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Prevalence of multi-drug resistant (MDR) pulmonary tuberculosis in a tertiary care rural hospital in Western Maharashtra, India

Introduction: The emergence of Multi-Drug Resistant tuberculosis (MDR-TB) has become a big issue in tuberculosis (TB) control programs in recent times. It is seen more frequently in patients already treated for TB, but has been now diagnosed in new TB patients as a primary infection. Present study was carried out to find out the prevalence of MDR-TB in new and previously treated pulmonary TB patients attending a tertiary care rural hospital. Material and Methods: The study included a total of 1154 suspected MDR-TB patients out of which 1052 were new patients while 102 had received anti-TB drugs in past. Sputum samples of these patients were tested using GeneXpert MTB/RIF assay to detect Mycobacterium tuberculosis infection and Rifampicin


Introduction
Tuberculosis (TB) continues to be a major health issue worldwide even after great efforts being taken for its control and prevention. Although Lung is the commonest site affected in TB, it can also affect other organ systems presenting as an extra-pulmonary TB. In pulmonary TB, the patients often present with an insidious clinical onset with symptoms such as lack of appetite, low-grade evening fevers, and night sweats. Respiratory symptoms such as cough which is initially dry and later on productive with purulent or mucous expectoration also develop. Hemoptysis and chest pain can also occur. 1 Infection with TB bacillus is found in almost 1/3 rd of world's population and every day nearly 5000 new cases are being added to this. 2 According to the 2017 Global TB report, in India the incidence of TB w as found to be approximately 28,00,000 cases which accounts for 1/4 th of total TB cases in the world. 3 India reports 1.9 million new cases of TB each year. Out of these, 0.8 million are open cases of TB with sputum smear positive for acid fast TB bacilli and have all the potential to infect their contacts. 4 As per the World Health Organisation (WHO), in India the death rate due to TB is nearly 28 per 100,000 population which is highest among all other communicable diseases and accounts for 26% of all avoidable adult deaths. 4 Although the WHO and governments across the world are taking great efforts for prevention and control of TB, the emergence and spread of Multi-drug resistant strains of TB bacillus has become a big hurdle in TB control programs. Presently MDR-TB is defined as resistance of TB bacilli to at least Rifampicin and Isoniazid (INH) which are the most important primary drugs used to treat TB, with or without resistance to other anti-tubercular drugs. 5 In 2016, multi-drug resistance was diagnosed in 3.9% of new patients of TB, whereas in patients who were treated  6 It is now defined as cases of tuberculosis resistant to Isoniazid and Rifampicin along with resistance to one of the fluoroquinolones (Ofloxacin, Levofloxacin or Moxifloxacin) and one of the injectable drugs (Kanamycin, Kapreomycin or Amikacin) which are drugs used to treat MDR-TB. 7 With early detection and appropriate treatment with second line anti-tubercular drugs, MDR-TB can be treated and emergence of XDR-TB can be prevented. With this background, the present study was conducted in a tertiary care rural hospital to find out the prevalence of multi-drug resistant pulmonary tuberculosis (MDR-TB) in all suspected patients (new as well as those patients who received anti-tubercular treatment in past).

Material and Methods
This prospective study was conducted in a tertiary care rural teaching hospital in rural part of western Maharashtra from January 2018 to April 2019. The study was approved by the Institutional ethics committee. Overall 1154 sputum smear positive as well as negative patients suspected to have drug resistant pulmonary tuberculosis were included in the study irrespective of age, sex and HIV status. Out of these, 1052 were new patients and 102 were previously treated patients.
Morning sputum samples were collected from these patients after giving proper instructions so as to obtain good quality sputum in a Falcon tube of 50 ml capacity. The samples were tested for detection of Mycobacterium tuberculosis and Rifampicin resistance using GeneXpert MTB/RIF assay. This method is more sensitive in detecting presence of TB bacilli in sputum samples than just smear and microscopic examination. It can detect both Mycobacterium tuberculosis DNA and genetic mutations associated with Rifampicin resistance simultaneously in a short span of just two hours using sputum samples. 8 Though conventional culture based drug sensitivity testing is considered as a gold standard investigation to detect MDR, the sensitivity and specificity of GeneXpert MTB/RIF assay is comparable with conventional methods. 9 GeneXpert MTB/RIF assay is a fully automated cartridge based molecular diagnostic test for TB. In this assay, about 3-5 ml of sputum sample is mixed with twice the volume of sample reagent. It is shaken vigorously and incubated at room temperature for 10 minutes. After 10 minutes it is again shaken vigorously and incubated for another 5 minutes. 2 ml of this processed sample is then added to GeneXpert cartridge which is then loaded in the device. The results are finally interpreted by the GeneXpert system based on fluorescent signals which are displayed on the system monitor after about 90 minutes. 9 This test has an advantage of short turnaround time, which makes it extremely useful for diagnosis of MDR-TB in a country like India with great burden of TB. Resistance only to Rifampicin (monoresistance) is very rare and almost all Rifampicin-resistant strains of TB bacilli are also resistant to other primary drugs, especially to Isoniazid. For this reason, Rifampicin resistance is considered as a surrogate marker for MDR-TB. 10 In the present study, the patients whose sputum sample showed positive results for M. tuberculosis by GeneXpert assay were considered as confirmed pulmonary TB cases. The samples showing additional result as Rifampicin resistance detected were considered as multi-drug resistant TB cases.

Results
During the study period, a total number of 1154 sputum samples from suspected drug resistant pulmonary TB patients (new as well as previously treated) were tested by GeneXpert assay irrespective of their age, sex and HIV status. Out of this, 357(30.94%) patients were tested positive for TB. Rifampicin resistance was detected in 64(5.55%) patients while 293(25.39%) patients had drug sensitive TB. The details of GeneXpert testing with distribution of MDR-TB in all suspected patients is shown in Figure 1.
Out of the total patients, 1052 were new patients, without any past history of TB or antitubercular drug treatment. 306(29.09%) of these new patients were detected as TB positive by GeneXpert assay and Rifampicin resistance was detected in 46(4.37 %) patients. 260(24.72%) patients had infection with drug sensitive TB bacilli. Table 1 shows the details of GeneXpert testing with distribution of MDR-TB in these newly suspected patients.  102 patients in this study were previously treated with anti-tubercular drugs and were suspected to have MDR-TB. Of these, M. tuberculosis was detected in sputum samples of 51(50%) patients. Rifampicin resistance was detected in 18(1 7.65%) of these isolates whereas 33(32.35%) isolates were sensitive to Rifampicin. The detailed result of GeneXpert testing with distribution of MDR-TB in these previously treated patients is shown in Table 2 .

Discussion
MDR-TB occurs because of development of resistance by Mycobacterium tuberculosis to both rifampicin and isoniazid with or without resistance to other anti-TB drugs. 5 The molecular mechanisms of resistance to these two drugs are now largely understood. Rifampicin acts on Mycobacterium tuberculosis by binding to the β -subunit of the RNA polymerase, inhibiting the elongation of messenger RNA. 11 Resistance to rifampicin is developed due to mutations in the rpoB gene that codes for the β -subunit of the RNA polymerase.
This results in conformational changes causing decreased affinity for the drug with development of resistance. 12 The Mechanism of action of isoniazid is inhibition of synthesis of mycolic acids in mycobacteria through the NADH-dependent enoyl-acyl carrier protein (ACP)-reductase, encoded by inhA gene. 10 Isoniazid resistance is associated with mutations in genes katG and inhA or its promoter region. 10 Multi-drug resistant tuberculosis, to a large extent a manmade problem, has emerged because of poor management of drug sensitive TB. Many new cases of MDR-TB are created each year because of multiple associated factors which include errors by treating physicians such as prescribing inappropriate or inadequate medications, poor compliance from patients, issues related to availability of drugs and many more. Because of this, TB bacilli which were initially fully susceptible to primary drugs develop spontaneous genetic mutations and acquire resistance. 13 The treatment of MDR-TB is costly, complicated and difficult. Though the drug resistant strains arise more commonly in patients who have been previously treated, sometimes they can cause primary infection in new patients without any prior history of TB. Therefore it is important not only to diagnose TB in a suspected patient, but also to detect multi drug resistance at first place. The availability of GeneXpert has revolutionised TB diagnostic services in recent times. This technique is more sensitive than smear examination alone for TB diagnosis and it also detects Rifampicin resistance in just 2 hours.
In the present study, MDR-TB was diagnosed in 5.55 % of overall MDR suspect patients using GeneXpert. In a study carried out by Bansal SK et al the MDR-TB prevalence was reported to be 6.3% in overall MDR suspects. 14 In case of new patients who never had past history of TB or antitubercular drug treatment, MDR-TB was detected in only 4.37% cases in our study. On the other hand, 17.65 % of previously treated patients were diagnosed to have MDR-TB. Almost similar findings were recorded in global estimates of TB in 2016, where 4.1% of newly suspected and 19% of previously treated TB cases were MDR. 15 Bansal SK et al have reported the prevalence of M DRTB as 1.9% in new cases and 10% in previously treated cases. 14 According to a multicentre study conducted in different parts of North India, the prevalence of MDR-TB was recorded as 9% in newly diagnosed cases of pulmonary TB which is higher than global prevalence rate of 3% -5%. 2 In o ne more study conducted in Punjab, 26% of newly diagnosed TB patients were found to have MDR-TB showing very high burden of drug resistant TB in the area. 16 In another study conducted by Sethi S et al, MDR-TB was found in 9.9% of newly diagnosed and 27.6% of previously treated patients. 17 According to the first National Drug Resistance Survey for TB, MDR was detected in 2.84% new TB patients and 11.60 % in previously treated patients. 3 So in all the studies including ours, MDR-TB was found to occur much more frequently in previously treated patients; however it does occur in new patients as a primary infection.

Conclusion
Previous drug treatment is perhaps the most imortant risk factor for the development of MDR-TB. Now a days, it is also being reported from newly diagnosed TB patients as a primary infection. If not diagnosed and treated early, it may spread rapidly and probably replace the drug sensitive strains. So detection of drug resistance is as important as mere diagnosis of TB and should be done when ever possible. All patients of TB with past history of anti-TB treatment, failure or not responding to treatment, defaulter patients with incomplete treatment should be strongly suspected to have MDR-TB. They should appropriately investigated and treated to limit the spread of MDR strains and improve the patient outcome.

Source of Funding
None.

Conflict of Interest
None.