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

The first patient was a four-year-old neutered male Savannah cat from Inverness, Scotland, who was presented to his general practice veterinarian with an intermittent cough and mild weight loss lasting three weeks. The cat was strictly indoor-only and had been fed a commercially available raw meat-based diet containing poultry and supplemental beef offal. Initial treatment with the anti-inflammatory drug meloxicam produced no response, and over the following two weeks the cough became more persistent while the cat grew increasingly lethargic with a reduced appetite.

Thoracic radiographs revealed a marked broncho-interstitial lung pattern, and a blind bronchoalveolar lavage performed under anaesthesia showed moderate neutrophilic inflammation but was negative on routine bacterial culture. An empirical course of the antibiotic marbofloxacin also failed to help. By the time the cat was referred to a specialist institution, he had lost 30 percent of his body weight. A computed tomography scan showed severe, diffuse nodular to confluent soft tissue lesions affecting all lung lobes, together with enlarged mediastinal lymph nodes. Fine-needle aspirates of consolidated lung tissue revealed severe pyogranulomatous inflammation, and Ziehl-Neelsen staining identified small numbers of acid-fast bacilli with the slender, curved morphology typical of mycobacteria. Culture subsequently confirmed Mycobacterium caprae.

Treatment began with a triple antibiotic protocol comprising pradofloxacin at 5 mg/kg, rifampicin at 10 mg/kg and azithromycin at 15 mg/kg, each given orally once daily in liquid formulations. An oesophagostomy tube was placed to help the owners administer the prolonged medication with minimal stress. The cat was hospitalised in isolation for eight days with strict infection control precautions, including personal protective equipment for staff. Public health officials were notified once the diagnosis was confirmed, but no further testing of in-contact people was deemed necessary. Clinically, the cat improved rapidly, gaining weight within a month and coughing far less frequently. Although repeat radiographs at three-monthly intervals showed only mild improvement, treatment was stopped after eleven months when lung changes had remained static for more than two months. The cat remains clinically normal a year after therapy ended.

The second patient was a three-year-old neutered female British Shorthair from Hampshire, England, who was fed the same raw meat-based diet product and presented with a one-month history of cough, weight loss and fever. Radiographs revealed a cavitating mass in the caudal left lung field, a large nodule in the left cranial lung and multifocal areas of dense consolidation throughout the remaining lung fields. Cytology of a bronchoalveolar lavage sample showed marked neutrophilic and macrophagic inflammation with epithelioid macrophages containing non-staining ghost bacilli, and Ziehl-Neelsen staining again revealed intracellular acid-fast organisms. Polymerase chain reaction testing at a mycobacterial reference laboratory detected DNA of the Mycobacterium tuberculosis complex, and sequencing confirmed M. caprae.

The same triple antibiotic regimen was prescribed, again using liquid formulations to support long-term daily compliance. The cat improved rapidly, gaining weight within a month, with coughing notably reduced by four months of therapy. Radiographs after five months showed significant improvement without complete normalisation, and after a further three months the findings were unchanged. Given the patient was clinically well and had completed nine months of treatment, therapy was discontinued. She remains clinically normal ten months after cessation.

The clinical significance of these cases extends beyond the rarity of the pathogen. In the United Kingdom, feline infections with the M. tuberculosis complex are usually caused by Mycobacterium bovis or Mycobacterium microti, and M. caprae has only recently been identified in domestic cats, previously documented in detail in a single post-mortem case. The treatment protocol used in both cats was adapted from human tuberculosis guidelines and consists of rifampicin, a fluoroquinolone and a macrolide continued for at least three months and for two months beyond resolution of clinical signs. Earlier retrospective work found that only 42 percent of treated cats achieved complete remission, often despite inappropriate therapy, whereas the updated multidrug approach has produced remission rates of approximately 80 percent.

Notably, neither cat achieved radiographic resolution despite extended therapy, yet both remained free of relapse. The authors draw an analogy with post-tuberculosis lung disease in humans, an increasingly recognised spectrum of structural and functional pulmonary abnormalities that persists after successful anti-mycobacterial therapy in 18 to 80 percent of treated patients. They suggest that stable radiographic changes could serve as a reasonable therapeutic endpoint in feline mycobacterial infections where initial lung damage is too severe to be expected to normalise.

The report also underscores the practical demands of treatment. Both cats required prolonged daily multidrug therapy with frequent follow-up visits, demanding substantial financial and emotional investment from their owners alongside excellent patient compliance. The authors caution that where such commitment is not feasible, euthanasia should be considered for severely affected animals, both for welfare reasons and to reduce the risk of antimicrobial resistance developing from inconsistent dosing. They further note that empirical treatment choices early in the diagnostic process, such as anti-inflammatory drugs, can complicate or worsen the clinical course and should be weighed carefully with owners.

Zoonotic considerations shaped the management of both cases. Transmission of M. bovis from cats to humans has been documented previously, although the risk is considered very low, and M. caprae transmission to humans has only been identified in proximity to infected goats, its reservoir host, never from cats. Because no in-contact humans had risk factors for zoonotic transmission, treatment of both cats was judged reasonable. The authors hope that raising awareness of this first reported successful treatment of M. caprae infection in UK domestic cats will aid patient management while mitigating zoonotic risks, and they highlight the oesophagostomy tube used in the first case as a practical aid that reduces stress and supports the owner-caregiver relationship throughout months of therapy.

Subject of Research: Successful treatment of Mycobacterium caprae tuberculosis infection in two domestic cats in the United Kingdom

Article Title: Successful Treatment of Mycobacterium caprae Infection in Two UK Cats

Article References: Clark, E., Gunn‐Moore, D., & O'Halloran, C. (2026). Successful Treatment of Mycobacterium caprae Infection in Two UK Cats. Veterinary Medicine and Science, 12(5), Article e71180. https://doi.org/10.1002/vms3.71180

Image Credits: AI Generated

DOI: 10.1002/vms3.71180

Keywords: Mycobacterium caprae, feline tuberculosis, zoonotic disease, veterinary medicine, triple antibiotic therapy, rifampicin, pradofloxacin, azithromycin, raw meat-based diet, post-tuberculosis lung disease, Mycobacterium tuberculosis complex, UK cats