Mycobacterium tuberculosis strain lineage in mixed tribal population across India and Andaman Nicobar Island

In India, the tribal population constitutes almost 8.6% of the nation’s total population. Despite their large presence, there are only a few reports available on Mycobacterium tuberculosis (M. tb) strain prevalence in Indian tribal communities considering the mobile nature of this population and also the influence of the mainstream populations they coexist within many areas for their livelihood. This study attempts to provide critical information pertaining to the TB strain diversity, its public health implications, and distribution among the tribal population in eleven Indian states and Andaman & Nicobar (A&N) Island. The study employed a population-based molecular approach. Clinical isolates were received from 66 villages (10 states and Island) and these villages were selected by implying situation analysis. A total of 78 M. tb clinical isolates were received from 10 different states and A&N Island. Among these, 16 different strains were observed by spoligotyping technique. The major M. tb strains spoligotype belong to the Beijing, CAS1_DELHI, and EAI5 family of M. tb strains followed by EAI1_SOM, EAI6_BGD1, LAM3, LAM6, LAM9, T1, T2, U strains. Drug-susceptibility testing (DST) results showed almost 15.4% of clinical isolates found to be resistant to isoniazid (INH) or rifampicin (RMP) + INH. Predominant multidrug-resistant (MDR-TB) isolates seem to be Beijing strain. Beijing, CAS1_DELHI, EAI3_IND, and EAI5 were the principal strains infecting mixed tribal populations across India. Despite the small sample size, this study has demonstrated higher diversity among the TB strains with significant MDR-TB findings. Prevalence of Beijing MDR-TB strains in Central, Southern, Eastern India and A&N Island indicates the transmission of the TB strains.


Introduction
Indian tribal population is one of the highly neglected groups of people due to geographical and cultural barriers in terms of health and associated pubic health services (Govt of India 2020-21). Tuberculosis (TB) claims about 2 million lives annually and it is the leading cause of mortality worldwide due to a single bacterial agent. The situation is particularly alarming in several developing countries including India, as India accounts for over 30% of the global burden of TB. Besides, the emergence and spread of drug-resistant strains and the synergistic association of these diseases with AIDS are posing major challenges for TB control. Due to the aforesaid reason, the World health organization (WHO) has announced tuberculosis as a Global emergency (WHO 2020). Recent report states that a widespread prevalence of pulmonary tuberculosis (PTB) extensively was seen across the zones and states in India. It indeed surged to conduct several studies at the zonal and various state level across India (Thomas et al. 2021). Preliminary data indicate the prevalence of TB among the tribal population is 703 per Azger Dusthackeer, Ashok Kumar and Sucharitha Kannappan Mohanvel have contributed equally to this work. 100,000 populations but there is an urgent need to access the incidence and strain diversity of Mycobacterium tuberculosis (Thomas et al. 2015). According to Rao et al. (2019), more than 70% of the tribal population resides in the central Indian states of the country. Madhya Pradesh in central India accounts for about 21.1% of the tribal population with 46 ethnic groups. To comprehend the transmission dynamics of M. tuberculosis strains and their distribution, molecular typing plays a significant role to scrutinize outbreaks in health care settings at a specific region/area (Ameke et al. 2021). Based on the WHO guidelines, TB resistance to at least two drugs (Isoniazid and Rifampicin) was characterized as multidrug-resistant tuberculosis (MDR-TB). MDR-TB epidemics were chiefly focused on transmission than drug resistance acquisition during treatment (Kendall et al. 2015;Sharma et al. 2017). Spoligotyping, Restriction fragment length polymorphism (IS6110-RFLP) typing/and Mycobacterial interspersed repetitive unite variable number tandem repeat (MIRU-VNTR) typing are the most used tools for epidemiological strain typing or molecular evolutionary studies of M. tuberculosis (Devi et al. 2021). Characterizing M. tuberculosis isolates by using Spoligotyping, is a PCRbased reverse hybridization blotting technique established by polymorphism (Gupta et al. 2014;Varma-Basil et al. 2017;Rahul 2019;Desikan 2012). Overall genomic differentiation in M. tuberculosis is designated by articulating via spoligotyping patterns which eventually depicts the diversity (Said et al. 2009). Our research intended to identify the types of M. tuberculosis strains prevalent across several parts of India, A&N Island tribal groups which seem to report different strains and the associated clusters across India. It is the foremost intervention upon mixed tribal populations across India, A&N Island. Several communal elements such as deprived medical support, pitiable socio-economic, and other medical risk aspects play a significant role featuring the mitigation of TB incidence (Togun et al. 2020). Additionally, the deterioration of the TB situation is mainly due to the progress of drug resistance in M. tuberculosis isolates. Our study aimed to estimate the prevalence of M. tuberculosis lineages among mixed tribal groups in tribal areas of Central, Southern, Eastern India, and A&N Island.

Study design
In order to study the prevalence of pulmonary tuberculosis among tribal populations in India from April 2015 to March 2020 in the published work of Thomas et al (2021) the large-scale national level tribal tuberculosis prevalence survey of India was carried, evolving different phase studies aged ≥ 15 years in tribal villages. Based on the population proportional to the estimated size (PPES) method, equal weightage was given to the entire country. Based upon the disease prevalence of 387/100,000 populations (Bhat 2009), 92,038 sample size were projected apparently, the country was divided into 6 zones, each with two or more states. Districts with > 70% tribal prime populations were recorded from each zone and itemized the villages details in these districts besides based on PPES. These states comprising East, West, North, South, Central, and North East of almost 88 villages totally selected from 17 states of India. In order to attain the minimum of 800 individuals selected randomly from village streets (Thomas et al. 2021). Our research report involves Phase I trial of Thomas et al. group, which covered 66 villages from 10 states and A&N island viz., Madhya Pradesh, Chhattisgarh, Jharkhand, Maharashtra, Odisha, Manipur, Tripura, Rajasthan, Telangana, and Nagaland, where in 78 clinical isolates of M. tuberculosis were isolated from tribal people living in these places and we have reported and analysed the type of lineages seen among these isolates.

Culture identification
The cultures received from the laboratories were sub-cultured on Lowenstein Jensen Media (LJ) and were identified using the MPT64 antigen detection test, growth on P-nitrobenzoic acid (PNB), and Ziehl-Neelsen staining to confirm Mycobacterium (Bacteriology and of. Standard Operating Protocol 2016, http:// www. nirt. res. in/ pdf/ bact/ SOP. pdf).

Drug resistance associated mutation
Those cultures identified as M. tuberculosis, further subjected to genotypic identification by Genotype MTBDR plus 96 kits to determine the susceptibility for RMP (rpoB) & INH (katG and inhA) by LPA assay (WHO 2007). When the cultures are found to be non-tuberculous mycobacteria (NTM), species identification was performed by a conventional method. Species growth identification done by checking for the growth in the presence of P-nitrobenzoic acid (PNB) by PNB inhibition test and 68 °C catalase test were performed as well (Agarwal et al. 2014).

Spoligotyping
Spoligotyping was performed as described by Kamerbeek et al. (1997). DRa and DRb primers were used for amplifying the direct repeat (DR) region in the genome of the M. tuberculosis complex. M. tuberculosis H37Rv strain and M. bovis BCG P3 chromosomal DNA were used as positive controls. The molecular biology grade water served as the negative control. The commercial membrane pre-coated with spacer-oligos, which represents the spacer region of known sequences, was used for hybridizing the amplified product. The membrane was incubated with streptavidin-peroxide and ECL to visualize the presence/absence of spacer on X-ray film as black squares. BioNumerics software version 6.6 (Applied Maths, Sint-Martens-Latem, Belgium) was used for spoligotyping through the neighbor-joining method with Jaccard`s distance and analysed the association between the different states and lineages.

Lineage dissemination
According to the lineage dissemination of the classification, the strain types were categorized into lineages 1 to 9 (L1 to L9). Based on this system of heterogeneous grouping, the distribution of strains belonging to lineages 1 to 4 were seen prevalent in Madhya Pradesh with more of L1 (n = 10), while L3 (n = 7) ranked next with one each of L4 and L2. In Odhisa, the predominant type was found to be L1 (n = 8). In A&N island L1 (n = 5) predominate followed by L2 (n = 3). In Chhattisgarh L3 lineage lead (n = 5) followed by L1 (n = 3). Whereas, the Northeast states comprising Manipur, Nagaland, and Tripura; overall exhibited L2 (n = 8) strain type followed by L3 (n = 6) ( Fig. 2).

Cluster examination
Almost 50 strains (64%) were isolated from men and 28 (26%) were women. The Male TB percentage exceeds the female case among the small sample size. Almost 16 different spoligotype patterns were identified in clinical isolates (n = 78) by the spoligotyping technique. Strains with the same spoligotype were considered as a cluster in the study. These 78 isolates were observed to belong to 8 clusters, ranging from 2 to 20 isolates per cluster, while 25 were found to be unique. Out of these 8 clusters, cluster 1 and cluster 8 were found to be MDR-TB Beijing strains, and cluster 3 was found to be mono resistant CAS1India strain from Madhya Pradesh (Table 1) (Fig. 3a, b). The major spoligotype families observed belonged to Beijing, CAS1_DELHI, EAI3_IND, and EAI5 family of    M. tuberculosis strains (n = 58, 74%), followed by Central Asian strain CAS2 (n = 2, 2.9%), EAI1_SOM (n = 3, 4.3%), and other strains viz; EAI3, EAI6_BGD1, LAM3, LAM6, LAM9, T1, T2, U are present solitary strains and cumulatively contribute 13% (Table 2).

Drug susceptibility testing
The drug susceptibility tests for INH and RMP were carried out for all the 78 clinical isolates. Results exhibited 15.4% (n = 12) of clinical isolates showing resistance to INH or RMP + INH, which are first-line anti-TB drugs. The results of our DST tests identified resistance to INH (mono drug) in 3.8% isolates and multidrug resistance in 11.5% of cases. While 2.6% MDR strain was recorded among the M. tuberculosis isolates from women subjects, 9% M. tuberculosis isolates from men were found to be MDR (Table. 3).

Mutation of rpoB, KatG and inhA
According to the LPA assay, 3 types of mutations were found in the rpoB gene among five different tribes. MUT3 (S531L) were found in TRI062 and TRI346, whereas 2 tribal clinical isolates (TRI326, TRI345) showed MUT1 (D516V) and MUT2 were seen in TRI339. Likewise, the katG gene exhibited a different mutation; MUT2 (S315T2) in TRI053 whereas other clinical samples showed similar mutations.

Discussion
Epidemiological analysis of the clinical isolates aided by spoligotyping, a PCR-based genotyping method was carried out to understand the lineage-wise distribution of M. tuberculosis among the tribal population. In this study, M. tuberculosis belonging to L1 to L4 was seen in various ratios among the states. The dominance of M. tuberculosis lineages that are more prone to acquire drug resistance complicates the TB control program to a greater extent in that region. We observed predominance of L1 in Odhisha, Andaman/Nicobar Island, and Madhya Pradesh; L2 was the dominant strain type in North-Eastern states, while L3 was found more in numbers at Chhattisgarh. Notably, L2 and L3 are omnipresent in all the reported states which implicates that the emergence of drug-resistant phenotypes in these tribal areas is more likely to occur requiring more stringent control measures focusing on the tribal population. According to Gagneux et al. (2007)   Complex clustering was visualized in spoligotyping data analysis. A total of eight clusters was noted by spoligotyping and these clusters were based on the state they are isolated and the type of their family. The number of strains per cluster ranged from 2 to 20. A total of 10 strains are designated as orphans that could not be brought under any clusters were also observed. The strain prevalent in each tribal area was the same as with the strain circulating in that respective state or the region. The number and types of the cluster in Madhya Pradesh were on the higher side and these need further investigation. The data from Madhya Pradesh showed that there exists a considerable amount of transmission among close tribal communities in India accentuating the need for constant screening of these communities to inter-communal spread among the tribes. It has two spoligotype clusters, one with CAS Delhi strain and the other with EAI strains, previous reports have shown a trend of decrease in EAI strains as we travel from south to north and increase in CAS Delhi strains as we travel from south to north of India (Singh et al. 2007). According to the above hypothesis, Madhya Pradesh should have an equal number of these strains and our data perfectly fits into the hypothesis. Since these are common strains in the community, we also predict that these strains have been transmitted from the general population to the tribal population. Similarly, we found that the cluster with Beijing strains (n = 13) is more predominant in the northeast region of India compared to other reports and also in the present study. This could be more related to the region being more geographically closer to china where Beijing strains are common as reported by Devi et al. (2015). The western part of India seems to report a high percentage of Beijing genotype (23%) besides associated with MDR-TB. The highest incidence of MDR-TB among the Beijing family of M. tuberculosis strains is also reported in the present study. This study report forecasts the Beijing strain prevalence over the border of eastern India and A&N Island.
Eight MDR (10.25%) M. tuberculosis was recognized in this work. Out of twelve drug-resistant isolates reported in this study including MDR-TB, five were from A&N Island which seems to be the foremost report from Nicobarese (ST292/EAI6_BGD1, ST64/LAM6, three from ST1/Beijing). Our report implicates that it would be a major challenge for the patients to travel to Port Blair for the initiation of MDR-TB treatment. It is extremely difficult and also a major risk for MDR-TB transmission. Moreover, three clinical strains were ST1/Beijing from Nagaland and Tripura respectively. The other 4 mono resistant clinical isolates were, 1 ST26 /CAS1_DELHI from Madhya Pradesh and Manipur each; ST702/EAI5 from Madhya Pradesh; ST292/ EAI6_BGD1 from A&N respectively. The recent report reveals 4.85% MDR and the percentage of INH resistance was found to be higher (14%) than MDR-TB in the Sahariya tribal population when compared to the non-tribal (6%) population (Prakash et al. 2016).

Conclusion
Based on the literature reviewed, compared to the common population, the prevalence of PTB among the tribal clusters are relatively seen at higher level amid the tribal groups at state and zonal level besides which were left unexplored due to the unapproachable perspective among the tribal populations which was set to call for site-specific interference plans. This study is the most likely first of its kind to report the M. tuberculosis strain lineage among mixed tribes belonging to various parts of India and Andaman & Nicobar Island. On the whole, our study sheds light on the increasing drug-resistance prone L2 lineage strains, especially the Beijing strains among the tribal population across the country and the inter-communal spread of TB between the various tribes and regions of the country.

Limitations
The Spoligotyping method used in this study is known to have a problem of homoplasy and this is one of the limitations of this study. Secondary line typing methods (IS6110-RFLP or MIRU-VNTRs) are to be employed to advance this research further. The major limitation of this study is the low sample size which could be addressed in the next phase of this tribal study to arrive at any conclusion in other domicile states of India.