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Detection of Rifampicin Mono-Resistant Mycobacterium Tuberculosis by Genexpert in Patients Attending Tertiary Care Teaching Hospital

Original Research Article In India incidence of TB is 27, 90,000, mortality is 4, 23,000 and incidence of MDRTB/RR 1, 47,000. Early detection of TB is the key to successful treatment and reduction of disease transmission. The aim of this study is to determine the Rifampicin resistant, Mycobacterium tuberculosis by Gene Xpert in patient attending TB chest hospital, Hyderabad. All the Samples, Pulmonary and Extra pulmonary, received from clinically suspected cases of Tuberculosis, IP and OP during the study period between March 2018 to October 2018 were processed in the department of Microbiology using Xpert MTB/RIF test version 4.8 (Cepheid) as per the manufacturer’s instruction. Result: Prevalence of total MTB culture positive was 34.2 % (754/2202) and Rifampicin resistant was 13.7 % (103/754) in the total samples processed. Conclusion: Early detection of TB is the key to successful treatment and reduction of disease transmission. Gene Xpert was found to be useful assay for rapid detection of MTB within 2 hours with additional advantage of identifying Rifampicin resistance with high sensitivity and specificity.


INTRODUCTION
Tuberculosis continues to be a major public health problem in India as well as worldwide. As of global TB 2017 report, incidence of TB in India is nearly 28, 00,000 (28 lakhs) which accounts for one fourth of global TB burden. Mortality due to TB is 4, 23,000. Incidence of MDR/ RR TB is 1, 47,000. The rate of MDR TB as per the First National Drug Resistance survey results is 2.84% among new TB patients and in previously treated is 11.6% [1].
Drug resistant Tuberculosis is a major public health problem that threatens progress made in TB cases and its control worldwide. As per, global tuberculosis 2017 worldwide report, there were 5, 58,000 new cases (range 4, 83,000-6, 39,000) of Rifampicin resistant TB. Among Rifampicin resistant TB cases, 3.5% new and 18% of previously treated tuberculosis cases and82% had multidrug resistant TB (MDR TB) [2].
Genetic mutations in tubercle bacilli that develop due to an inadequate or poorly administered treatment regimens, weak services program that led to delay detection and initiation of effective treatment of drug-resistant TB and non-adherence to treatment lead to development and spread of Drug resistant Tuberculosis [3]. Differentiation of NTM from mycobacterium tuberculosis is difficult with microscopy.
Due to paucibacillary state, extrapulmonary specimen's microscopy results are often negative. Culture which is the gold standard for TB diagnosis often leads to considerable delays, compromising patient care [4].
CBNAAT is a mycobacterium tuberculosis complex specific, cartridge base nucleic acid amplification assay, having fully integrated and automated amplification and detection using real time PCR, providing results within 100 minutes. It is highly specific test which uses 3 specific primers and 5 unique molecular probes to target the rpo B gene of Mycobacterium tuberculosis. No cross reactions were observed with many other bacterial species, including a comprehensive panel of Mycobacteria, therefore excluding NTM. Critical validation trails done in 4 distinctly diverse settings showed, a single CBNAAT test detects 92.2% of culture positive patients, with a specificity of 99% (as compared to 59.9% sensitivity of a single direct sputum smear) [5].
The Xpert assay is highly rapid, sensitive and specific in diagnosis of both pulmonary and extra pulmonary tuberculosis [6][7][8]. Early detection of TB is the key to successful treatment and reduction of disease transmission. As little data is available on prevalence of TB and MDRTB/RR in south India the present study was done with an aim to determine the Prevalence of Rifampicin mono-resistant Mycobacterium tuberculosis among TB patients attending TB Chest, a tertiary care teaching Hospital.

MATERIALS AND METHODS
A prospective study was conducted from March 2018 to October 2018 at tertiary care teaching Hospital. The study was ethically approved by the institute and ethical committee.
Patients having clinical, radiological features suggestive of TB, old cases of TB with recurrence, failure of ATT after 1 month, defaulters, all age groups, both sexes, PLHIV(people living with HIV) were included in the study. Patients who provided inadequate specimen that is heavily bloodstained or contaminated with PCR inhibitors like formalin, xanthochromic CSF samples were excluded.
From each presumptive pulmonary TB patient 2-4 ml of sputum was collected. In case of presumptive extrapulmonary TB 2-4 ml of pus, lymph node aspirates, CSF, pleural fluid or peritoneal fluid samples were collected. Samples were collected in a sterile, dry wide necked and leak proof container and were labeled with unique sample number, date, time of collection. Samples were immediately processed for gene Xpert MTB Rifampicin assay. The samples were inspected for quality and appropriate samples reagent was added to the sample in ratio 2:1 and lid was closed. Sample was then shaken for 10 to 20 minutes and incubated for approximately 10 minutes. Sample was shaken again and further incubated for 5 minutes. The liquid samples were then transferred into the gene Xpert cartridge in the gene Xpert MTB/Rifampicin assay system. The computerized system was then operated according to the standard operating procedure to carry out analysis. Results were automatically generated after 2 hours, indicating if MTB was detected or not detected, where MTB was detected, the gene Xpert automatically generated result indicating if the MTB is Rifampicin resistant or not resistant [9].       [11] were similar to present study MTB infection prevalence.

Out
In the present study, prevalence of Rifampicin resistant in MTB positive cases was found to be 13. Though Mumbai reports a very high incidence of Rifampicin resistance of 66.8%as seen in Chowgule et al. study, 1998 [14].
In our study, prevalence of Rifampicin resistance MTB cases high predominance was seen in the age group 0 to 20 years (17.2%)which was similar to the study done by Villegas [16]. The likely reasons for predominance of females over males might be due to, majority of females are illiterate and are home makers who does not even have easy access to healthcare facilities, poor nutritional status which might be due to social and health seeking behaviour difference.
In the present study, the prevalence of Rifampicin resistant MTB cases was higher in pulmonary (14.1%) cases when compared to extra pulmonary (11.4%) TB cases. The study done by the Chakra borty et al. found 13.7% from pulmonary and8.6% from extra-pulmonary cases [17], and study done by the Praveen B. Gautam et al. found27.6% from pulmonary and 21% from extra-pulmonary cases [10] which were supportive to present study findings. Predominance of rifampicin resistance among pulmonary cases over extra-pulmonary cases can be explained by the spread of infection through aerosols inhalation, which has high chances of spreading the infection in the community. In case of extra-pulmonary cases, spread of infection through hematogenous route from primary focus like lungs of the patient or direct inoculation of Mycobacterium at the site of infection as a result of trauma, injury and surgery can occur.
In the present study only 2 patients out of 103 Rifampicin resistant MTB were HIV seropositive. This is in accordance to the study done by Gautam

CONCLUSION
Molecular technique has revolutionized the diagnosis of TB (both PTB and EPTB) as well as MDR TB and TB in PLHIV. CBNAAT (Xpert MTB / Rifampicin) can be used in field condition, sub district level, where 24 hours electricity is available as it requires minimum training and biosafety. Rifampicin resistance is considered as surrogate marker of MDR TB as practically all rifampicin resistant bacilli are resistant to INH. However, it should be remembered that a positive result suggests but a negative result do not exclude TB as well as MDR TB. At present CBNAAT has not totally replaced the traditional smear and culture for TB.WHO recommends CBNAAT for diagnosis of TB and detection of rifampicin resistance in presumptive vulnerable paediatric age group, smear negative with x-ray suggestive of TB, and all diagnosed TB patients non-responders to treatment, DRTB contacts, previously treated TB, TB HIV co infection and newly diagnosed TB. Early detection of TB is the key to successful treatment and reduction of disease transmission