dedication.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]