INTRODUCTION
The emergence of drug-resistant tuberculosis (TB) poses a great threat to control TB worldwide by finding rifampicin (RIF) resistance (RR). We can have an indication of isoniazid resistance because it will act as a surrogate marker of multidrug resistance. Hence, it is very important to study the prevalence of RIF-RR region-wise and finding out any changing pattern of resistance over time in a particular region. Pulmonary TB (PTB) is a chronic infectious airborne disease caused by a group of acid-fast bacteria called the Mycobacterium TB complex. PTB remains a major public health issue as one-third of the world’s population has latent TB with 10% of this population developing active disease during their lifetime.[1]Mycobacterium TB (MTB) is an important public health problem because of the high mortality that it causes. The main strategy to control this problem is the rapid diagnosis of MTB infection and the identification of high-risk cases. One of the latest techniques is GeneXpert MTB/RIF has been used to detect PTB cases.[2,3] RIF is arguably the most important drug in the treatment of TB. Infection by RIF-RR TB requires long therapy with less effective and more toxic second-line drugs. In recent years, cartridge-based nucleic acid amplification test (CBNAAT) has been recommended by World Health Organization as a rapid diagnostic test for the detection of TB and RIF-RR.[4]
The objective of this study was to use CBNAAT to determine the prevalence of RIF-RR MTB among PTB cases at Dr. Rajendra Prasad Government Medical College, Kangra at Tanda.
METHODS
Study area
This retrospective study was conducted over 4 years (from January 2018 to December 2021) at Dr. Rajendra Prasad Government Medical College, Kangra at Tanda, Himachal Pradesh.
Study population
Specimens were collected from all age group who was suspected to PTB. Samples were collected in sterile containers from each patient for diagnosis of PTB. All samples were stored at 4°C until processed by standard laboratory protocol. The specimens were processed within 24 h at the laboratory. GeneXpert MTB/RIF assay (Cepheid, Sunnyvale, CA, USA) was used for the identification of Mycobacterium. A 1 mL unconcentrated specimen was used (without centrifuge) for GeneXpert MTB/RIF assay. Drug susceptibility testing of isolates to RIF was determined using the GeneXpert MTB/RIF assay.
Data analysis
Data collected were analyzed and processed with Excel and Word Software (Microsoft).
Analysis of samples by GeneXpert Mycobacterium tuberculosis/rifampicin assay
The assay was performed using version 4 cartridges according to the manufacturers’ recommendations. Briefly, the sample reagent (containing NaOH and isopropyl alcohol) was added at a 2:1 ratio to the clinical specimen to kill the mycobacteria and liquefy the samples. Fluids were processed directly by the addition of a 2:1 volume of SR buffer. The sample-SR mixture was shaken vigorously and incubated for 10 min before being shaken again and kept at room temperature for another 10 min. Two milliliter of the digested material was transferred to the cartridge. Cartridges were inserted into the GeneXpert machine, and the automatically generated results were read after 90 min. Resistance was expressed as the percentage of colonies that grew on critical concentrations of the drug (40 μg/mL for RIF). The interpretation was made according to the usual criteria for resistance, i.e., 1% for all drugs. In case the results were reported as invalid, error, or no result, the sample was reprocessed and rerun, if sufficient material was available.[5–7]
RESULTS
Of the total 11,774 clinically suspected PTB specimens were collected, in which sputum (9963), bronchoalveolar lavage (BAL) (157), and gastric fluid specimens (1654) were collected. 2358/11,774 (20%) samples were MTB positive and 9416/11,774 (80%) were MTB negative. Out of which 2307 samples were from sputum, 45 were from gastric aspirate and 6 were from BAL were MTB positive [Table 1 and Figure 1].
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Table 1: Sample-wise distribution among Mycobacterium tuberculosis positive and negative sample
Figure 1: Distribution of pulmonary sample. BAL: Bronchoalveolar lavage
Among 2358 MTB-positive samples, 884, 660, 421, and 393 samples were MTB-positive in the year of 2018, 2019, 2020, and 2021, respectively [Figure 2], and 1629/2358 (69.1%) were male and 729/2358 (30.9%) were female [Table 1]. Of the total 2358 MTB-positive samples, 2240/2358 (95%) samples were RIF sensitive, in which 1553 (65.9%) were males and 687 (29.1%) were females, 76/2358 (3.2%) samples were RIF-RR, in which 51 (2.2%) were males and 25 (1%) were females, and 42/2358 (1.8%) samples were RIF indeterminate, in which 25 (1%) were males and 17 (0.8%) were females [Table 2 and Figure 3].
Figure 2: Distribution of MTB positive and negative samples. MTB: Mycobacterium tuberculosis
Table 2: Distribution of rifampicin sensitive, resistant, and indeterminate among Mycobacterium tuberculosis-positive sample
Figure 3: Distribution of RIF sensitive, resistant, and indeterminate among Mycobacterium tuberculosis-positive sample. RIF: Rifampicin
DISCUSSION
In the present study, the prevalence of MTB was 20% (2358/11,774) detected by GeneXpert assay. Among 2358 MTB positive, 37.5% (884/2358) were in 2018, 28% (660/2358) were in 2019, 17.9% (421/2358) were in 2020, and 16.7% (393/2358) were detected in 2021 [Table 1] which is gradually decrease from 2018 to 2021 might be because of proper implementation of the TB control program and extra precaution taken during COVID pandemic. Among 2358 MTB-positive samples, 95% (2240/2358) samples were RIF sensitive, 3.2% (76/2358) samples were RIF-RR, and 1.8% (42/2358) samples were RIF indeterminate by GeneXpert MTB/RIF assay.
Males were predominantly infected with RIF-RR TB (RR-TB). Disparity in gender distribution of RR-TB could have been so due to the fact that men are more exposed to factors that lead to RIF-RR such as overcrowding in marketplaces, poor adherence to treatment, smoking, and alcoholism which make them more susceptible. This is also consistent with global trends in TB by gender.[8] In the present study, the gender-based prevalence among MTB positive gave a 1629/2358 (69.1%) were male and 729/2358 (30.9%) were female, among which 1553 males and 687 females were RIF sensitive, 51 males and 25 females were RIF-RR, and 2 5 males and 17 females were RIF intermediate [Table 2]. This result is approximately equal to a study done by Abdoulaye et al. 2022 and Combo Georges et al. 2020, in which male was largely predominant i.e., 75.2% and 70.73%, respectively.[9,10] A study conducted by Pokheriyal et al., 2021, Dehradun reported that 94.11% (16/17) were RIF sensitive and 5.89% (1/17) were RIF-RR;[11] which is slightly higher than the present study. A study conducted by Patel et al. 2019 Ahmadabad reported 4.5% (15/333) RIF-RR by CBNAAT.[12] A study done by Jain and Agrawal 2019 from Madhya Pradesh showed samples positivity rate were 41.87% (727), of which 39.17% (680) RIF sensitive and 2.7% (47) resistant among PTB;[13] which is similar to the present study. A study by Reddy and Alvarez-Uria 2017 Andhra Pradesh;[4] Singh et al., Patna 2020;[5] and Shilpa Nadagir et al., Karnataka;[14] reported that 9.2% (171/1851), 5.6% (10/146), and 8.75% (12/137) prevalence for RIF-RR by CBNAAT, respectively. A study by Gautam et al., 2018, Uttar Pradesh reported that 26.1% (44/168) were RIF-RR which is higher to the present study and most of them were male i.e., 38 while 6 were female.[15] These high rates of RIF-RR in MTB by Uttar Pradesh study may be attributed to nonadherence to treatment regimens and improper TB control practices. A study done by Christopher et al. 2019 reported 4.9% showed RIF-RR;[16] which is similar to the present study. TB burden countries such as India as this GeneXpert MTB/RIF assay will help in rapid diagnosis of RIF-RR among suspected PTB cases.
CONCLUSION
It was concluded from our study that the rate of detection of MTB was found more in males and over the year, the detection rate was decreased in sputum samples, however, such a pattern was not observed in other samples, namely in gastric aspirates and BAL samples. The rate of RIF-RR was 3.2% of total samples which was again found more in males. No particular pattern of the rate of detection of RIF-RR was found over the year. The overall positivity rate was 20%, and the rate of positivity decreased from 32% to 14% over the 4 years in sputum samples. Hence, the GeneXpert assay was found to be a very important tool to detect RIF-RR among suspected PTB patients.
Limitation of the study
The limitation of this study is that we have not included microscopy and culture techniques among suspected PTB patients.
Ethical clearance
This is a study that used laboratory-based diagnostic data. The study was authorized by the in charge of the laboratory.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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