Phylogenomic and genomic analysis reveals unique and shared genetic signatures of Mycobacterium kansasii complex species.

Species belonging to the Mycobacterium kansasii complex (MKC) are frequently isolated from humans and the environment and can cause serious diseases. The most common MKC infections are caused by the species M. kansasii (sensu stricto), leading to tuberculosis-like disease. However, a broad spectrum of virulence, antimicrobial resistance and pathogenicity of these non-tuberculous mycobacteria (NTM) are observed across the MKC. Many genomic aspects of the MKC that relate to these broad phenotypes are not well elucidated. Here, we performed genomic analyses from a collection of 665 MKC strains, isolated from environmental, animal and human sources. We inferred the MKC pangenome, mobilome, resistome, virulome and defence systems and show that the MKC species harbours unique and shared genomic signatures. High frequency of presence of prophages and different types of defence systems were observed. We found that the M. kansasii species splits into four lineages, of which three are lowly represented and mainly in Brazil, while one lineage is dominant and globally spread. Moreover, we show that four sub-lineages of this most distributed M. kansasii lineage emerged during the twentieth century. Further analysis of the M. kansasii genomes revealed almost 300 regions of difference contributing to genomic diversity, as well as fixed mutations that may explain the M. kansasii's increased virulence and drug resistance.

Hi-plex deep amplicon sequencing for identification, high-resolution genotyping and multidrug resistance prediction of Mycobacterium leprae directly from patient biopsies by using Deeplex Myc-Lep.

BACKGROUND: Expansion of antimicrobial resistance monitoring and epidemiological surveillance are key components of the WHO strategy towards zero leprosy. The inability to grow Mycobacterium leprae in vitro precludes routine phenotypic drug susceptibility testing, and only limited molecular tests are available. We evaluated a culture-free targeted deep sequencing assay, for mycobacterial identification, genotyping based on 18 canonical SNPs and 11 core variable-number tandem-repeat (VNTR) markers, and detection of rifampicin, dapsone and fluoroquinolone resistance-associated mutations in rpoB/ctpC/ctpI, folP1, gyrA/gyrB, respectively, and hypermutation-associated mutations in nth. METHODS: The limit of detection (LOD) was determined using DNA of M. leprae reference strains and from 246 skin biopsies and 74 slit skin smears of leprosy patients, with genome copies quantified by RLEP qPCR. Sequencing results were evaluated versus whole genome sequencing (WGS) data of 14 strains, and versus VNTR-fragment length analysis (FLA) results of 89 clinical specimens. FINDINGS: The LOD for sequencing success ranged between 80 and 3000 genome copies, depending on the sample type. The LOD for minority variants was 10%. All SNPs detected in targets by WGS were identified except in a clinical sample where WGS revealed two dapsone resistance-conferring mutations instead of one by Deeplex Myc-Lep, due to partial duplication of the sulfamide-binding domain in folP1. SNPs detected uniquely by Deeplex Myc-Lep were missed by WGS due to insufficient coverage. Concordance with VNTR-FLA results was 99.4% (926/932 alleles). INTERPRETATION: Deeplex Myc-Lep may help improve the diagnosis and surveillance of leprosy. Gene domain duplication is an original putative drug resistance-related genetic adaptation in M. leprae. FUNDING: EDCTP2 programme supported by the European Union (grant number RIA2017NIM-1847 -PEOPLE). EDCTP, R2Stop: Effect:Hope, The Mission To End Leprosy, the Flemish Fonds Wetenschappelijk Onderzoek.

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.

A Murine Model of Mycobacterium kansasii Infection Reproducing Necrotic Lung Pathology Reveals Considerable Heterogeneity in Virulence of Clinical Isolates.

Among non-tuberculous mycobacteria, Mycobacterium kansasii is one of the most pathogenic, able to cause pulmonary disease indistinguishable from tuberculosis in immunocompetent susceptible adults. The lack of animal models that reproduce human-like lung disease, associated with the necrotic lung pathology, impairs studies of M. kansasii virulence and pathogenicity. In this study, we examined the ability of the C57BL/6 mice, intratracheally infected with highly virulent M. kansasii strains, to produce a chronic infection and necrotic lung pathology. As a first approach, we evaluated ten M. kansasii strains isolated from Brazilian patients with pulmonary disease and the reference strain M. kansasii ATCC 12478 for virulence-associated features in macrophages infected in vitro; five of these strains differing in virulence were selected for in vivo analysis. Highly virulent isolates induced progressive lung disease in mice, forming large encapsulated caseous granulomas in later stages (120-150 days post-infection), while the low-virulent strain was cleared from the lungs by day 40. Two strains demonstrated increased virulence, causing premature death in the infected animals. These data demonstrate that C57BL/6 mice are an excellent candidate to investigate the virulence of M. kansasii isolates. We observed considerable heterogeneity in the virulence profile of these strains, in which the presence of highly virulent strains allowed us to establish a clinically relevant animal model. Comparing public genomic data between Brazilian isolates and isolates from other geographic regions worldwide demonstrated that at least some of the highly pathogenic strains isolated in Brazil display remarkable genomic similarities with the ATCC strain 12478 isolated in the United States 70 years ago (less than 100 SNPs of difference), as well as with some recent European clinical isolates. These data suggest that few pathogenic clones have been widely spread within M. kansasii population around the world.

Whole-Genome Sequences of Mycobacterium abscessus subsp. massiliense Isolates from Brazil.

The Mycobacterium abscessus complex comprises multidrug-resistant, opportunistic, and rapidly growing pathogens responsible for severe infections. Here, we report the genome composition of four Mycobacterium abscessus subsp. massiliense isolates from three sources: two from the lung of a cystic fibrosis patient, one from a mammary cyst, and one from a gutter system.

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.

Exploring the "Latin American Mediterranean" family and the RDRio lineage in Mycobacterium tuberculosis isolates from Paraguay, Argentina and Venezuela.

BACKGROUND: The Latin American & Mediterranean (LAM) spoligotype family is one of the most successful genotype of Mycobacterium tuberculosis worldwide and particularly prevalent in South-America. Within this family, a sublineage named Region of Difference Rio (RDRio) was reported initially in Brazil and is characterized by a genomic deletion of about 26.3 kb. This lineage seems to show a specific adaptation to the Euro-Latin American population. In this context, we sought to evaluate the LAM family and the presence of the RDRio genotype in samples from three Latin American countries including Paraguay, Venezuela and Argentina. To detect LAM strains reliably we applied a typing scheme using spoligotyping, 12 loci MIRU-VNTR, the Ag85C103 SNP and the regions of difference RDRio and RD174. IS6110-RFLP results were also used when available. RESULTS: Genotyping of 413 M. tuberculosis isolates from three Latin-American countries detected LAM (46%) and the ill-defined T clade (16%) as the most frequent families. The highest clustering rate was detected in the sample population from the city of Caracas in Venezuela. We observed considerable differences in the presence of the RDRio lineage, with high frequency in Caracas-Venezuela (55%) and low frequency in Buenos Aires-Argentina (11%) and Paraguay (10%). The molecular markers (RD174, Ag85C103, MIRU02-MIRU40 signature) of the RDRio lineage were essentially confirmed. For the LAM family, the most polymorphic loci were MIRU40, MIRU31, MIRU10, MIRU26, MIRU16 and the least polymorphic MIRU24, MIRU20, MIRU04, MIRU23. CONCLUSIONS: Our results suggest a differential adaptation of LAM-sublineages in neighboring populations and that RDRio strains spread regionally with different rates of distribution. The Ag85C SNP and RDs (RD174, RDRio) tested in this study can in fact facilitate molecular epidemiological studies of LAM strains in endemic settings and low-income countries.

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.

Genetic Clustering of Tuberculosis in an Indigenous Community of Brazil.

We conducted a population-based study of tuberculosis (TB) from 2009 to 2015 in an indigenous community of Brazil, the largest in the country, to investigate risk factors associated with recent TB transmission. The clinical isolates of Mycobacterium tuberculosis were genotyped by IS6110-RFLP (restriction fragment length polymorphism) and spoligotyping analysis. Among 67 isolates typed by RFLP, 69% fell into fifteen clusters, and 91% of TB cases with shared IS6110-RFLP pattern were diagnosed within 2 years of another case in the cluster. Individual risk factors associated with genetic clustering were domestic overcrowding (odds ratio [OR]: 6.10; 95% confidence interval [CI]: 1.50-24.88) and low social class (OR: 3.72; 95% CI: 1.00-13.98). Most reported contacts (76%) were identified within the household of the index TB case, but most of the genetic clustering of M. tuberculosis occurred outside of household (79%). Expanded contacts investigation and prophylaxis outside of household should be considered as a priority for TB control programs in this population.

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.

Genetic diversity of the Mycobacterium tuberculosis Beijing family in Brazil and Mozambique and relation with infectivity and induction of necrosis in THP-1 cells.

INTRODUCTION: The success of the Mycobacterium tuberculosis Beijing (MtbB) lineage in different geographical regions has been attributed to high transmission, increased virulence, drug resistance and rapid adaptation to the host. In some countries of secondary MtbB dispersion like South Africa and Peru, rising prevalence of the Beijing strains is registered. However, in neighboring countries to affected regions such as Mozambique and Brazil, respectively, the prevalence of these strains is still low and this could be due to biological particularities of the circulating MtbB strains and/or differentiated host susceptibility. OBJECTIVE: To characterize genetically and phenotypically MtbB strains isolated in Brazil (n = 8) and Mozambique (n = 17). METHODS: This is a descriptive study of genotypes of the MtbB isolates, determined by spoligotyping, MIRU-VNTR typing, analysis of the IS6110 copy number in the NTF region and screening for mutations in mutT2, mutT4, rpoB, katG and pks 15/1 genes. Virulence-associated properties of the studied isolates were verified in the in vitro model of infection of human THP-1 cells. RESULTS: The genotypes defined by the 24VNTRs were distinct for all isolates included in this study and presented an HGDI of 0.997. The VNTR patterns with seven copies of MIRU26 and seven copies of QUB26, representative for the previously described MtbB genotype B0, dominant in Russia, were detected in 38.5% of the studied isolates. In addition, all isolates presented RD105 deletion and a 7 bp insertion in pks15/1 gene. Almost all tested strains belonged to the RD181 sublineage, with the exception of two strains from Mozambique of RD150 sublineage. Combined analysis of the NTF region integrity and mutations in mutT genes showed that 62.5% and 47% of isolates obtained in Brazil and Mozambique, respectively, were of the ancestral genotype. The virulence index of the ancient isolates, evaluated in the THP-1 cells, was significantly lower than that of the modern genotype group. CONCLUSIONS: These data demonstrate genotype particularities of the Beijing strains isolated in Brazil and Mozambique, two countries of low prevalence of the MtbB lineage in local Mtb populations. In contrast to the neighboring countries with high prevalence of the MtbB strains of modern sublineage, significant proportions of the isolates obtained in Brazil and Mozambique were presented by the strains of the ancient sublineage. Our data suggest that lower virulence of the ancient strains, compared with the modern strains, could be involved in the slow spread of the MtbB strains in some regions.

Strain classification of Mycobacterium tuberculosis isolates in Brazil based on genotypes obtained by spoligotyping, mycobacterial interspersed repetitive unit typing and the presence of large sequence and single nucleotide polymorphism.

Rio de Janeiro is endemic for tuberculosis (TB) and presents the second largest prevalence of the disease in Brazil. Here, we present the bacterial population structure of 218 isolates of Mycobacterium tuberculosis, derived from 186 patients that were diagnosed between January 2008 and December 2009. Genotypes were generated by means of spoligotyping, 24 MIRU-VNTR typing and presence of fbpC103, RDRio and RD174. The results confirmed earlier data that predominant genotypes in Rio de Janeiro are those of the Euro American Lineages (99%). However, we observed differences between the classification by spoligotyping when comparing to that of 24 MIRU-VNTR typing, being respectively 43.6% vs. 62.4% of LAM, 34.9% vs. 9.6% of T and 18.3% vs. 21.5% of Haarlem. Among isolates classified as LAM by MIRU typing, 28.0% did not present the characteristic spoligotype profile with absence of spacers 21 to 24 and 32 to 36 and we designated these conveniently as "LAM-like", 79.3% of these presenting the LAM-specific SNP fbpC103. The frequency of RDRio and RD174 in the LAM strains, as defined both by spoligotyping and 24 MIRU-VNTR loci, were respectively 11% and 15.4%, demonstrating that RD174 is not always a marker for LAM/RDRio strains. We conclude that, although spoligotyping alone is a tool for classification of strains of the Euro-American lineage, when combined with MIRU-VNTRs, SNPs and RD typing, it leads to a much better understanding of the bacterial population structure and phylogenetic relationships among strains of M. tuberculosis in regions with high incidence of TB.

Insights into the population structure of Mycobacterium tuberculosis using spoligotyping and RDRio in a southeastern Brazilian prison unit.

Tuberculosis (TB) is still a serious public health problem, continuing to be an important threat for confined populations. We used spoligotyping to estimate the genotypic clades of Mycobacterium tuberculosis isolates from inmates in two blocks in a southeastern Brazilian prison unit, with TB incidence rate of 8185/100.000. The Latin American Mediterranean (LAM) clade is well represented in the country, and the LAM specific molecular markers, RD(Rio) large sequence polymorphism and the SNP on the Rv3062 [ligB(1212)], were used to characterize spoligotype signatures from prison isolates. Typing of RD(Rio) and ligB increase LAM clade from 66.7% (n=72/108) to 69.4% (n=75). The LAM2 SIT17 (n=23) and SIT179 (n=12) signatures comprised one third of all isolates, followed by Haarlem (11.5%, n=12), T (8.7%, n=9) and X (5.7%, n=6) clades. Strains with unknown signatures represented 5.5% (n=6), and four (3.7%) did not match any lineage. We observed RD(Rio) among 64 (59.2%) isolates, and 54 (50%) were of the LAM clade. In particular, the LAM2/RD(Rio) sub-lineage was significantly associated with clustering (p=0.02) and its frequency was higher (32%) when compared to that of the previous general TB cases in RJ (4.29%). Overall cluster frequency defined by spoligotyping/IS6110-RFLP was 62%. The two evolutionary markers helped to evaluate some LAM signature misconceptions and demonstrate that LAM2/RD(Rio) was found with high frequency, hitherto being unnoticed. All these data, allied to high clustering, imply that public health measures to minimize the escalation of TB in prison is essential, and both spoligotyping as well as RD(Rio) would be useful tools to monitor the effects of the measures with respect to M. tuberculosis lineage variation.