Tuberculosis (TB) is a highly infectious disease caused by Mycobacterium tuberculosis that infects the lungs at any stage of lifecycle, and it is a major cause of death globally. HIV (human immunodeficiency virus) and TB are co-partners in crime with multidrug resistance as the major cause for the unavailability of effective treatment regimen 1. TB infection is transmitted from person to person through air by inhalation of aerosols from coughs or sprays droplets of active diseased persons. According to the World Health Organization (WHO), the death rate caused by TB is higher as compared to that caused by other diseases triggered by a single infectious agent. Worldwide, in the year of 2017,  M. tuberculosis caused around 1.3 million deaths and 10 million new TB cases out of which 5.8 million was men, 3.2 million women and 1.0 million children. 2 Multidrug resistant TB (MDR-TB) results from resistance to one of the two most active first-line drugs rifampicin (RMP) and isoniazid (INH)3.

In the year of 2017, around 558,000 multidrug resistance cases were registered out of which in India (24%), China (13%) and Russian Federation (10%)2 (WHO report, 2017). This was caused by inappropriate treatment, lack of knowledge towards complete treatment; indigence and improper healthcare facilities 4. Peoples with low immune system are at the high risk. Mutations in the 81bp RRDR (rifampicin resistance determining region) of rpoB gene are the main reason for M. tuberculosis resistance to rifampicin. More than 95% mutations only occur in this 81 bp hotspot region (codons 507 to 533).56 The Proper treatments with optimal time and drug dosing is a key factor to prevent drug resistance along with maximum efficacy. Rifampicin interacts with β subunit of RNA polymerase (RNAP) to arrest DNA directed RNA synthesis in M. tuberculosis. Spacer oligonucleotide typing also called as spoligotyping is a PCR based method of genotyping which is commonly used in detection of M.tuberculosis complex. This method amplifies the whole DR region (direct repeats) to check the DNA polymorphism in the spacer sequences. As the DR region is highly conserved in M. tuberculosis, it is used to differentiate different strains 7. In the case of disease outbreaks or emergency, Spoligotyping is very rapid and simple as compared to other genotyping methods 89. Mutations in the rpoB gene can cause bacterial resistance to rifampicin which can use as a surrogate marker for the detection of multidrug resistance 10.

In this study, we used PCR and sequencing techniques to identify point mutations within the 81 bp hotspot RRDR in clinical samples collected from different health care centers in Hyderabad. These samples were also subjected to spoligotyping to identify the drug resistant strains.

All the rifampicin-resistant strains had missense mutations. Sequence analysis detected a single or multiple mutations in the RRDR region of the rpoB gene at codons 516, 512 and 531. However, few MDR-TB isolates with HIV co-infection did mot harbour mutations in the RRDR hotspot region. We have observed that apart from these hotspot mutations other mutations at codons 512, 514, 517 and 526 were observed, indicating that high frequency of mutations occurs in HIV co-infected individuals. DNA extracted from 9 samples was checked on the agarose gel stained with ethidium bromide for confirmation of positive bands. All samples showed positive bands. The extracted DNA samples were further used for rpoB amplification. After amplification all products were checked on 1.5% agarose gel for 411 bp positive amplification (Figure 1).

Sequencing of the PCR purified samples was performed by capillary electrophoresis. The results were screened for mutations in the rpoB gene with reference to M. tuberculosis H37Rv strain sequence. In drug resistance pattern, there is an amino acid substitution or amino acid change at the codon position 531 in 81 bp hotspot region of RRDR in the rpoB gene. It is known that the most common mutation in the rpoB gene is at codon 531 with serine to leucine substitution (TCG to TTG) 14. All samples were aligned with M. tuberculosis  H37Rv reference sequence for mutation screening in rpoB gene out of which only one sample (Sample-9) shows the mutation at codon 531 (Figure 2). In Figure 3, the highlighted nucleotide of the Sample-9 represents the T nucleotide while that in the remaining samples represents the C nucleotide.  Figure 4 shows the nucleotide sequence chromatogram. Figure 4a shows no change in the nucleotide at codon position 531 (TCG) while Figure 4b shows the nucleotide change at the codon position 531 (TCG to TTG i.e. Serine to Leucine). We have also screened for all possible codons for associated mutations (Figure 2) but we didn’t find any mutations except the Sample-9 shows serine to leucine mutation (TCG to TTG), thus indicates MDR resistance.

The same DNA samples used for sequencing were analyzed by spoligotyping. All the extracted DNA samples were used for amplification of DR region. After amplification all products were checked on 1.5% agarose gel (Figure 5). Spoligotyping of 9 samples shows distinct spoligopatterns (Figure 6). Out of the 9 samples, 4 samples (44.5%) belongs to M. tuberculosis EAI3 family, 2 samples (22.2%) belongs to M. tuberculosis CAS and the others samples belongs to M. tuberculosis EAI5 (11.1%), M. tuberculosis Beijing (11.1%), and M. bovis-BCG (11.1%). The families and lineage were identified and assigned using TB-VIS online tool. In this study, 5 samples were from Indo-Oceanic lineages (55.5%), 1 sample from M. bovis (11.1%), 1 sample from East Asian (Beijing) (11.1%) and 2 samples from unknown lineages (22.2%). The details of spoligotyping defined lineage and families of study samples were shown in Table 1 and Table 2.

The samples used in this study were also determined for Phylogenetic analysis using Maximum Likelihood method 13 and distance matrix constructed using MEGA7software tool. In Figure 7, the numbers at the nodes are the bootstrap values based on percentage. The numbers of base substitutions per site between sequences are shown in Figure 8. The analyses were conducted using the maximum composite likelihood model for study samples.

In this study, we used DNA sequencing for the screening of MDR-TB from the collected clinical samples from different healthcare centres in Hyderabad and Spoligotyping for lineage and families’ identification. Majority of the MDR-TB isolates had mutations in the rpoB gene, and 88% of these mutations were located in three codons (531, 526 and 516) at the 81 bp hotspot region which contributes to drug resistance 15.

The rpoB gene is considered as a reliable marker accounting for more than 95% drug resistant strains of tuberculosis but other genes like katG (in more than 70 drug resistant strains) are also a suitable biomarker for MDR resistance. Screening for other genes is required for further confirmations.

As per increasing incidence of TB patients in developing countries and the emergence of MDR-TB, DNA sequencing is a very simple, cost-effective, and rapid method for the diagnosis before clinical treatment for patients. Sequencing along with Spoligotyping improves the identification of M. tuberculosis (MTB) isolates. The conventional methods like sputum microscopy and chest X-ray for detection and diagnosis for antibiotic sensitivity are time consuming as compared to molecular methods 5.

The results from this study suggest that mutations in the rpoB gene hotspot may not be the only factors associated with rifampicin resistance in TB co-infected with HIV. Mutations at other sites may also contribute to drug resistance. Therefore, GenexPert focusing on targeted hotspot regions may miss out mutations at other sites. Further studies about these novel mutations in addition to those in the hotspot region in the TB-HIV co-infected population would add more diagnostic markers for the reliable prediction of drug resistance. This will provide insight into developing novel diagnostic tools for the detection of M. tuberculosis co-infected with HIV in a high TB-HIV endemic area like India.