Detection of Mutations in 81-bpRifampin Resistance Determining Region (RRDR) of rpoB gene in Mycobacterium tuberculosis using GeneXpert MTB/RIF in Clinical Specimens from Quetta, Pakistan

1Center for Advanced Studies in Vaccinology and Biotechnology (CASVAB), University of Balochistan, Quetta, Pakistan 2Department of Zoology, Sardar Bahadur Khan Women’s University, Quetta, Balochistan, Pakistan 3 Bolan Medical College, Quetta, Pakistan 4Department of Microbiology, University of Balochistan, Quetta, Pakistan Article Information Received 22 May 2019 Revised 30 July 2019 Accepted 14 September 2019 Available online 01 June 2020


INTRODUCTION
T he emergence of multi-drug resistant tuberculosis (MDR-TB) has become a significant obstacle to global TB control. With the worldwide spread of M. tuberculosis (MTB) strains resistant to both isoniazid and rifampicin (RIF), MDR-TB has become a major public health problem posing formidable challenges due to its complex diagnostic O n l i n e

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resistant MTB strains also exhibit resistance to isoniazid (Drobniewski et al., 1998;Sadri et al., 2016). Rifampicin binds the β-subunit of MTB RNA polymerase (rpoB) and thus inhibits RNA synthesis. RIF-resistance mechanisms usually involve missense mutations in the 81 base pairs (codons 507 to 533) hot-spot region of the rpoB gene referred to as Rifampincin-Resistance-Determining Region (RRDR) and 95% of RIF-resistant strains have mutations within this 81-bp RRDR (Ramaswamy and Musser, 1998;Van Der Zanden et al., 2003). The rapid spread of MDR/RR-TB particularly in new patients is challenging the success of tuberculosis control programs mostly in low-income countries, including Pakistan. According to WHO report, 2018, the global incidence of TB was 10.0 million in 2017 with 160,684 cases of MDR/ RR-TB. An estimated 558,000 people developed TB who showed RIF-resistance (RR-TB), and among these 82% had MDR-TB (WHO, 2018). Pakistan ranks fifth among 30 most tuberculosis affected countries and forth among the 27 countries with high burden of MDR-TB. In Pakistan 525,000 people developed TB and 54,000 deaths occurred among HIV-negative and 2200 among HIV-positive in 2017. Approximately 27,000 MDR/RR-TB cases emerged in Pakistan of which 16% were previously treated cases and 4.2% were new cases (WHO, 2018).
Studies conducted in diverse geographical areas have shown that the burden of MDR-TB and the mutations responsible for drug resistance vary from country to country and region to region (Purwar et al., 2011). However, data regarding prevalence of RR/MDR-TB and rpoB gene mutations is scarce at Quetta, Balochistan. Therefore, this study was conducted to determine the prevalence of rifampicin resistance and rpoB gene mutations among the suspected TB cases in this region using GeneXpert MTB/ RIF. Knowledge of the pattern of mutations present in RIFresistant isolates could provide insight into the epidemiology of RIF-resistant MTB isolates of this particular area.

Ethical consideration
The current study was approved by the Research and Ethics Review Committee of University of Balochistan, Quetta. All the participants gave their informed consent.

Patients selection
The individuals with the symptoms of pulmonary and extra-pulmonary tuberculosis attending outpatient department (OPD) of Fatima Jinnah General and Chest Hospital, Quetta were recruited in the study. Patients' data including gender, age, MDR-contact, TB history etc. were also recorded.

Clinical specimens
A total of 2300 specimens were obtained from patients suspected to have TB infection. These specimens were screened for the routine mycobacteriological diagnostic tests at Provincial TB Reference Laboratory (BSL-3), Fatima Jinnah General and Chest Hospital, Quetta. All the specimens were examined for infection of M. tuberculosis, resistance to rifampicin as well as detection of mutations in the RRDR rpoB gene by GeneXpert MTB/RIF. The patients' data and samples' characteristics (consistency and volume) were recorded. The samples collected were sputum, bronchoalveolar lavage (BAL), cerebral spinal fluid, plural fluid, gastric aspirate, ascetic fluid colon biopsy, pus, and urine. The specimens were directly collected in sterile containers and labeled with patient ID while other forms of specimens were collected by specialists and sent to the Laboratory. All the precautionary measures were adopted during sample collection. Specimens were processed immediately for laboratory testing.

Laboratory procedures
All samples were decontaminated using N-acetyl-L-cysteine (NALC)-NaOH technique except the CSF samples and followed by concentrating the samples by centrifugation for 15 minutes at 3000g using the standard protocol recommended by Center for Disease Control and Prevention (Kent and Kubia, 1985). For fluorescent microscopy, concentrated smears were prepared from decontaminated specimen sediments using standard protocol (Kent and Kubia, 1985), stained with Auramine-O and visualized under fluorescent microscope using 200X and 400X magnification for the presence of M. tuberculosis.
GeneXpert MTB/RIF (Cepheid) was performed in accordance with the manufacturer's guide. Briefly, the Sample Reagent (SR) was mixed with the decontaminated specimen in a 3:1 ratio and agitated for 15 minutes. The mixture was then introduced into the cartridge and loaded into the GeneXpert instrument. M. tuberculosis genomic DNA was extracted by sonication with subsequent DNA amplification by PCR. Moreover, the Xepert detects RIFresistance cases conferred due to mutations in the M. tuberculosis rpoB gene using fluorescent probes know as molecular beacons (Fig. 1). The RIF-resistance detection is based on the amplicon hybridization with 5 overlapping probes complementary to 81-bp core region (RRDR) of rpoBMTB gene (El-Hajj et al., 2001).

Statistical analysis
Data analysis was performed using SPSS-20. Descriptively, the percentages and frequencies were O n l i n e

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calculated for various variables. Chi-square test was used to investigate the associated factors of TB infection and RIF-resistance. P value <0.05 was considered statistically significant.

RESULTS
Total of 2300 clinical specimens were collected from suspected TB patients during the study period. Of whom, 2032(88.3%) specimens were pulmonary and 268 (11.7%) were extra-pulmonary. The respiratory samples comprised sputum, gastric aspirates and bronchoalveolar lavage (BAL) while the non-respiratory samples included pleural fluid, pericardial fluid, ascetic fluid, cerebrospinal fluid (CSF), colon biopsy, urine and pus. The age of the patients ranged from 2 months to 107 years with the mean of 42.2 ± 22.8 years. Of the total, 1175 (51.1%) were males and 1125 (48.9%) were females with the sex ratio of 1:1.1.
From 2300 TB suspected cases, M. tuberculosis was found to be in 899 (39.1%) cases as detected by GeneXpert MTB/RIF. Among 899 confirmed patients, the rifampicin resistance was detected in 46 (5.1%) cases. RIF-resistance is conferred by rpoB gene mutations present within 81-bp RRDR overlapped by 5 different Probes A, B, C D, and E in GeneXpert. Of the 46 RIF-resistant patients, most of the cases (78.3%, n=36) harbored mutations in Probe E whereas mutations in Probe A, B, D were observed 2.2% (n=1), 4.3% (n=2), and 6.5% (n=3) cases, respectively. Three cases had mutations in all Probes and one had mutations in 2 Probes A and B. No patient with RIFresistance was found to have Probe C related mutations. Among the RIF-resistant patients, 45.7 % were males and 54.3% were females. About half of the proportions of RIFresistant patients were aged 40-59 (Table I). 42 patients had MDR contact and eight patients were co-infected with HIV.

DISCUSSION
Drug resistance in M. tuberculosis seems to result from the stepwise acquisition of new mutations in the genes for various drug targets (Heymet al., 1994). Emergence of MDR-TB is a serious challenge for clinicians; it arises mostly due to rpoB gene mutations (Miller et al., 1994). The frequency of mutations in MTB rpoB gene varies geographically (Adikaram et al., 2012). Mutations in rpoB gene have been reported earlier in Asian countries, which are generally related with a high level of RIF-resistance (Bahrmand et al., 2009). Despite Pakistan being a highly TB endemic area, few studies are available regarding molecular characterization of rpoB mutations in MDR-TB patients. The information regarding the prevalence of these mutations might be helpful for better therapy and management of MDR-TB patients. In the current study, we determined the prevalence of rpoB gene mutations conferring RIF-resistance in M. tuberculosis strains among TB patients.
Rifampicin resistance was mainly related with mutations in the 81-bp region within rpoB gene (Van Rie et al., 2001;Telenti et al., 2003). Studies based on DNA sequencing have indicated that above 95% of the RIFresistant strains harbor mutations within the rpoB gene in 81-bp core region (Cavusoglu et al., 2002). Automated DNA sequencing has characterized greater than 50 mutations within this region, most of which possess point mutations at codons 516, 526 or 531 (Ahmad et al., 2002).
In our study the predominant genetic mutations in the 81-bp RRDR of rpoB gene were found in codons 531 (78.3%), 526 (6.5%), 513 (4.3%), 511 (2.2%), whereas no mutations was observed in codon 522. On the GeneXpert MTB/RIF assay these codons were represented by Probes E, D, B, A and C, respectively. A study by Mboowa et al.

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Mutations in rpoB Gene of Mycobacterium tuberculosis (2014) in Kampala, Uganda found these frequencies 531 (58%), 513 (25%), 526 (8%), 511 (8%), and none for codon 522, while Ullah et al. (2016) in Khyber Pakhtoonkhwa, Pakistan also reported the most common mutations in codon 531 (77%) followed by codons 513 (10.8%), 526 (8.3%), 511 (1.2%), and 522 (1.5%). Khan et al. (2013) in Punjab, Pakistan found mutations in codons 531 (52%), 516 (15%), 512 (7%) and 526 (7%). These studies indicate that the most common mutation conferring RIF-resistance is associated with codon 531. Previous studies have shown the sensitivity of GeneXpert MTB/RIF to be 94.4-100% and that of specificity 98.3-100% for the detection of RIFresistance (Moure et al., 2011). RIF-resistance related with Probe C was not detected in this study, it could be attributed to the fact that this specific site within RRDR may probably be less susceptible to genetic mutations conferring drug resistance or Probe C related RIF-resistance is absent in our setting.
We found that GeneXpert detected 46 (5.1%) RIFresistant TB cases out of 899 confirmed TB patients in Balochistan which is similar with the study by Masenga et al. (2017) who reported 5.9% RIF-resistant cases in Zambia. Several studies from different regions of Punjab, Pakistan reported 6%, 11.3% and 11.5% isolates resistant to at least one TB drug that is higher in comparison with our finding (Javaid et al., 2008;Qazi et al., 2014;Ullah et al., 2016). These variations in results could be due to different sample sizes in these studies.
In this study, the prevalence of RIF-resistance was almost similar in males and females (45.7 % vs 54.3 %). These findings are consistent with the study by Nair et al. (2016) in India who reported the similar risk of RIFresistance among men and women. This could be due to the fact that both males and females are equally exposed to factors which cause RIF-resistance. However, we observed that more females were infected with MTB as compared with males with statistically significant difference (41.9%vs 36.4%, p= 0.008). In our study the frequency of TB patients was highest in the age group 20-39 years, which is in agreement with other studies conducted in Nairobi (Ndungu et al., 2013) and Pakistan (Munir et al., 2015), where the highest TB infection was observed in age groups of 18-34 and 21-50 years, respectively.
Balochistan, a province of Pakistan, has over thirty million population and shares a common border with Iran and Afghanistan. To the best of our knowledge, it is the first study to report the prevalence of rifampicin resistant TB cases and determine the frequency of rpoB gene mutations within 81-bp RRDR in this province.
In conclusion, GeneXpert MTB/RIF detected 46 rifampicin resistant cases out of 899 TB patients caused by rpoB gene mutations in the 81-bp RRDR. The most frequent rpoB gene mutation was observed in codon 531/533 (Probe E, 78.3%) while the least was detected within the sequence 511 (Probe A, 2.2%). Such studies on mutations can be useful for the development of novel therapeutics for the TB treatment.

Statement of conflict of interest
The authors declare there is no conflict of interest.