European Journal of Cardiovascular Medicine

Tuberculosis (TB) is considered as one of the ancient diseases that has afflicted human lives and has caused significant morbidity and mortality. India is among the high burden countries which accounted for 87% of world TB in 2023. In 2023, TB returned to being the world’s leading cause of death from a single infectious agent.¹ More than 10 million people continue to fall ill with TB every year and the number has been rising steadily following COVID -19 pandemic. According to the Global TB Report 2023, an estimated 10.8 million incidents of TB were reported in 2024.¹ 

Drug resistance tuberculosis (DR-TB) is a major threat in efforts to end TB globally. Multidrug resistance tuberculosis (MDR-TB) is defined as a disease caused by Mycobacterium tuberculosis that is resistant to at least isoniazid and rifampicin with or without other first line anti-tubercular drugs.² MDR-TB occurs primarily due to poor adherence to TB medications, irregular and inappropriate use of drugs, interruption in drug supplies and easy accessibility of drug without prescription.² As per WHO Global TB report 2024, MDR TB is found to be 2.5% among new TB cases and 13% among previously treated cases in India.⁴

Rifampicin resistance (RR)is considered as surrogate marker for MDR TB.⁵ Rifampicin mono‑resistances are rare and are generally associated with isoniazid resistance. Thus, about 90% of Rifampicin‑resistant Pulmonary Tuberculosis (PTB) cases are multi‑drug‑resistant TB (MDR‑TB).⁶ Rapid diagnosis of TB and detection of drug resistance is utmost important for containing TB transmission and improving patient care. Though culture is considered as gold standard for diagnosis of TB, owing to its turn-around time of around 8 weeks causes diagnostic delay and enhances transmission.⁷ In early 2011, WHO has recommended GeneXpert MTB/RIF assay for diagnosing tuberculosis and multidrug-resistant tuberculosis (MDR TB) which has caused significant contribution in TB diagnostics. Moreover, Gene Xpert MTB/RIF is a cost-effective molecular method unlike others like line probe assay and liquid culture diagnostics and does not require any specialised training or biosafety measures.⁸ It is an automated cartridge-based real-time DNA-based test that can detect both TB and resistance to rifampicin in less than 2 hours, and it is based on molecular beacon technology to detect rifampicin resistance. The number of positive beacons and the time to their detection allow the test to distinguish among the following results: no TB; TB detected, rifampicin resistance detected; TB detected, no rifampicin resistance detected; TB detected, rifampicin resistance indeterminate and an invalid result.⁹

 As epidemiology of TB in India is diverse and there is paucity of study as regards to TB from this peripheral district of West Bengal, the study aimed to find out the prevalence and associated risk factors of rifampicin resistant TB among presumptive pulmonary tuberculosis patients by GeneXpert MTB/RIF assay in our hospital.

Study design and setting: A prospective cross-sectional study was conducted in a tertiary care hospital in West Bengal. The study was conducted for a period of 6 months from June 2024 to November 2024 in the Department of Microbiology, Jhargram Government Medical College and Hospital (JGMCH).

Study participants: A total of 829 presumptive pulmonary tuberculosis patients who attended the Chest and Respiratory Medicine department of JGMCH during the study period were included. Presumptive pulmonary tuberculosis refers to patients with any of the symptoms and signs suggestive of tuberculosis including cough>2 weeks, fever>2 weeks, significant weight loss, haemoptysis, any abnormality in chest radiograph.

Inclusion criteria:

1)Presumptive pulmonary tuberculosis patients suspected of MDR-TB who attended the tertiary care centre during the study period.

2) Presumptive pulmonary tuberculosis who were negative on microscopy by ZN stain.

3) Patients willing to participate in the study.

Exclusion criteria:

1)Extrapulmonary tuberculosis patients

2) Patients from whom adequate amount of sample could not be obtained.

3) Patients who did not consented for the study.

Laboratory procedure

Samples were collected as per the guidelines of Revised National Tuberculosis Control Program (RNTCP), India. Patients from whom sputum samples could not be obtained, bronchoalveolar lavage fluid was considered. Samples were then immediately processed for Xpert MTB/Rif assay by GeneXpert system (Cepheid, Sunnyvale, CA, USA). GeneXpert MTB/Rif assay is a cartridge based nucleic acid amplification test (CBNAAT) used for simultaneous and rapid detection of Mycobacterium tuberculosis and rifampicin resistance. Sample reagent buffer was then added to the sample in the ratio of 2:1 using a sterile pipette and then the lid was closed. Sample was then agitated twice before transferring 2 ml of the inactivated material to the test cartridge. The test cartridge was then loaded in GeneXpert device as per manufacturer’s instructions and finally results were interpreted from the measured fluorescence signals and displayed after 2 hours. Clinical history, demographic details and laboratory results of the patients were noted down using a standard questionnaire.

Statistical analysis: Data collected were analysed using Microsoft Excel software. Descriptive statistics was used to summarize the sociodemographic variables associated with pulmonary tuberculosis patient and value less than 0.05 was considered statistically significant. Odd’s ratio was determined to find out the factors associated with rifampicin resistance.

Ethical consideration: Ethical approval for the study was obtained from from Institutional Ethics Committee as per memo no IEC/2024/02 dated 11.06.2024.

Among 829 study participants included in our study, 548 (66.10%) were males and 281(33.9%) were females. The mean age of the study participants was 46.81 years with a minimum age of 7 years and maximum age of 84 years. Out of 829 presumptive pulmonary tuberculosis participants, prevalence of pulmonary tuberculosis was 24.84% (206) and rifampicin resistance tuberculosis was 0.84% (7) as detected by Xpert MTB/RIF assay (Figure 1).

Figure 1: Prevalence of MTB and RR-TB in presumptive pulmonary TB patients

Out of 206 diagnosed MTB patients, a higher prevalence of pulmonary TB was observed among males (28.10%) as compared to females (18.50%) and the difference was statistically significant (p<0.05). The highest proportion of TB positive patients were in the age group 21-40 years. Pulmonary TB was detected more in previously treated patients (35.95%) and the association was found to be statistically significant. (p<0.05) (Table 2).

Table 1: Sociodemographic variables associated with diagnosed pulmonary TB patients

The prevalence of RR-TB among diagnosed PTB patients was 3.39% (7/206) in our study. Rifampicin resistance was observed in 2.59% of male participants and 5.76% of female study participants and the crude odds ratio (COR) was less than 1. Rifampicin resistance was not reported in participants less than 20 years of age. RR-TB was more among alcoholics, in the tribal community and in people with previous history of TB treatment and COR was more than 1 in all denoting significant association (Table 3).

Table 2: Association of RR-TB with socio-demographic variables

In this study,57.14% (4/7) of RR-TB patients had very low Ct values indicating high bacterial load. In contrary, most of the TB patients where Rifampicin resistance was not detected showed either medium Ct value or high Ct value indicating low bacterial load. (Table 4).

Table 3: Rifampicin resistance and Ct value status among diagnosed MTB cases

Tuberculosis is a chronic infectious disease caused by the bacterium Mycobacterium tuberculosis and has caused a significant global health challenge.¹⁰ India accounts for 27% of global TB cases, the highest TB burden in the world.¹ In the present study prevalence of Mycobacterium tuberculosis was found to be 24.84%. Two different studies from African subcontinent reported prevalence of PTB as 11.6% and 6.17% respectively.^11,12 While another study from Nigeria reported a higher prevalence of 29.2%.¹³A study by Gautam et al¹⁴ from Uttar Pradesh, India reported a higher prevalence of 32.9% where as a study from West Bengal reported prevalence of 25.77%¹⁵ which concurs with our study. The difference in prevalence may be due to different geographical location, study design, variation in socio-demographic factors, opportunities for TB screening, diagnostic method used and general awareness among the public.

A higher positivity of pulmonary tuberculosis of 28.10% was found among males as compared to females (18.50%) and the global scenario concurs with our study.^16,17 In contrast, a study from Amhara region¹⁸ reported higher prevalence of TB among female patients. This may be due to more outdoor activities resulting in more exposure to crowded areas and more chances of transmission.

Since 29.37% of our district in West Bengal comprises of tribes as per 2011 census, we tried to find out the prevalence in tribal and non-tribal community. Tribal community were identified by a combination of factors which included linguistic differences, certain surnames exclusive to them, identification by community members and self-identification. There was not much difference in prevalence of TB in tribal and non-tribal community in our study. Prevalence of TB among tribal community was found to be 25.26%. Though tribal population is considered a risk group for TB¹⁹ our study could not arrive at a definite conclusion. A more descriptive study including a larger sample size would suggest a better picture in this region.

Emergence of drug resistance TB has become a significant public health problem and has contributed to high mortality. India has an estimated DR-TB incidence of 8 per 100,000 population.¹ DR-TB is one significant threat in achieving “End TB Initiative” and hinders effective management of TB. In the present study, prevalence of RR-TB was found to be 3.39% among confirmed TB cases. Our study concurs with previous studies from Karnataka and Himachal Pradesh where Rifampicin resistance among MTB patients was 4% and 3.2% respectively^20,21. Two other studies from different parts of India reported higher prevalence of 5.6% and 8.75% respectively.^22,23 Acquisition of resistance is mainly due to non-compliance to TB treatment on the part of patients or poor supervision of TB control programs.

The study showed that no RR-TB was reported in less than 20 years of age. This is in contrast to a previous study from West Bengal where RR-TB was found to be higher among 11-20 years of age.¹⁵ Our study shows more likelihood of development of RR-TB in previously treated patients (COR:14.23,95%CI = 1.67-120.86).Our finding corroborates with other studies.^24,25The high resistance might be due to level of TB control practices in different study population as well as non-compliance to adequate treatment resulting in selective pressure that favours the multiplication of mutant organisms which emerge as resistant clones. These clones multiply in the presence of suboptimal dose to become predominant, leading to recrudescence and emergence of drug resistance.²⁶

In this study alcoholics were 1.6 times more likely to develop Rifampicin resistant

TB(COR=1.68,95%CI=0.366-7.711). This is quite a well-established factor as alcoholism brings down body’s immune response. Also, alcohol interferes with isoniazid, the primary drug used to treat TB thus bringing down its effectiveness. Our finding was supported by a previous study from Uganda.²⁷Few other studies emphasises that effect of alcohol on MDR TB is inconclusive.²⁸

Xpert MB/RIF assay which is utilised globally for rapid diagnosis and detection of RR also provides quantitative estimation of TB bacillary load in cycle threshold values (Ct value). High Ct value indicates low bacillary load and vice-versa.²⁹In our study, most of the RR-TB samples had either very low or medium Ct values. A study from Lancet³⁰ published in the year 2020 emphasizes the fact that high bacillary load can be attributed to MDR where as low bacillary load may be associated with false rifampicin resistance. The study advocated repeat testing in case rifampicin resistance is detected with high Ct value.

Limitation

There were certain limitations in our study. Firstly, it would have been appropriate if both rifampicin and isoniazid resistance could have been detected by culture methods as that would have provided the real scenario of MDR-TB in this region. Secondly, larger sample with other factors like previous contact with TB patients would have given a better picture of prevalence.

The prevalence of RR -TB in our study was 3.34%. The low prevalence does not rule out the risk of active transmission in the community. Moreover, in a peripheral region like ours there is always reluctance among the people in accessing healthcare services along with lack of awareness which may hinder the rapid diagnosis and thus containment of DR-TB. Thus, emphasis should be made on strengthening diagnostics and creating awareness. Strict supervision of TB cases for compliance to treatment is compulsory to prevent drug resistance.

Funding: None

Conflict of interest: None declared

Acknowledgement: The authors are thankful to Principal of our institute, Dr Susmita Bhattacharyya for allowing us to carry out the study. We would also like to acknowledge the laboratory technologists of Department of Microbiology and staffs of Chest Medicine for their support in carrying out the study.

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