Genetic Typing of Tuberculosis Isolates in Kerala Reveals Rare BCG Disease Cases

Tuberculosis remains one of the oldest and most stubborn infectious diseases known to medicine, yet the bacterium behind it is not a single entity. What clinicians call the tuberculosis bacillus is in fact a group of closely related organisms, the Mycobacterium tuberculosis complex (MTBC), which includes M. tuberculosis, M. bovis, M. africanum, M. orygis and the attenuated vaccine strain M. bovis BCG. Telling these species apart is far more than an academic exercise. It shapes therapy, because M. bovis is intrinsically resistant to pyrazinamide, a cornerstone first-line drug, and it shapes public health, because some members of the complex jump between animals and humans. A new study from a tertiary care centre in Kerala, India, published in BMC Infectious Diseases, now provides the first published species-level map of MTBC isolates from this southern Indian state, and its findings carry lessons for laboratories and clinicians well beyond the region.

The research team, led by Parasmal Suresh and Aiswarya R. Nath of the Amrita School of Nanosciences and Molecular Medicine and Lalitha Biswas, with microbiologists at the Amrita Institute of Medical Sciences and Research Centre in Kochi, undertook a retrospective analysis of 239 archived MTBC clinical isolates collected between 2017 and 2024. Kerala had a conspicuous gap in the literature: despite its strong health infrastructure and active tuberculosis control programme, no published data existed on which MTBC species circulate in the state. The study was designed to close that gap using molecular tools that can resolve differences invisible to routine phenotypic testing, and it was supported by funding from the Kerala Biotechnology Commission under the Kerala State Council for Science, Technology and Environment.

The central technique of the study was polymerase chain reaction followed by restriction fragment length polymorphism analysis, or PCR-RFLP, targeting the gyrB gene. The logic is elegant. The gyrB gene, which encodes the B subunit of DNA gyrase, accumulates small sequence differences among MTBC members while remaining conserved enough within each species to serve as a reliable marker. When the amplified gyrB fragment is cut with restriction enzymes, each species produces a characteristic pattern of fragment sizes on a gel, a molecular fingerprint that allows speciation without the need for expensive whole genome sequencing. This approach has become a workhorse in reference laboratories because it is rapid, inexpensive and applicable directly to cultured isolates grown in liquid media such as the Mycobacterial Growth Indicator Tube.

To complement the gyrB analysis, the researchers deployed PCR assays targeting regions of difference, genomic segments that have been deleted from particular lineages during the evolution of the complex. Two of these segments were central to the work. Region of difference 1, or RD1, is absent from M. bovis BCG but present in wild-type M. bovis and M. tuberculosis, and it encodes key virulence factors including the ESAT-6 and CFP-10 proteins. Region of difference 4, or RD4, is likewise deleted in BCG but retained in wild-type M. bovis. By combining RD1 and RD4 PCR results, the team could distinguish the vaccine strain from its wild, disease-causing relatives, a distinction with direct clinical consequences because BCG infections are treated differently and carry different epidemiological implications.

The headline result was unambiguous. Of the 239 isolates, 232, or 97.1 percent, were identified as M. tuberculosis, the classic human-adapted pathogen. The remaining seven isolates, 2.9 percent, were M. bovis BCG. Notably, no wild-type M. bovis and no M. orygis were detected anywhere in the cohort. This absence of zoonotic species is itself informative. In many parts of the world, particularly where cattle husbandry and unpasteurized dairy consumption are common, M. bovis accounts for a meaningful share of human tuberculosis, and M. orygis, first described in antelopes but increasingly recognized in human patients across South Asia, has emerged as a zoonotic concern. Kerala’s data suggest that, at least among patients presenting to this tertiary centre, human-to-human transmission of M. tuberculosis overwhelmingly dominates the local tuberculosis landscape.

The seven BCG isolates, however, tell a more surprising and clinically urgent story. Four were recovered from children who had received BCG vaccination, including three infants and one four-year-old child. The remaining three came from adult patients who had received intravesical BCG therapy, a standard immunotherapy in which the live attenuated vaccine strain is instilled directly into the bladder to treat non-muscle-invasive bladder carcinoma. In both settings, the very organism intended to protect or to heal had instead caused disease. Among the paediatric cases, some infections were disseminated, spreading beyond the site of vaccination, a rare but well-documented complication in infants with undiagnosed immunodeficiencies and one that demands prompt recognition.

Why does this matter for treatment? The answer lies in drug susceptibility. M. bovis BCG, like wild-type M. bovis, is naturally resistant to pyrazinamide, the enzyme target of which is defective in these lineages. A patient with BCG disease who is treated with a standard four-drug regimen that includes pyrazinamide is effectively receiving an inactive drug, prolonging therapy and risking failure. When a laboratory identifies an isolate only as an MTBC member, or relies on commercial tests that do not distinguish species, this intrinsic resistance can be mistaken for acquired drug resistance, triggering unnecessary regimen changes and misclassification of the patient. Species-level identification therefore directly guides clinicians toward appropriate, pyrazinamide-sparing combinations for BCG and M. bovis infections.

The study also looked forward. In a parallel in silico analysis, the team examined published gyrB sequences to identify a single nucleotide polymorphism that could enable specific detection of M. orygis, the zoonotic species that has been increasingly reported from human cases in the region and that standard commercial identification probes often miss. Although no M. orygis was found in this cohort, developing a targeted molecular assay based on such a SNP would allow laboratories across South Asia to screen their collections cheaply and determine whether this emerging pathogen is circulating undetected. The authors frame this as groundwork for future surveillance, an acknowledgment that absence of evidence in one cohort does not establish absence of the organism in the wider population.

Methodologically, the study illustrates the value of archiving clinical isolates. Because the team could revisit seven years of stored cultures, they were able to apply molecular techniques retrospectively and generate a dataset that no single prospective study could have assembled quickly. The work was approved by the Institutional Ethics Committee of Amrita Institute of Medical Sciences, and because only archived, de-identified isolates and clinical data were used, the requirement for individual informed consent was waived. The isolates had been cultured from a range of specimen types, including respiratory samples such as bronchoalveolar lavage fluid, cerebrospinal fluid and other extrapulmonary sources, reflecting the broad spectrum of tuberculosis presentations seen at a tertiary referral centre.

The broader message of the Kerala study resonates with a growing international consensus that tuberculosis diagnostics must move beyond a one-size-fits-all identification of the complex. Molecular epidemiology has repeatedly shown that the members of MTBC differ in host preference, virulence, drug susceptibility and public health significance, and that these differences are best captured by genotyping methods ranging from PCR-RFLP and region-of-difference PCR to whole genome sequencing. For a state like Kerala, with a large cattle-owning rural population and high BCG vaccine coverage at birth, the finding that human M. tuberculosis accounts for nearly all disease is reassuring, while the detection of BCG disease in vaccinated infants and bladder cancer patients is a reminder that even the attenuated vaccine strain can become a pathogen. Species-level molecular diagnosis, the authors conclude, is not a luxury but a necessity, one that guides correct treatment, recognizes vaccine-associated and iatrogenic disease, and builds the surveillance foundation needed to detect zoonotic tuberculosis should it emerge in the region.

Subject of Research: Molecular species identification of Mycobacterium tuberculosis complex clinical isolates in Kerala, India

Article Title: Molecular characterization of Mycobacterium tuberculosis complex isolates at a tertiary care centre in Kerala, India

Article References: Suresh, P., Nath, A. R., Kumar, A., Cletus, H., Venugopal, A., Biswas, R., Varghese, L., Bhaskaran, P. N., & Biswas, L. (2026). Molecular characterization of Mycobacterium tuberculosis complex isolates at a tertiary care centre in Kerala, India. BMC Infectious Diseases. https://doi.org/10.1186/s12879-026-14527-5

Image Credits: AI Generated

DOI: 10.1186/s12879-026-14527-5

Keywords: Mycobacterium tuberculosis complex, M. bovis BCG, tuberculosis, PCR-RFLP, gyrB, zoonotic tuberculosis, Kerala, India, pyrazinamide resistance, region of difference, molecular diagnostics, tertiary care