Insights into transmission dynamics of Mycobacterium tuberculosis complex in Nepal

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis complex (MTBC) in humans and animals. Numbers of multi drug resistance TB (MDR-TB), extrapulmonary TB (EPTB) and zoonotic TB cases are increasingly being reported every year in Nepal posing a major public health problem. Therefore, the Government of Nepal should act immediately to strengthen the screening facilities across the country to be able to identify and treat the TB infected patients as well as detect zoonotic TB in animal species. Endorsement of One Health Act by the Government of Nepal is an opportunity to initiate the joint programs for TB surveillance among human and animal species using one health approach to reduce the TB burden in Nepal.

Warty cutaneous tuberculosis of the nose: A rare localization

Cutaneous tuberculosis is a rare, extra-pulmonary form of tuberculosis caused by mycobacteria of the tuberculosis complex. It is characterized by clinical polymorphism often posing a difficult diagnostic challenge. Herein, we report a case of cutaneous tuberculosis in its warty form located on the nose. This was a 57-year-old patient who was infected in the classroom three months previously while taking lessons from a woman with pulmonary tuberculosis. A facial examination revealed a blackish, papillomatous patch invading almost the entire nose, with a keratotic surface spreading over the wings of the nose. The diagnosis of verrucous tuberculosis was reached on the basis of epidemiological, clinical, and paraclinical arguments. Under anti-tuberculosis treatment for six months, the lesion had healed without sequelae. The diagnosis of verrucous cutaneous tuberculosis must be established in the presence of any chronic and crusty lesion. The management responds to the treatment protocol for all forms of tuberculosis.

Probing Differences in Gene Essentiality Between the Human and Animal Adapted Lineages of the Mycobacterium tuberculosis Complex Using TnSeq

Members of the Mycobacterium tuberculosis complex (MTBC) show distinct host adaptations, preferences and phenotypes despite being >99% identical at the nucleic acid level. Previous studies have explored gene expression changes between the members, however few studies have probed differences in gene essentiality. To better understand the functional impacts of the nucleic acid differences between Mycobacterium bovis and Mycobacterium tuberculosis, we used the Mycomar T7 phagemid delivery system to generate whole genome transposon libraries in laboratory strains of both species and compared the essentiality status of genes during growth under identical in vitro conditions. Libraries contained insertions in 54% of possible TA sites in M. bovis and 40% of those present in M. tuberculosis, achieving similar saturation levels to those previously reported for the MTBC. The distributions of essentiality across the functional categories were similar in both species. 527 genes were found to be essential in M. bovis whereas 477 genes were essential in M. tuberculosis and 370 essential genes were common in both species. CRISPRi was successfully utilised in both species to determine the impacts of silencing genes including wag31, a gene involved in peptidoglycan synthesis and Rv2182c/Mb2204c, a gene involved in glycerophospholipid metabolism. We observed species specific differences in the response to gene silencing, with the inhibition of expression of Mb2204c in M. bovis showing significantly less growth impact than silencing its orthologue (Rv2182c) in M. tuberculosis. Given that glycerophospholipid metabolism is a validated pathway for antimicrobials, our observations suggest that target vulnerability in the animal adapted lineages cannot be assumed to be the same as the human counterpart. This is of relevance for zoonotic tuberculosis as it implies that the development of antimicrobials targeting the human adapted lineage might not necessarily be effective against the animal adapted lineage. The generation of a transposon library and the first reported utilisation of CRISPRi in M. bovis will enable the use of these tools to further probe the genetic basis of survival under disease relevant conditions.

Re-decontamination of liquid mycobacterial cultures: Additional Mycobacterium tuberculosis yield in the era of Xpert MTB/RIF Ultra in Cape Town, South Africa

A retrospective review of liquid mycobacterial cultures was performed at a laboratory in South Africa from 01 January 2018 to 31 December 2018 to assess the increased yield in detecting Mycobacterium tuberculosis complex following sample re-decontamination. Only 9 of 99 (9%) re-decontaminated samples were culture positive for M. tuberculosis complex. Xpert MTB/RIF Ultra, concurrently performed on 7 of the 9 samples, detected M. tuberculosis complex in all but 1 sample. Re-decontamination of non-sterile samples did not increase the M. tuberculosis complex yield enough to offset the financial costs and additional labour in a laboratory that utilises the Xpert MTB/RIF Ultra system as a first-line diagnostic modality.

A case report of transmission and disease caused by Mycobacterium caprae and Mycobacterium bovis in Lima, Peru

Background The Tuberculosis (TB) burden in Peru is significant with respect to both disease morbidity and mortality. Furthermore the recent diversification of farming enterprise to include a wide range of animal species has necessitated the consideration of members of the Mycobacterium Tuberculosis Complex (MTBC) with the potential for zoonotic transmission. M. bovis and M. caprae, a lesser known member of the MTBC exhibit an exceptionally wide host spectrum in animals and are capable of causing disease in humans. M. bovis has a predictable resistance profile which includes resistance to pyrazinamide. Thus, failure to identify M. bovis as the causative agent in reported TB cases leads to higher levels of treatment failure and contributes to the transmission of drug-resistant TB. Case presentation Reported here are the clinical presentations, investigations and treatment histories of two patients identified from a population level genotyping study in Lima, Peru that were at the time of treatment thought to be M. tuberculosis patients but in retrospect were spectated using whole genome sequencing as M. caprae and M. Bovis. Conclusions The cases reported here constitute convincing evidence that M. caprae and M. bovis are causative agents of TB infection in humans in Peru and underscore the importance of species-level MTBC member identification to effectively control and treat zoonotic TB. Furthermore these cases highlight the challenges of using clinical risk factors to identify cases of zoonotic TB in humans as their clinical presentation and transmission history is often difficult to distinguish from anthroponotic TB.

MILK CONTAMINATION BY Mycobacterium tuberculosis complex, IMPLICATIONS FOR PUBLIC HEALTH IN AMAZONAS, BRAZIL

In Brazil, contamination of raw milk with Mycobacterium tuberculosis complex (MTC) has been reported in several states. In Amazonas, the rate of consumption of raw milk and its derivatives is the highest in the country. The state has the highest prevalence of tuberculosis in both humans and livestock. Therefore, we assessed the contamination of cattle and buffalo milk with MTC in the state of Amazonas, focusing on Mycobacterium bovis (M. bovis) , the species most commonly originating in cattle and buffalo. In 2019, 250 samples of raw milk (91 cattle; 159 buffalo) were collected from three milk plants, before the processing, in the state of Amazonas. The samples were placed into 21 pools then analyzed using Shotgun Metagenomic Sequencing and taxonomic classification by Kraken-2 and MegaBLAST. To confirm the identity of Mycobacterial species found, BLASTN was used to identify specific genomic positions at the TbD1 and RD1 regions and flanking RD4 region. MTC species genetic material were identified in all pools of raw milk. Genetic material consistent with M. bovis were identified in seven pools of raw milk (1 cattle and 6 buffalo). Milk from buffalo presented significantly higher MTC reads than milk from cattle. The common practice of consumption of raw milk and its derivatives in Amazonas presents an imminent risk to public health. Urgent measures to prevent transmission of foodborne tuberculosis are needed in the Amazon region. Greater efforts and resources should also be directed towards elimination of bovine tuberculosis disease in cattle and buffalo herds in the state of Amazonas and all of Brazil.

Construction of lux-based promoter-reporter platforms in Mycobacterium bovis BCG for screening of drug repurposing small-molecule compounds as new anti-tuberculosis drugs

The emergence of multidrug-resistant strains and hyper-virulent strains of Mycobacterium tuberculosis are big therapeutic challenges for tuberculosis (TB) control. Repurposing bioactive small-molecule compounds has recently become a new therapeutic approach against TB. This study aimed to construct a rapid screening system to identify novel anti-TB agents from a library of small-molecule compounds. In this study, a total of 320 small-molecule compounds were used to screen for their ability to suppress the expression of a key virulence gene, phoP, of M. tuberculosis complex using luminescence (lux)-based promoter-reporter platforms. The minimum inhibitory and bactericidal concentrations on drug-resistant M. tuberculosis and cytotoxicity to human macrophage were determined. RNA-sequencing (RNA-seq) was conducted to determine the drug mechanisms of the selected compounds as novel antibiotics or anti-virulent agents against the M. tuberculosis complex. Six compounds displayed bactericidal activity against M. bovis BCG, in which Ebselen demonstrated the lowest cytotoxicity to macrophage and was considered as a potential antibiotic for TB. Another ten compounds did not inhibit the in vitro growth of the M. tuberculosis complex but down-regulated the expression of phoP specifically. Of them, ST-193 and ST-193 (hydrochloride) showed low cytotoxicity and could dysregulate the entire phoP-associated gene network, and thus identified as potential anti-virulence agents for M. tuberculosis. This study provides a rapid screening platform coupled with a systematic validation and eventually suggested one potential antibiotic and two anti-virulence agents for M. tuberculosis infections.

Prevalence of bovine tuberculosis and characterization of the members of the Mycobacterium tuberculosis complex from slaughtered cattle in Rwanda

Background: Bovine tuberculosis (bTB) is an endemic disease in Rwanda, but little is known about its prevalence and causative mycobacterial species. The disease causes tremendous losses in livestock and wildlife and remains a significant threat to public health. Materials and methods: A cross-sectional study employing a systematic random sampling of cattle (n=300) with the collection of retropharyngeal lymph nodes and tonsils (n=300) irrespective of granulomatous lesions was carried out in six abattoirs to investigate the prevalence and identify mycobacterial species using culture, acid-fast bacteria staining, polymerase chain reaction, and GeneXpert assay. Individual risk factors and the origin of samples were analysed for association with the prevalence. Findings: Of the 300 samples, six were collected with visible TB-like lesions. Our findings demonstrated the presence of Mycobacterium tuberculosis complex (MTBC) in 1.7% (5/300) of sampled slaughtered cattle. M. bovis was isolated from 1.3% (4/300) animals while one case was caused by a rifampicin-resistance (RR) M. tuberculosis. Non-tuberculous mycobacteria were identified in 12.0% (36/300) of the sampled cattle. There were no significant associations between the prevalence and abattoir category, age, sex, and breeds of slaughtered cattle. Conclusions: This study is the first in Rwanda to isolate both M. bovis and RR M. tuberculosis in slaughtered cattle indicating that bTB is prevalent in Rwanda with a low prevalence. The isolation of RR M. tuberculosis from cattle indicates possible zooanthroponotic transmission of M. tuberculosis and close human-cattle contact. To protect humans against occupational zoonotic diseases, it is essential to control bTB in cattle and raise the awareness among all occupational groups as well as reinforce biosafety at the farm level and in the abattoirs.