Assessing the Genetic Diversity of Mycobacterium tuberculosis Strains in Kerala, India: A Comprehensive Study

Introduction


INTRODUCTION
The COVID-19 pandemic has disrupted TB services, with newly diagnosed tuberculosis cases decreasing from 7.1 million in 2019 to 5.8 million in 2020 [1].Studies conducted during the pandemic have found a link between the pandemic and the impact on the immune system's response to TB, which may contribute to an increase in TB cases.This potential consequence poses a significant challenge to achieving the WHO's End TB Strategy goal to reduce TB incidence by 80% and TB deaths by 90% between 2015 and 2030 [2].India bears a substantial portion of the global TB burden, accounting for approximately 27% of total cases [1].Rapid molecular diagnostic tests, antibiotics treatment, and identifying and monitoring individuals who have been in close contact with someone who has TB are crucial steps for halting further transmission and controlling tuberculosis.In 2019, Kerala, a state in South India, reported 72 tuberculosis cases per 100,000 population, representing data from before the COVID-19 pandemic [3].
Studies have shown differences in virulence factors and immune system responses among different M. tuberculosis strains or lineages.Certain strains may exhibit varying degrees of transmissibility and diseasecausing capabilities, contributing to the complex dynamics of TB transmission and pathogenesis [6].Data from India and South Africa suggest that individuals with concurrent infection with M. tuberculosis and SARS-CoV-2 have an increased risk of death due to complications from both infections.Additionally, reports indicate a substantial reduction in reported TB cases and completed treatment cycles in these countries by approximately one million in 2020, amidst broader healthcare disruptions and lockdowns imposed during the pandemic [7].
Some strains of M. tuberculosis, such as the Beijing genotype, have been linked to the development of drugresistant TB, although the relationship is not fully understood [8].M. tuberculosis has a diverse genetic population worldwide, with six distinct strains: Indo-Oceanic, East Asian, East African-Indian, Euro-American, West African I, and West African II, though the distinctions between these strains can be ambiguous in some cases [8].
Molecular epidemiology techniques like spoligotyping, restriction fragment length polymorphism (RFLP), and Mycobacterial interspersed repetitive units-Variable number of tandem repeats (MIRU-VNTRs), have been employed to investigate the genetic diversity and transmission dynamics of M. tuberculosis strains, contributing to a better understanding of their relationships [9].These methods help researchers understand the predominant strains of M. tuberculosis present in a specific geographic region and how they spread among individuals [5].Spoligotyping is a technique used to identify genetic relationships between different strains of M. tuberculosis based on DNA polymorphism in the direct repeat (DR) locus.It is a robust method with an intra-laboratory reproducibility over 90% in well-trained laboratories, owing to its reliance on a single PCR amplification, ability to differentiate main lineages and sublineages within the M. tuberculosis complex, and cost-effectiveness [10,11].
Spoligotyping relies on a specific region of the M. tuberculosis genome called the DR locus.This region consists of multiple short, identical DNA sequences (DRs) interspersed with unique spacer sequences of varying lengths.The spoligotyping technique involves amplifying this entire DR locus using multiplex or nested PCR.This process essentially creates copies of the target DNA region, allowing for further analysis.The PCR products, representing different spacers and DRs, are hybridized to a membrane with 43 covalently bound synthetic oligonucleotides.These oligonucleotides represent the polymorphic spacers identified in M. tuberculosis H37Rv [spacers 1 -19, 22-32, and 37-43] and M. bovis BCG [spacers 20-21 and 33-36].The hybridization signals are detected by chemiluminescence through biotin labeling of one of the primers used for PCR amplification and a streptavidin-peroxidase conjugate system, which binds to biotin-labeled PCR products, and then visualized by autoradiography.Individual strains are differentiated based on their unique pattern of absent spacers within the complete set of 43 spacers [10].The missing spacers are likely caused by deletions in the bacterial DNA through mechanisms such as homologous recombination, transposition, or other forms of genetic recombination.The DR region is a preferred site for IS6110 insertion, which can lead to deletions and genetic variation [12].Spoligotyping is a well-established and widely applied molecular typing technique in tuberculosis studies for investigating outbreaks, transmission dynamics, and population structure [13].It allows researchers to simultaneously identify and distinguish between distinct genetic variants or sublineages within the M. tuberculosis complex [11].
Despite the known presence of drug-resistant tuberculosis (DR-TB) in Kerala, only a limited number of studies have investigated the genetic diversity and transmission dynamics of multidrug-resistant M. tuberculosis isolates from this southern Indian state.This contrasts with the large number of studies that have investigated the molecular epidemiology of DR-TB in northern India.Furthermore, only a few investigations have aimed to identify and assess the genetic mutations associated with drug resistance genotypes in the general population.This study employed spoligotyping to investigate the molecular epidemiology of M. tuberculosis strains isolated from patients with tuberculosis attending a tertiary care hospital in central region of Kerala.The findings will contribute to understanding the predominant lineages of MTBC circulating in this region.

MATERIAL AND METHODS
Study setting and sample collection.The study was conducted at a tertiary care center in the central region of Kerala.A total of 404 pulmonary and extrapulmonary specimens, excluding blood, were collected from November 2015 to October 2017.These specimens were sent for routine mycobacterial culture and drug susceptibility testing (DST) using the MGIT system, in accordance with the established protocols of the Mycobacteriology Section.Blood samples were not included in this study as they were not received for culture during the study period.Patient data collection.Demographic information (age, sex, place of residence, birthplace, previous episodes of TB disease, and underlying medical conditions such as diabetes, HIV, or lung disease) were collected and recorded from the patients' medical records.
Sample processing and decontamination.The samples were processed within 48 h of collection using the N-acetyl-L-cysteine-NaOH digestiondecontamination procedure [14] to minimize the risk of M. tuberculosis contamination at the Mycobacteriology Section of the Microbiology Department.This procedure was performed in a Biosafety Level 3 [BSL3] laboratory with strict adherence to World Health Organization (WHO) guidelines for working with M. tuberculosis to protect personnel and prevent environmental contamination.
Culture and confirmation of M. tuberculosis.All specimens were subjected to direct smear microscopy using Ziehl-Neelsen [ZN] staining for acid-fast bacilli detection, followed by N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) digestion and decontamination for non-sterile specimens.However, samples from sterile sites (cerebrospinal fluid (CSF), synovial fluid, and ascitic fluid) were directly inoculated after concentration by centrifugation onto BBL Mycobacteria Growth Indicator Tube (MGIT BD BACTECTM) containing 7 mL of modified Middle Brook 7H9 broth, supplemented with an enrichment supplement and a mixture of antimicrobials (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin) to inhibit contaminants and non-target microorganisms.The MGIT tubes and Lowenstein-Jensen's medium were incubated at 37 ºC to ensure maximum recovery rates.The tubes were examined daily for up to 8 weeks of incubation using a BACTEC TM Micro MGIT device equipped with a 365 nm wavelength UV light source fluorescence detector for signs of mycobacterial growth, including granular appearance and fluorescence.All positive tubes were confirmed for the presence of acid-fast bacilli (AFB) by Z-N staining and subcultured onto a blood agar plate to rule out contamination and confirm the purity of the isolated M. tuberculosis strains.The smears were examined for AFB and the characteristic cord factor of MTBC [15].Confirmed MTBC isolates were further identified using the BD MGIT MTBC identification test [TBC ID].
Phenotypic DST for antimicrobial resistance to the first-line anti-TB drugs was performed using the BACTEC™ 960  DNA extraction [10].Bacterial DNA extraction was performed using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's instructions.Broth culture samples were centrifuged at 6000 x g (8000 rpm) for 1 min to pellet the bacterial cells.The resulting pellet was resuspended in the minimal volume of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and MilliQ water to ensure a sufficient amount of DNA for downstream applications, and to ensure high-quality DNA extraction using the QIAamp Mini Kit protocol.
Spoligotyping.Spoligotyping was performed on confirmed MTBC isolates to determine their genetic variation.Genomic DNA extracted from the isolates was used to amplify the highly polymorphic DR region, specifically the spacer oligonucleotides (spacers) between the DR elements.This amplification was achieved using labeled primers and the Mapmygenome Spoligotyping Kit (Hyderabad) following the manufacturer's standardized protocol with the specifically designated primers of DRa and DRb regions [10].Conventional PCR was performed with the following conditions: an initial denaturation step at 94 ⁰C for 3 min, followed by 25 amplification cycles of a denaturation step at 94 ⁰C for 1 min, an annealing step at 55 °C for 1 min, and an extension step at 72 ⁰C for 30 sec [10].Following the PCR amplification steps, a final extension step at 72 °C for 7 min was performed to ensure full-length extension of all PCR products.The PCR reactions were then stored at 4°C for subsequent spoligotyping analysis.To monitor for potential contamination, a DNA-free water control was included in each PCR run.M. tuberculosis H37Rv and M. bovis-BCG strain were included as positive amplification controls in all PCR assays to verify the presence of the expected amplicon.These strains are known to produce the specific amplicon targeted by the primers used.Following amplification, the PCR products were separated by agarose gel electrophoresis (AGE) through a 1.5% agarose gel at 100 volts (V) and 500 milliamps (mA) for 45 min.This process enables the separation and detection of the amplified DNA fragments.The amplified DNA fragments were then subjected to spoligotyping, a membrane-based technique used to identify distinct DNA sequence variations within the DR region of MTBC.In this method, spacer probes with sequences complementary to the spacer sequences between the DR regions of MTBC were hybridized into the amplified DNA fragments.The membrane was then incubated at 60 C for 1 h, followed by two washes with a buffer solution containing 2 times the concentration of saline-sodium phosphate-EDTA (SSPE) buffer and 0.5% sodium [ DOI: 10.61186/JoMMID.12.1.42] [ Downloaded from jommid.pasteur.ac.ir on 2024-06-13 ] 2024 Vol. 12 No. 1 dodecyl sulfate (SDS) for 10 min to remove nonspecifically bound probes.The membrane was incubated with streptavidin-conjugated peroxidase for 1 h and washed twice in a buffer solution with a pH of 7.4 and containing 2 times the concentration of SSPE and 0.5% SDS for 10 min.Attachment of biotin molecules to the PCR products was achieved by incorporating a biotinylated primer during the amplification step.According to the manufacturer's instructions, the biotinylated PCR products were hybridized with 43 spacer oligonucleotides covalently bound to a membrane, each with a specific sequence corresponding to a particular spacer region.The membrane was then incubated with the enhanced chemifluorescence system (ECF) (GE Healthcare, UK) substrate, a detection system that uses chemifluorescence to produce a fluorescent signal, for 10 min.Next, the resulting signal pattern indicating the binding of biotinylated PCR products to the spacer oligonucleotides was visualized using a developer solution to reveal the chemifluorescent signal and a fixer solution to stabilize the signal.Finally, the membrane was exposed to Hyperfilm, a high-sensitivity autoradiography film, to capture the chemiluminescent signal emitted from the chemifluorescent substrate bound to the hybridization pattern.
Spoligotyping results were visualized as black squares representing hybridization signals on autoradiography film, indicating the presence or absence of specific spacer oligonucleotides in the analyzed MTBC strains.These patterns were then converted into binary codes consisting of 43 digits, each representing the presence (1) or absence (0) of a corresponding spacer oligonucleotide.This binary code information was used for further analysis using the online TB-VIS tool [15] to determine the specific families and lineages of the M. tuberculosis strains, as different strains possess unique combinations of these 43 spacer oligonucleotides, which determine their specific families and lineages [16].
Male patients had a mean age of 56 years (± 20 standard deviation) compared to females with a mean age of 46 years (± 22 standard deviation).The age group with the highest number of cases (61-80 years) was similar for both genders.Among the 404 processed specimens, 224 (55.4%) originated from males and 180 (44.6%) from females, with males having a higher proportion of positive cultures as shown in Figure 4.7.This trend continued among positive cultures, with 27 (56.3%)isolates from males and 21 (43.8%) from females.
Antibiotic susceptibility test results.Among the 48 M. tuberculosis isolates, 20 (41.7%) were susceptible to all five first-line anti-TB drugs, while three (6.2%)exhibited resistance to at least one of the five first-line anti-TB drugs.The highest rate of monoresistance was towards streptomycin [STR] [n=7, 14.9 %], followed by isoniazid [INH] [n=6, 12.5%] and pyrazinamide [PZA] [n=4, 8.3 %].Among the total of 48 culture-positive TB cases, two [4.1%] were found to be MDR-TB.The two MDR isolates were obtained from pulmonary TB cases.Additionally, 12.5% (6/48) of the culture-positive cases displayed resistance to three or more of the five first-line anti-TB drugs.
Spolityping patterns.Among the MTBC isolates, the Indo-oceanic family was the most prevalent, followed by the East Asian lineage or Beijing family.Notably, one isolate belonged to the M. bovis lineage.It is important to note that the lineages of seven isolates could not be determined due to either unknown or untypeable genetic profiles or technical limitations or insufficient data.Analysis of the isolates revealed the following distribution: 36.1% (n=17) belonged to the M. tuberculosis East-African-Indian (EAI3) family, followed by 27.7% (n=13) in the M. tuberculosis EAI5 family and 21.2% (n=10) in the M. tuberculosis Central Asian (CAS) family.M. tuberculosis Beijing isolates constituted 8.5% (n=4), while the family 33 lineage and M. bovis-BCG family each accounted for 4.3% (n=2) and 2.1% (n=1) of the isolates, respectively (Table 1).Notably, one isolate grew in culture but failed to amplify during PCR.
The M. tuberculosis isolate discussed earlier was classified to lineages using the TB-VIS online tool.The isolate was identified as belonging to either the Central Asian (CAS) or East African-Indian (EAI) lineage.
Among the five multidrug-resistant (MDR) and Rifampicin-resistant (RR) M. tuberculosis isolates, three belonged to the M. tuberculosis EAI3 family, one to the M. tuberculosis EAI5 family, and one to the M. tuberculosis Beijing family.

DISCUSSION
Analyzing the frequencies and transmission patterns of various M. tuberculosis genotypes is crucial for developing effective strategies for TB control and vaccine development [17].Genotyping techniques, including Spoligotyping, MIRU-VNTR, and IS6110 Restriction Fragment Length Polymorphism [IS6110-RFLP] are essential for understanding the genetic diversity of M. tuberculosis isolates [18].Although RFLP typing is widely used for mycobacterial cultures, its application is limited by the slow growth rate of M. tuberculosis, which requires 20-40 days to produce sufficient DNA for analysis.This delay hinders its utility in clinical settings, particularly when investigating potential nosocomial transmission of tuberculosis [19].Spoligotyping has a significant advantage over RFLP due to its ability to work with minimal DNA requirements, making it a potential tool for direct analysis of clinical specimens, potentially bypassing the need for time-consuming culturing processes [13].However, further investigation is needed to definitively confirm the usefulness of spoligotyping for direct analysis of clinical specimens.Previous studies have reported satisfactory results when using spoligotyping on various clinical specimens.For instance, a specific study reported a specificity of 98% and a sensitivity of 96% for spoligotyping [19].
The present study revealed that the East African-Indian (EAI) family (63.8%) was the most dominant spoligotype family, accounting for 63.8% of the isolates, followed by the Central Asian strain (CAS) family (21.2%), the Beijing family (8.5%), and family 33 (4.3%).Additionally, our study detected the presence of M. bovis-BCG, which accounted for 2.1% of the total strains analyzed.Our results align with previous studies, showing that M. tuberculosis lineages 1 (Indo-Oceanic or EAI) and 3 (Central Asian or CAS) are the most prevalent in India [20].In contrast, lineages 2 (East Asian or Beijing) and 4 (Euro-American) exhibit a wider global distribution, with a higher prevalence in Europe, Africa, and other parts of the world [20][21][22].Research has revealed that lineage 3 is prevalent in northern and northwestern India, while lineage 1 is prominent in the southern region, with lower frequencies observed in other areas [8,17,18].Unlike lineage 1, lineage 2 exhibits a more even distribution across India, with an overall prevalence of 17%, although it is more prevalent in certain northeastern states.This finding is consistent with the fact that most strains circulating in South India, particularly in Kerala, belong to lineage 1 [Indo-Oceanic] [23].
Consistent with previous studies [24,25], the East African Indian (EAI) lineage emerged as the predominant genotype in this region of India.Singh et al. (2015) reported the ancient EAI lineage as predominant in South India, with a prevalence of 44% in the Chennai and 38.3% in Hyderabad [26].This genotype, initially identified in Guinea Bissau, West Africa [27], likely migrated to the Asian mainland during the migration of modern humans out of Africa, which is supported by studies suggesting a African origin of the EAI lineage [28,29].Shanmugham et al. (2011) previously reported a high prevalence (60%) of the EAI family in the Tiruvallur district of South India, which is approximately 50 km from Chennai [17].Our study revealed that 62.4% of the isolates belonged to the EAI lineage, which is consistent with the findings of Thomas et al. (2011) who identified EAI as the second most prevalent lineage in Hyderabad, India [25].This finding suggests a geographical trend, where the prevalence of the EAI lineage decreases and the Central Asian strain (CAS) becomes more prominent as one moves northwards in India, indicating a possible spatial distribution pattern of M. tuberculosis lineages in the country.In contrast, studies in North India report lower EAI lineage prevalence, ranging from 3.8% in Delhi [12] to 10% overall [7], indicating a geographical variation in EAI prevalence.In our study, 7.7% (3 out of 17) of EAI strains were MDR-TB, which is higher than the 10.3% observed in non-EAI strains, indicating a disparity in MDR-TB prevalence between the two groups, contradicting previous findings [29].
The CAS family, originating primarily in the Middle East and Central Asia, exhibits a significant presence in South Asia (21.2%) and is especially prevalent in India (75%) [30,31].This lineage is also found in neighboring countries in this region, including Iran (19.1%) and Pakistan (56.5%) [32,33].The CAS lineage has been reported in various other regions, although at lower prevalence rates compared to South Asia, including Africa (5.3%), Central America (0.1%), Europe (3.3%),Far-East Asia (0.4%), and North America (3.3%) [34].The CAS lineage, considered a more recent lineage, predominates in India's western, central, and northern regions, including urban centers such as Mumbai and Delhi, and states such as Punjab and Uttar Pradesh (including Agra), consistent with prior findings [30,35].

Table 1 .
Description of the major M. tuberculosis lineages and spoligotyping profiles [n=47]

Table 2 .
Distribution of spoligotypes based on the location of TB infection (pulmonary or extrapulmonary)

Table 3 .
Drug susceptibility pattern of the predominant spoligotype families