Investigating drug resistance of Mycobacterium leprae in the Comoros: an observational deep-sequencing study.

BACKGROUND: Despite strong leprosy control measures, including effective treatment, leprosy persists in the Comoros. As of May, 2022, no resistance to anti-leprosy drugs had been reported, but there are no nationally representative data. Post-exposure prophylaxis (PEP) with rifampicin is offered to contacts of patients with leprosy. We aimed to conduct a countrywide drug resistance survey and investigate whether PEP led to the emergence of drug resistance in patients with leprosy. METHODS: In this observational, deep-sequencing analysis we assessed Mycobacterium leprae genomes from skin biopsies of patients in Anjouan and Mohéli, Comoros, collected as part of the ComLep (NCT03526718) and PEOPLE (NCT03662022) studies. Skin biopsies that had sufficient M leprae DNA (>2000 bacilli in 2 µl of DNA extract) were assessed for the presence of seven drug resistance-associated genes (ie, rpoB, ctpC, ctpI, folP1, gyrA, gyrB, and nth) using Deeplex Myc-Lep (targeted next generation deep sequencing), with a limit of detection of 10% for minority M leprae bacterial populations bearing a polymorphism in these genes. All newly registered patients with leprosy for whom written informed consent was obtained were eligible for inclusion in the survey. Patients younger than 2 years or with a single lesion on the face did not have biopsies taken. The primary outcome of our study was the proportion of patients with leprosy (ie, new cases, patients with relapses or reinfections, patients who received single (double) dose rifampicin-PEP, or patients who lived in villages where PEP was distributed) who were infected with M leprae with a drug-resistant mutation for rifampicin, fluoroquinolone, or dapsone in the Comoros. FINDINGS: Between July 1, 2017, and Dec 31, 2020, 1199 patients with leprosy were identified on the basis of clinical criteria, of whom 1030 provided a skin biopsy. Of these 1030 patients, 755 (73·3%) tested positive for the M leprae-specific repetitive element-quantitative PCR (qPCR) assay. Of these 755 patients, 260 (34·4%) were eligible to be analysed using Deeplex Myc-Lep. 251 (96·5%) were newly diagnosed with leprosy, whereas nine (3·4%) patients had previously received multidrug therapy. 45 (17·3%) patients resided in villages where PEP had been administered in 2015 or 2019, two (4·4%) of whom received PEP. All seven drug resistance-associated targets were successfully sequenced in 216 samples, 39 samples had incomplete results, and five had no results. No mutations were detected in any of the seven drug resistance-related genes for any patient with successfully sequenced results. INTERPRETATION: This drug resistance survey provides evidence to show that M leprae is fully susceptible to rifampicin, fluoroquinolones, and dapsone in the Comoros. Our results also show, for the first time, the applicability of targeted sequencing directly on skin biopsies from patients with either paucibacillary or multibacillary leprosy. These data suggest that PEP had not selected rifampicin-resistant strains, although further support for this finding should be confirmed with a larger sample size. FUNDING: Effect:Hope, The Mission To End Leprosy, the Fonds Wetenschappelijk Onderzoek, the EU.

Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis.

Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific Mycobacterium leprae targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of the M. leprae genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and Mycobacteria, showing no cross-reactivity. Intra- and inter-operator Cp variation was evaluated using dilution curves of M. leprae DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects M. leprae DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment.

Characterization of Mycobacterium tuberculosis var. africanum isolated from a patient with pulmonary tuberculosis in Brazil.

Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC), including Mycobacterium tuberculosis var. tuberculosis (MTB) and Mycobacterium tuberculosis var. africanum (MAF). While MTB is isolated worldwide, MAF is almost completely restricted to the African continent, and despite the historical proximity between Brazil and Africa during the slave trade, no case of TB being caused by MAF has been reported in Brazil to date. We hereby describe the first case of TB caused by MAF in Brazil comparing its genome against the published ones. A female patient who had never visited Africa presented with clinical symptoms typical of pulmonary TB. Based on 16S rRNA gene sequencing, the cultured isolate was identified as belonging to MTBC and partial sequence of the hsp65 gene was identical to that of MAF. This was confirmed by genotyping based on detection of Single Nucleotide Polymorphism (SNP), Region of Difference (RD) and spoligotyping. The isolate presented the Shared International Typing (SIT) 181. In the whole-genome comparison against MAF genomes available on published EMBL-EBI European Nucleotide Archive (ENA), the Brazilian genome (MAFBRA00707) was identified as belonging to Lineage 6 and clustered with isolates from The Gambia. This is the first report of the isolation of MAF from a patient from Brazil, without evidence of having any contact with an African index case.

Antiretroviral therapy-induced paradoxical worsening of previously healed Mycobacterium haemophilum cutaneous lesions in advanced HIV infection.

Mycobacterium haemophilum is a nontuberculous mycobacterium that causes localized or disseminated disease, mainly in immunocompromised hosts. We report the case of a 35-year-old HIV-infected woman who presented with several enlarging cutaneous lesions over the arms and legs. Histopathological examination revealed the diagnosis of a cutaneous mycobacterial disease. Mycobacterial analyses unveiled M. haemophilum infection. Six months after completion of a successful antimycobacterial treatment, she developed an immune reconstitution inflammatory syndrome (IRIS). This paradoxical relapse presented as tenderness, redness and swelling at the precise sites of the healed lesions and took place in the setting of significant recovery of the CD4 cell count (from 05 to 318 cells/mm 3 ). Microbiological analyses of these worsening lesions were negative, and they spontaneously remitted without the initiation of a novel antimycobacterial treatment cycle. M. haemophilum infection should always be considered as a cause of skin lesions in immunocompromised subjects. Physicians should be aware of the possibility of IRIS as a complication of successful antiretroviral therapy in HIV-infected patients with M. haemophilum infection.

Frequency of the Mycobacterium tuberculosis RDRio genotype and its association with multidrug-resistant tuberculosis.

BACKGROUND: In recent decades, Mycobacterium tuberculosis with the RDRio genotype, frequently isolated from tuberculosis patients in Rio de Janeiro, has become part of the Latin American - Mediterranean (LAM) family and has been associated with multidrug-resistant tuberculosis (MDR-TB). The aim of this study was to investigate the frequency of M. tuberculosis RDRio in the state of Minas Gerais, Brazil, and its relationship with MDR-TB. METHODS: For convenience, 172 susceptible and 63 MDR M. tuberculosis isolates were taken from pulmonary samples from patients diagnosed between January 2007 and December 2011. The DNA extracted from these isolates was analyzed by spoligotyping, PCR-RFLP to characterize fbpC103/Ag85C103, multiplex PCR to detect RDRio and RD174, and MIRU-VNTR 24 loci. RESULTS: Among the 235 isolates, the RDRio pattern was identified in 122 (51.9%) isolates (IC 0.45-0.58), with 100 (42.5%) wild-type and 13 (5.5%) mixed pattern isolates, whereas RD174 was identified in 93 of the 122 RDRio positive samples (76.3%). The LAM family and the LAM9 lineage were the most frequently identified among the isolates in this study. Among the 63 MDR isolates, 41 (65.1%) were RDRio and 28 (44.4%) RD174. CONCLUSION: The association of both deletions with MDR proved to be statistically significant, corroborating the few reports that have associated RDRio with MDR.

Whole genome sequence of Mycobacterium kansasii isolates of the genotype 1 from Brazilian patients with pulmonary disease demonstrates considerable heterogeneity.

Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil.

Genetic diversity of Mycobacterium tuberculosis isoniazid monoresistant and multidrug-resistant in Rio Grande do Sul, a tuberculosis high-burden state in Brazil.

Tuberculosis (TB) remains a major public health problem in the world and Brazil is among the countries with the highest incidence and prevalence rates, and Rio Grande do Sul, a Brazilian state, occupy a prominent position. Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) further aggravates this scenario, making it more difficult to treat and control the disease. Isoniazid monoresistance (IMR) may increase the risk of progression to MDR-TB and treatment failure. However, most drug resistance molecular tests only focus on detecting rifampicin (RIF) resistance.In the present study, we characterized a total of 63 drug resistant isolates of M. tuberculosis (35 MDR, 26 IMR and two isolates monoresistant to rifampicin [RMR]) of the Rio Grande do Sul state by MIRU-VNTR (24 loci), spoligotyping, presence of RDRio, fbpC103, pks15/1 and sequencing of the katG, rpoB and inhA genes. We observed a higher proportion of the LAM family 30/63 (47.61%). In IMR, mutations were found in the katG gene (98% at codon 315) in 72.5%, and mutations in the promoter region of the inhA gene in 6.25% of the isolates. In MDR-TB and RMR-TB isolates, 92.1% had mutations in the rpoB gene (57% at codon 531). The presence of a 12 bp insertion between codons 516 and 517 of the rpoB gene in MDR-TB isolates was found in five isolates. In conclusion, we observed that the highest frequency of IMR-TB and MDR-TB strains belong to the LAM and Haarlem genotypes in Rio Grande do Sul state. A significant number of isolates previously characterized as Mycobacterium pinnipedi2 through spoligotyping were found to belong to the M. tuberculosis LAM family. This was responsible for a number of significant cases and the molecular profile of this strain and the pattern of mutations related to drug resistance were analyzed. These findings may contribute to a better understanding about the spread of M. tuberculosis resistant in southern of Brazil.

Whole genome sequence of Mycobacterium kansasii isolates of the genotype 1 from Brazilian patients with pulmonary disease demonstrates considerable heterogeneity

Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil.

Genetic diversity of Mycobacterium tuberculosis from Pará, Brazil, reveals a higher frequency of ancestral strains than previously reported in South America.

There is only scarce information available on genotypic diversity of the Mycobacterium tuberculosis complex (MTBC) clinical isolates circulating in the Northern part of Brazil, a relatively neglected region regarding research on tuberculosis. We therefore characterized 980 MTBC clinical isolates from the state of Pará, by spoligotyping and data was compared with patterns from around the world, besides analyzing drug susceptibility, and collecting sociodemographic data. We also performed 24 loci MIRU-VNTR typing to evaluate phylogenetic inferences among the East-African-Indian (EAI) lineage strains. The Geographic Information System analyses were performed to generate a descriptive visualization of MTBC strain distribution in the region. A total of 249 different spoligopatterns primarily belonging to evolutionary recent Euro-American lineages, as well as Central-Asian, Manu and ancestral EAI lineages, were identified, in addition to strains with reportedly unknown lineage signatures. The most frequent lineages were Latin American Mediterranean, T and Haarlem. Interestingly, EAI lineage strains were found in a significantly higher proportion in comparison with previous studies from South America. Regarding EAI lineage, the absence of spacers 4-9 and 23-24 co-related to 24 loci MIRU-VNTRs may suggest a close evolutionary relationship between such strains in Pará and those prevalent in Mozambique, which might have contributed to the genetic diversity of MTBC strains in this region.

Molecular Typing of Mycobacterium bovis from Cattle Reared in Midwest Brazil.

Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), the pathogen responsible for serious economic impact on the livestock sector. In order to obtain data on isolated M. bovis strains and assist in the control and eradication program for BTB, a cross sectional descriptive molecular epidemiology study in the Brazilian Midwest was conducted. Through spoligotyping and 24-loci MIRU-VNTR methods, 37 clinical isolates of M. bovis circulating in the region were analyzed, 10 isolated from the state of Mato Grosso, 12 from the state of Mato Grosso do Sul and 15 from the state of Goiás. The spoligotyping analysis identified 10 distinct M. bovis profiles (SB0121 n = 14, SB0295 n = 6, SB0140 n = 6, SB0881 n = 3, SB1144 n = 2, SB1145 n = 2, SB0134 n = 1, SB1050 n = 1, SB1055 n = 1, SB1136 n = 1) grouped in six clusters and four orphan patterns. The MIRU-VNTR 24-loci grouped the same isolates in six clusters and 22 unique orphan patterns, showing higher discriminatory power than spoligotyping. When associating the results of both techniques, the isolates were grouped in five clusters and 24 unique M. bovis profiles. Among the 24-loci MIRU-VNTR evaluated, two, ETR-A and QUB 11b loci, showed high discriminatory ability (h = ≥ 0.50), while MIRU 16, MIRU 27, ETR-B, ETR-C, Mtub21 and QUB 26 loci showed moderate ability (h = 0.33 or h = 0.49) and were the most effective in evaluating the genotypic similarities among the clinical M. bovis isolate samples. Herein, the 29 patterns found amongst the 37 isolates of M. bovis circulating in the Brazilian Midwest can be due to the animal movement between regions, municipalities and farms, thus causing the spread of various M. bovis strains in herds from Midwest Brazil.

Genome Comparison of an Ancestral Isolate and a Modern Isolate of Mycobacterium tuberculosis of the Beijing Lineage from São Paulo, Brazil.

Mycobacterium tuberculosis of the Bejing subtype (MtbB) is transmitted efficiently in high burden countries for this genotype. A higher virulence was associated with isolates of the "modern" Beijing genotype sub-lineages when compared to "ancient" ones. Here, we report the full genomes of the strain representing these two genotypes from Brazil, a country with a low incidence of MtbB.

Sequence and structural characterization of tbnat gene in isoniazid-resistant Mycobacterium tuberculosis: identification of new mutations.

The present study was carried out to investigate the presence of polymorphism in the N-acetyltransferase gene of 41 clinical isolates of Mycobacterium tuberculosis, that were resistant to isoniazid (INH) with no mutations in the hot spots of the genes previously described to be involved in INH resistance (katG, inhA and ahpC). We observed single nucleotide polymorphisms (SNPs) in ten of these, including the G619A SNP in five isolates and an additional four so far un-described mutations in another five isolates. Among the latter SNPs, two were synonymous (C276T, n=1 and C375G, n=3), while two more non-synonymous SNPs were composed of C373A (Leu→Met) and T503G (Met→Arg) were observed in respectively one and two isolates. Molecular modeling and structural analysis based in a constructed full length 3D models of wild type TBNAT (TBNAT_H37Rv) and the isoforms (TBNAT_L125M and TBNAT_M168R) were also performed. The refined models show that, just as observed in human NATs, the carboxyl terminus extends deep within the folded enzyme, into close proximity to the buried catalytic triad. Analysis of tbnat that present non-synonymous mutations indicates that both substitutions are plausible to affect enzyme specificity or acetyl-CoA binding capacity. The results contribute to a better understanding of structure-function relationships of NATs. However, further investigation including INH-sensitive strains as a control group is needed to get better understanding of the possible role of these new mutations on tuberculosis control.

Estudo de associação entre o(s) polimorfismo(s) de base única no gene que codifica para a N-acetiltransferase de M. tuberculosis e a resistência a Isoniazida / Study of association between o(s) polymorphism(s) of only base in the gene that codifies for the N-acetyltransferase de M. tuberculosis and the resistance the Isoniazida

A isoniazida é uma das principais drogas usadas no tratamento e quimioprofilaxia da tuberculose. Os mecanismos moleculares de resistência à mesma, são complexos e freqüentemente estão associados à presença de mutações nos genes katG, inhA, ahpC e kasA; porém, 25-50% dos isolados clínicos resistentes à isoniazida (INH) não apresentam mutações nestes genes indicando o envolvimento de outros genes e ou fatores. Recentemente, foi identificado em Mycobacterium tuberculosis um gene denominado nat, o qual apresenta um polimorfismo de base única (SNP) na posição 619 (619 G>A) e cujo produto, a N-acetiltransferase (NA T), aparentemente está envolvido na acetilação da isoniazida in vitm. Dados da literatura sugerem a associação da variante mutante 619A com resistência à isoniazida; assim, o objetivo inicial do nosso trabalho foi validar esta associacão, em isolados de M. tuberculosis de pacientes do Brasil, através de genotipagem por PCR-RFLP e seqüenciamento.

Adicionalmente, a utilização do seqüenciamento como estratégia de genotipagem permitiu a identificação de tres novos SNPs no gene nat dos isolados estudados, sendo duas não sinônimas (C20T e A233G), identificadas em isolados resistentes a INH e uma sinônima (T312C) em um isolado sensível a INH. Para a genotipagem do SNP 619, 380 isolados clínicos de pacientes tratados para tuberculose pulmonar de diferentes regiões do Brasil foram utilizados, sendo 198 resistentes e 183 sensíveis a INH. Entre eles, 20 (5,2%) apresentaram a variante mutante 619A, porém, a freqüência desta mutação foi significativamente maior nos isolados resistentes que nos sensíveis (8,6% e 16% respectivamente; p = 0,002, OR = 5,73, IC 1.55 - 25,02). A análise isolada das amostras resistentes estratificadas de acordo com o tipo de resistência (multidroga resistentes e monoresistente a isoniazida) não mostrou diferença significativa com relação à freqüência da variante 619A (p= 0,06). Sessenta e seis por cento dos isolados que apresentaram a variante mutante 619A apresentaram também mutações em outros genes associados com a resistência a INH.

A análise adicional dos isolados de M. tuberculosis por Spoligotyping, demonstrou que a variante mutante 619A não é característica de uma família específica, pois a mesma foi detectada em seis diferentes famílias (família S, LAM, Haarlem, X, T e LAM3/S convergente), além de dois isolados com perfis não definidos. O seqüenciamento do gene nat em todas as micobactérias pertencentes ao Complexo M. tuberculosis mostrou que este gene é conservado entre as cepas do complexo. Adicionalmente, uma outra mutação anteriormente não descrita, G751A, foi observada exclusivamente nos isolados de M. africanum subtipo la provenientes de uma coleção de isolados clínicos e cepas de referência. Com objetivo de melhor classificar isolados provenientes de pacientes com T8 de Gana, submetemos os mesmos a caracterização genética utilizando um conjunto de marcadores recém descritos ou descritos pela primeira vez neste trabalho, inclusive nat751 e RD711390, o ultimo supostamente especifico para M. africanum subtipo I b. Numa análise combinada de isolados de referência e isolados clínicos, confirmamos tanto a especificidade destes novos marcadores quanto a eficiência deste na classificação atual do Complexo Mycobacterium tuberculosis.