Single-tube protocol for culture-independent spoligotyping of Mycobacterium tuberculosis based on MeltArray.

IMPORTANCE: Spacer oligonucleotide typing (spoligotyping), the first-line genotyping assay for Mycobacterium tuberculosis (MTB), plays a fundamental role in the investigation of its epidemiology and evolution. In this study, we established a single-tube spoligotyping assay using MeltArray, a highly multiplex polymerase chain reaction (PCR) approach that runs on a real-time PCR thermocycler. The MeltArray protocol included an internal positive control, gyrB, to indicate the abundance of MTB via the quantification cycle and 43 spacers to identify the spoligotype via melting curve analysis. The entire protocol was completed in a single step within 2.5 hours. The lowest detectable copy number for the tested strains was 20 copies/reaction and thus sufficient for analyzing both culture and sputum samples. We conclude that MeltArray-based spoligotyping could be used immediately in low- and middle-income countries with a high tuberculosis burden, given its easy access, improved throughput, and potential applicability to clinical samples.

Mycobacterium tuberculosis Beijing in the State of Nuevo Leon, Mexico.

Tuberculosis remains a serious threat to human health as an infectious disease in Mexico. Data about the genotypes of circulating Mycobacterium tuberculosis isolates (MTB) in the State of Nuevo Leon, Mexico are scarce. We aimed to determine the genotypes of circulating MTB belonging to the Beijing lineage recovered from patients in the State of Nuevo Leon, Mexico. A total of 406 MTB isolates from this state were genotyped using the spoligotyping method and 18-locus MIRU-VNTR. Lineage classification and MTB transmission analysis were performed. Based on the spoligotyping analysis, we found 24 strains belonging to the Beijing genotype that were characterized phylogenetically. The MIRUs showed greater discriminatory power than the standard RFLP-IS6110 method; therefore, the greatest allelic diversity among the Beijing strains was observed with MIRU10, MIRU31, MIRU39, MRU40, and MIRU 26. MVLA analysis showed a profile variation between Beijing and non-Beijing strains. The minimum spanning tree (MST) showed that 79% (19) of the strains are related. All Beijing strains exhibited the deletion of region TbD1, which is a characteristic of modern strains. The application of spoligotyping and MIRU-VNTR-18 methods together proved to be more sensitive, discriminatory, and rapid than the standard method for the epidemiological analysis of Mycobacterium Beijing isolates. This study is one of the first to describe the genomic diversity of M. Beijing in the State of Nuevo Leon, Mexico.

A first insight into tuberculosis transmission at the border of Ecuador and Colombia: a retrospective study of the population structure of Mycobacterium tuberculosis in Esmeraldas province.

Objective: Tuberculosis (TB) is a major public health concern in Ecuador and Colombia, considering that both countries are high-burden TB settings. Molecular epidemiology is crucial to understand the transmission dynamics of Mycobacterium tuberculosis complex (MTBC) and to identify active transmission clusters of regional importance. Methods: We studied the potential transmission of TB between Colombia and Ecuador through the analysis of the population structure of MTBC lineages circulating in the Ecuadorian province of Esmeraldas at the border with Colombia. A total of 105 MTBC strains were characterized by 24-loci MIRU-VNTR and spoligotyping. Results: MTBC lineage 4 is only present in Esmeraldas; no MTBC strains belonging to Lineage 2-sublineage Beijing were found despite its presence in other provinces of Ecuador and, in Colombia. Genotyping results revealed a high degree of diversity for MTBC in Esmeraldas: Neither active transmission clusters within this province nor including MTBC strains from Colombia or other provinces of Ecuador were found. Conclusion: Our data suggest that tuberculosis dynamics in this rural and isolated area may be not related to highly transmitted strains but could be influenced by other health determinants that favor TB relapse such as poverty and poor health system access. Further studies including a larger number of MTBC strains from Esmeraldas are necessary to test this hypothesis.

Prevalence, drug resistance, and genotypic diversity of the RDRio subfamily of Mycobacterium tuberculosis in Ecuador: a retrospective analysis for years 2012-2016.

Introduction: A major sublineage within the Mycobacterium tuberculosis (MTB) LAM family characterized by a new in-frame fusion gene Rv3346c/55c was discovered in Rio de Janeiro (Brazil) in 2007, called RDRio, associated to drug resistance. The few studies about prevalence of MTB RDRio strains in Latin America reported values ranging from 3% in Chile to 69.8% in Venezuela, although no information is available for countries like Ecuador. Methods: A total of 814 MTB isolates from years 2012 to 2016 were screened by multiplex PCR for RDRio identification, followed by 24-loci MIRU-VNTR and spoligotyping. Results: A total number of 17 MTB RDRio strains were identified, representing an overall prevalence of 2.09% among MTB strains in Ecuador. While 10.9% of the MTB isolates included in the study were multidrug resistance (MDR), 29.4% (5/17) of the RDRio strains were MDR. Discussion: This is the first report of the prevalence of MTB RDRio in Ecuador, where a strong association with MDR was found, but also a very low prevalence compared to other countries in Latin America. It is important to improve molecular epidemiology tools as a part of MTB surveillance programs in Latin America to track the transmission of potentially dangerous MTB stains associated to MDR TB like MTB RDRio.

Strain identification of Mycobacterium tuberculosis in multidrug-resistant tuberculosis (MDR-TB) patients at Undata Hospital, Palu, Central Sulawesi.

MDR-TB is a tuberculosis disease resistant to the two most effective anti-TB drugs, rifampin and isoniazid. MDR-TB is a threat to TB control. This study aims to identify the Mycobacterium tuberculosis strain in MDR-TB patients at Undata Hospital in Palu, Central Sulawesi. This type of research is descriptive and observational with a cross-sectional design. The study was conducted in Palu City, Donggala Regency, and Sigi Regency from April-June 2021. The sample in this study consisted of 22 patients who had undergone MDR-TB treatment from 2019 to 2020. The results showed that 55% of the patients with MDR-TB lived in Palu City, 27% in Donggala Regency, and 18% in Sigi Regency. Out of the 22 patients, 13 were men (59%) and nine were women (41%). Based on the results of the examination of Mycobacterium tuberculosis culture, three samples from patients with MDR-TB were positive for M. tuberculosis. The results of the spoligotyping examination showed that the strain belonged to the Beijing family. Identifying the type of M. tuberculosis strain through spoligotyping examination should be carried out in TB patients who have not undergone MDR-TB treatment or those who have failed treatment and are still found to be positive for M. tuberculosis bacteria.

Identification and molecular characterization of Mycobacterium bovis DNA in GeneXpert® MTB/RIF ultra-positive, culture-negative sputum from a rural community in South Africa.

This study investigated the presence of Mycobacterium bovis (M. bovis) DNA in archived human sputum samples previously collected from residents who reside adjacent to the M. bovis-endemic Hluhluwe-iMfolozi wildlife park, South Africa (SA). Sixty-eight sputum samples were GeneXpert MTB/RIF Ultra-positive for M. tuberculosis complex (MTBC) DNA but culture negative for M. tuberculosis. Amplification and Sanger sequencing of hsp65 and rpoB genes from DNA extracted from stored heat-inactivated sputum samples confirmed the presence of detectable amounts of MTBC from 20 out of the 68 sputum samples. Region of difference PCR, spoligotyping and gyrB long-read amplicon deep sequencing identified M. bovis (n = 10) and M. tuberculosis (n = 7). Notably, M. bovis spoligotypes SB0130 and SB1474 were identified in 4 samples, with SB0130 previously identified in local cattle and wildlife and SB1474 exclusively in African buffaloes in the adjacent park. M. bovis DNA in sputum, from people living near the park, underscores zoonotic transmission potential in SA. Identification of spoligotypes specifically associated with wildlife only and spoligotypes found in livestock as well as wildlife, highlights the complexity of TB epidemiology at wildlife-livestock-human interfaces. These findings support the need for integrated surveillance and control strategies to curb potential spillover and for the consideration of human M. bovis infection in SA patients with positive Ultra results.

Population structure of Mycobacterium tuberculosis in El Oro: A first insight into Ecuador-Peru tuberculosis transmission.

BACKGROUND: Tuberculosis (TB) is a major public health concern in Ecuador and Peru, both settings of high burden of drug resistance TB. Molecular epidemiology tools are important to understand the transmission dynamics of Mycobacterium tuberculosis Complex (MTBC) and to track active transmission clusters of regional importance. This study is the first to address the transmission of TB between Peru and Ecuador through the population structure of MTBC lineages circulating in the Ecuadorian border province of "El Oro". METHODS: A total number of 56 MTBC strains from this province for years 2012-2015 were included in the study and analyzed by 24-loci MIRU-VNTR and spoligotyping. RESULTS: Genotyping revealed a high degree of diversity for MTBC in "El Oro", without active transmission clusters. MTBC L4 was predominant, with less than 2% of strains belonging to MTBC L2-Beijing. CONCLUSIONS: These results may suggest that TB dynamics in this rural and semi-urban area would not be linked to highly transmitted strains like MTBC L2-Beijing from Peru, but related to TB relapse; although further studies with larger MTBC cultures collection from recent years are needed. Nevertheless, we recommend to reinforce TB surveillance programs in remote rural settings and border regions in Ecuador.

Molecular epidemiology and drug resistance of Mycobacterium tuberculosis in a tertiary care hospital in northeastern Mexico.

INTRODUCTION: Tuberculosis (TB) is a re-emerging disease considered a public health concern. In the present study, we analyzed the epidemiology and drug resistance of Mycobacterium tuberculosis strains isolated from patients with pulmonary TB. METHODOLOGY: Mycobacterium tuberculosis isolates (n = 190) were obtained from patients with pulmonary TB admitted to Dr. José Eleuterio González University Hospital (UH). Each M. tuberculosis isolate was analyzed by spoligotyping (spacer oligonucleotide typing) and MIRU-VNTR (Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeat). Drug resistance was evaluated using the Anyplex™ II MTB/MDR/XDR assay. RESULTS: The predominant spoligotypes observed were X1 (SIT 119, n = 46), T1 (SIT 53, n = 40), H3 (SIT 50, n = 13), Beijing (SIT 1, n = 11), and EAI2-Manila (SIT 19, n = 8). MIRU-VNTR analysis showed that the locus QUB-26 had the highest allelic variability. The observed drug resistance included monoresistance to rifampicin (2.6%; n = 5), isoniazid (3.2%; n = 6), and fluoroquinolones (1.6%; n = 3) as well as multidrug resistance (5.3%; n = 10). All of the Beijing strains were susceptible. Regarding comorbidities, 13.7% (26/190) of the patients were co-infected with TB and HIV (TB+HIV+), and 31.6% (55/190) had TB along with diabetes (TB + diabetes). CONCLUSIONS: The most prevalent lineages were X1 (SIT 119; 24.3%) and T1 (SIT 53; 21%). An alarming proportion (12.6%) of M. tuberculosis isolates presented drug resistance. To effectively manage TB, continuous surveillance of regional strain dissemination, drug resistance profiles, and TB-associated comorbidities is crucial.

Genetic diversity of Mycobacterium tuberculosis isolates from the central, eastern and southeastern Ethiopia.

Introduction: The population structure of Mycobacterium tuberculosis complex (MTBC) in Ethiopia is diverse but dominated by Euro-American (Lineage 4) and East-African-Indian (Lineage 3) lineages. The objective of this study was to describe the genetic diversity of MTBC isolates in Central, Eastern and Southeastern Ethiopia. Methods: A total of 223 MTBC culture isolates obtained from patients referred to Adama and Harar TB reference laboratories were spoligotyped. Demographic and clinical characteristics were collected. Results: Six major lineages: Euro-American (Lineage 4), East-African-Indian (Lineage 3), East Asian (Lineage 2), Indo-Oceanic (Lineage 1), Mycobacterium africanum (Lineage 5 and Lineage 6) and Ethiopian (Lineage 7) were identified. The majority (94.6 %) of the isolates were Euro-American and East-African-Indian, with proportions of 75.3 % and 19.3 %, respectively. Overall, 77 different spoligotype patterns were identified of which 42 were registered in the SITVIT2 database. Of these, 27 spoligotypes were unique, while 15 were clustered with 2-49 isolates. SIT149/T3_ETH (n = 49), SIT53/T1 (n = 33), SIT21/CAS1_Kili (n = 24) and SIT41/Turkey (n = 11) were the dominant spoligotypes. A rare Beijing spoligotype pattern, SIT541, has also been identified in Eastern Ethiopia. The overall clustering rate of sub-lineages with known SIT was 71.3 %. Age group (25-34) was significantly associated with clustering. Conclusion: We found a heterogeneous population structure of MTBC dominated by T and CAS families, and the Euro-American lineage. The identification of the Beijing strain, particularly the rare SIT541 spoligotype in Eastern Ethiopia, warrants a heightened surveillance plan, as little is known about this genotype. A large-scale investigation utilizing a tool with superior discriminatory power, such as whole genome sequencing, is necessary to gain a thorough understanding of the genetic diversity of MTBC in the nation, which would help direct the overall control efforts.

Whole genome sequencing improves the discrimination between Mycobacterium bovis strains on the southern border of Kruger National Park, South Africa.

Background: Mycobacterium bovis forms part of the Mycobacterium tuberculosis complex and has an extensive host range and zoonotic potential. Various genotyping methods (e.g., spoligotyping) have been used to describe the molecular epidemiology of M. bovis. Advances in whole genome sequencing (WGS) have increased resolution to enable detection of genomic variants to the level of single nucleotide polymorphisms. This is especially relevant to One Health research on tuberculosis which benefits by being able to use WGS to identify epidemiologically linked cases, especially recent transmission. The use of WGS in molecular epidemiology has been extensively used in humans and cattle but is limited in wildlife. This approach appears to overcome the limitations of conventional genotyping methods due to lack of genetic diversity in M. bovis. Methods: This pilot study investigated the spoligotype and WGS of M. bovis isolates (n = 7) from wildlife in Marloth Park (MP) and compared these with WGS data from other South African M. bovis isolates. In addition, the greater resolution of WGS was used to explore the phylogenetic relatedness of M. bovis isolates in neighbouring wildlife populations. Results: The phylogenetic analyses showed the closest relatives to the seven isolates from MP were isolates from wildlife in Kruger National Park (KNP), which shares a border with MP. However, WGS data indicated that the KNP and MP isolates formed two distinct clades, even though they had similar spoligotypes and identical in silico genetic regions of difference profiles. Conclusions: Mycobacterium bovis isolates from MP were hypothesized to be directly linked to KNP wildlife, based on spoligotyping. However, WGS indicated more complex epidemiology. The presence of two distinct clades which were genetically distinct (SNP distance of 19-47) and suggested multiple transmission events. Therefore, WGS provided new insight into the molecular epidemiology of the M. bovis isolates from MP and their relationship to isolates from KNP. This approach will facilitate greater understanding of M. bovis transmission at wildlife-livestock-human interfaces and advances One Health research on tuberculosis, especially across different host species.

Spoligotyping, genotyping, and spatial distribution of Mycobacterium bovis in cattle in the state of Bahia, Brazil / Genotipagem por spoligotyping e distribuição espacial de Mycobacterium bovis em bovinos no estado da Bahia, Brasil

Mycobacterium bovis is responsible for bovine and buffalo tuberculosis, an important zoonotic disease with global distribution. The knowledge of the distribution and the precise identification of this disease, including advanced diagnoses such as spoligotyping, allows choosing the best strategies to fight the disease's progress. The present work aimed to investigate mycobacteria's presence, genotype their strains, and evaluate tuberculosis cases' spatial distribution from suggestive lesions in carcasses of bovine and buffalo inspected in slaughterhouses under an official inspection regime in the state of Bahia, Brazil. The study investigated 453,417 animals. Among these, 31 (0.007%) from 17 municipalities were suspected of tuberculosis. Among the culture medium growth, 95% of these were categorized as alcohol-acid resistant bacilli (BAAR). All isolates were subjected to spoligotyping and 95% were confirmed as M. bovis (SB0120, SB0121, SB0852, SB0828, SB0295, SB0881, SB1648, SB6119, SB0140, SB1055). The strain SB0120 was the most prevalent, and this profile has been described in cases of human tuberculosis by M. bovis, highlighting the zoonotic potential of this profile. This study also identified strains never reported in Bahia, highlighting a distinctive pattern from other parts of Brazil, besides mixed infections. Besides, to identify strains never before described in the state, highlighting a distinctive pattern in Brazil (SB6119 and SB0852, respectively). An unpublished profile was identified and inserted in the international database (Mbovis.org), named SB2715.(AU)

O Mycobacterium bovis é o responsável pela tuberculose bovina e bubalina, doença zoonótica importante e com distribuição global. O conhecimento da distribuição e a identificação precisa dessa enfermidade, incluindo diagnósticos mais avançados como o spoligotyping, permite escolher as melhores estratégias de combate ao avanço da doença. O presente trabalho objetivou investigar a presença de micobactérias, genotipar suas estirpes e avaliar a distribuição espacial dos casos de tuberculose a partir de lesões sugestivas nas carcaças de bovinos e bubalinos inspecionadas em frigoríficos sob regime de inspeção oficial no estado da Bahia. Foram investigados 453.417 animais dentre os quais 31 (0,007%) foram suspeitos de doença e provenientes de 17 municípios. Após o crescimento em meio de cultura, 95% foram categorizados como bacilos álcool-ácido resistentes (BAAR). Todos os isolados foram submetidos à spoligotyping e 95% foram confirmados M. bovis (SB0120, SB0121, SB0852, SB0828, SB0295, SB0881, SB1648, SB6119, SB0140, SB1055). A cepa SB0120 foi a mais prevalente e este perfil vem sendo descrito na literatura com casos de tuberculose humana por M. bovis ressaltando o potencial zoonótico deste perfil. Este estudo também identificou cepas nunca relatadas no estado da Bahia, destacando um padrão distinto de outras partes do Brasil, além da existência de infecções mistas. Permitiu ainda relatar linhagens nunca antes descritas no estado com destaque para um padrão novo no Brasil (SB6119 e SB0852 respectivamente). Um perfil inédito identificado foi identificado e inserido no banco de dados internacional (Mbovis.org), nomeado SB2715.(AU)

Spoligotyping, genotyping, and spatial distribution of Mycobacterium bovis in cattle in the state of Bahia, Brazil

ABSTRACT: Mycobacterium bovis is responsible for bovine and buffalo tuberculosis, an important zoonotic disease with global distribution. The knowledge of the distribution and the precise identification of this disease, including advanced diagnoses such as spoligotyping, allows choosing the best strategies to fight the diseases progress. The present work aimed to investigate mycobacterias presence, genotype their strains, and evaluate tuberculosis cases spatial distribution from suggestive lesions in carcasses of bovine and buffalo inspected in slaughterhouses under an official inspection regime in the state of Bahia, Brazil. The study investigated 453,417 animals. Among these, 31 (0.007%) from 17 municipalities were suspected of tuberculosis. Among the culture medium growth, 95% of these were categorized as alcohol-acid resistant bacilli (BAAR). All isolates were subjected to spoligotyping and 95% were confirmed as M. bovis (SB0120, SB0121, SB0852, SB0828, SB0295, SB0881, SB1648, SB6119, SB0140, SB1055). The strain SB0120 was the most prevalent, and this profile has been described in cases of human tuberculosis by M. bovis, highlighting the zoonotic potential of this profile. This study also identified strains never reported in Bahia, highlighting a distinctive pattern from other parts of Brazil, besides mixed infections. Besides, to identify strains never before described in the state, highlighting a distinctive pattern in Brazil (SB6119 and SB0852, respectively). An unpublished profile was identified and inserted in the international database (Mbovis.org), named SB2715.

RESUMO: O Mycobacterium bovis é o responsável pela tuberculose bovina e bubalina, doença zoonótica importante e com distribuição global. O conhecimento da distribuição e a identificação precisa dessa enfermidade, incluindo diagnósticos mais avançados como o spoligotyping, permite escolher as melhores estratégias de combate ao avanço da doença. O presente trabalho objetivou investigar a presença de micobactérias, genotipar suas estirpes e avaliar a distribuição espacial dos casos de tuberculose a partir de lesões sugestivas nas carcaças de bovinos e bubalinos inspecionadas em frigoríficos sob regime de inspeção oficial no estado da Bahia. Foram investigados 453.417 animais dentre os quais 31 (0,007%) foram suspeitos de doença e provenientes de 17 municípios. Após o crescimento em meio de cultura, 95% foram categorizados como bacilos álcool-ácido resistentes (BAAR). Todos os isolados foram submetidos à spoligotyping e 95% foram confirmados M. bovis (SB0120, SB0121, SB0852, SB0828, SB0295, SB0881, SB1648, SB6119, SB0140, SB1055). A cepa SB0120 foi a mais prevalente e este perfil vem sendo descrito na literatura com casos de tuberculose humana por M. bovis ressaltando o potencial zoonótico deste perfil. Este estudo também identificou cepas nunca relatadas no estado da Bahia, destacando um padrão distinto de outras partes do Brasil, além da existência de infecções mistas. Permitiu ainda relatar linhagens nunca antes descritas no estado com destaque para um padrão novo no Brasil (SB6119 e SB0852 respectivamente). Um perfil inédito identificado foi identificado e inserido no banco de dados internacional (Mbovis.org), nomeado SB2715.

Evaluación de spoligotyping a partir de baciloscopías como metodología alterna e independiente de cultivo para la genotipificación de Mycobacterium tuberculosis / Evaluation of spoligotyping-smear sputum as an alternative and culture-independent methodology for Mycobacterium tuberculosis genotyping

INTRODUCCIÓN: Tuberculosis (TBC) sigue siendo la segunda causa de muerte por una enfermedad infecto-contagiosa después del síndrome de inmunodeficiencia humana adquirida (SIDA). Actualmente, el escenario de técnicas y metodologías de laboratorio para la identificación y drogo-sensibilidad está cambiando gradualmente. Se han recomendado e introducido ensayos rápidos basados en la amplificación de ácidos nucleicos (NAAT's) que se desarrollan mediante la reacción de polimerasa en cadena (RPC). Bajo este principio, se destaca spoligotyping -una herramienta de genotipificación y epidemiología molecular en TBC- estandarizada a partir de aislados bacterianos, que permite el estudio del genoma de Mycobacterium mediante la amplificación de 43 secuencias cortas no repetitivas, localizadas en la región de repetición directa (RD1). OBJETIVO: Evaluación de spoligotyping a partir de baciloscopías, como una opción independiente de cultivo, para la caracterización de Mycobacterium tuberculosis a partir de muestras de esputo en pacientes del Instituto Nacional Cardiopulmonar de Tegucigalpa, Honduras. MÉTODO: De 37 pacientes con cultivo (y baciloscopía) positivos para M. tuberculosis, se obtuvieron 50 muestras de expectoración. Se realizó estudio microbiológico y molecular en muestras respiratorias conteniendo ADN de micobacterias, a partir de baciloscopías, concentrados y cultivo, para la identificación y análisis genotípico a través de la técnica de spoligotyping. RESULTADOS: El spoligotyping fue positivo en 37/37 de muestras de cultivo positivo (S: 100%), en 36/37 (S: 97,3%) de muestras con baciloscopía positiva y en 6/10 (S: 60%) de muestras de concentrado de esputo. La intensidad de la baciloscopía positiva tuvo una relación directa con la sensibilidad de spoligotyping. DISCUSIÓN: El fusionar el potencial de una herramienta útil en epidemiología molecular para analizar muestras de ADN proveniente de baciloscopías, visualiza una plataforma diagnóstica y genotípica para países en vías de desarrollo como una alternativa innovadora y altamente sensible en la hibridación de oligonucleótidos específicos a partir material genético en baciloscopías (P+, P++, P+++), pero requiere mejorar la concordancia entre patrones genéticos obtenidos, comparables con el uso estandarizado de aislados de cepas de M. tuberculosis.

BACKGROUND: Tuberculosis (TB) remains as the second cause of death by an infectious disease preceded by the acquired immune deficiency syndrome (AIDS). Currently, laboratory techniques and methodologies of diagnosis and drug susceptibility testing are constantly changing. Therefore, it has been recommended the introduction of rapid assays based on the amplification of nucleic acids test (NAAT's) through a polymerase chain reaction (PCR). Based on this principle, outstands spoligotyping - as a genotype and molecular epidemiology tool in tuberculosis - it is standardized to use isolated bacteria for the study of Mycobacterium genome through the amplification of 43 non-repetitive sequences, located at the direct repetitive region 1 (RD1). AIM Evaluation of spoligotyping from acid fast staining smears as an independent option from bacterial isolation to characterize Mycobacterium tuberculosis by using sputum samples from TB patients from National Cardiopulmonary Institute in Tegucigalpa, Honduras. METHOD: Of 37 patients with positive culture (and smear microscopy) for M. tuberculosis, 50 expectoration samples were obtained. Microbiological and molecular tests were performed in respiratory samples containing mycobacterial DNA from sputum smears, concentrates and solid culture, for identification and genotype analysis by spoligotyping technique. RESULTS: Spoligotyping was positive in 37/37 of positive culture samples (S: 100%), in 36/37 (S: 97.3%) of smear-positive samples and in 6/10 (S: 60%) of concentrate samples sputum. The intensity of positive smear microscopy had a direct relationship with the sensitivity of spoligotyping. DISCUSSION: This study combined the potential of a molecular epidemiology tool to analyse DNA from sputum samples in smears acid fast staining, it visualizes diagnosis and genotyping platform in developing countries gathering innovation and high sensitivity in the hibridization of specific olignonucleotides from positive smears (P+, P++, P+++). However, the low specificity showed the need to improve better agreement among genetic patterns compared to the standardized bacterial isolation from M. tuberculosis strains.

Isolation and genotyping of Mycobacterium bovis in suggestive lesions of tuberculosis in cattle slaughtered in the state of Ceará, Brazil / Isolamento e tipificação genética de Mycobacterium bovis em lesões sugestivas de tuberculose em bovinos abatidos no estado do Ceará

Bovine tuberculosis (BTB) is a zoonosis caused by the bacterium Mycobacterium bovis, which induces the development of nodular and granulomatous lesions in various animal tissues. The recognition of these suggestive gross lesions during postmortem sanitary inspection in slaughterhouses provides a presumptive diagnosis, which requires the use of complementary tests to confirm the disease. This study aimed to verify the occurrence of BTB in cattle slaughtered in slaughterhouses in the state of Ceará, Brazil, using bacteriological and molecular methods. To this end, suggestive lesions were analyzed on carcasses condemned by the "Serviço de Inspeção Estadual" (SIE). The samples were submitted to microbiological analysis using culture media and specific staining followed by spoligotyping molecular technique for identification and genotyping of the mycobacteria. Occurrence of lesions suggestive of BTB was verified in bovine carcasses (0.071%) from different municipalities of the state. These lesions were located mainly in the lung (95.12%), lymph nodes (58.53%), and liver (36.58%). Microbiological culture showed bacterial isolation (17.94%), with the growth of colonies showing morphological and tannic characteristics belonging to genus Mycobacterium spp. Genetic polymorphism analysis identified M. bovis in all isolates, which were discriminated into six spoligotypes (SB0121, SB0295, SB1064, SB0120, SB0870, and SB0852). These profiles have been described in Brazil and several areas of the world, except for profiles SB1064 and SB0852, which were described in the country for the first time. The results show that the association of the diagnostic methods used was the basis for the first study on identification of mycobacteria found in the state, which may provide a database for the epidemiological study of BTB in the state of Ceará.(AU)

A tuberculose bovina (TB) é uma zoonose causada pelo Mycobacterium bovis, o qual induz ao desenvolvimento de lesões nodulares e granulomatosas em vários tecidos do animal. O reconhecimento dessas lesões macroscópicas sugestivas durante a inspeção sanitária post mortem em matadouros fornece um diagnóstico presuntivo, sendo necessário a utilização de testes complementares para confirmação da doença. O objetivo deste trabalho foi verificar a ocorrência da TB em animais abatidos em matadouros-frigoríficos no estado do Ceará através da utilização de métodos bacteriológicos e moleculares. Para tanto, foram analisadas lesões sugestivas de TB em carcaças condenadas pelo Serviço de Inspeção Estadual (SIE). As amostras foram submetidas à análise microbiológica, utilizando meios de cultivo e de coloração específicos, seguida pela técnica molecular spoligotyping para identificação e tipificação genética da micobactéria. Verificou-se a ocorrência de lesões sugestivas de TB em carcaças bovinas (0,071%) oriundas de diferentes municípios do estado do Ceará. Essas lesões estavam localizadas principalmente no pulmão (95,12%), linfonodos (58,53%) e fígado (36,58%). O cultivo microbiológico obteve isolamento bacteriano (17,94%), com o crescimento de colônias apresentando características morfológicas e tintoriais pertencentes ao gênero Mycobacterium spp. A análise do polimorfismo genético identificou a presença de M. bovis em todos os isolados, que foram discriminados em seis espoligotipos (SB0121, SB0295, SB1064, SB0120, SB0870 e SB0852), descritos no Brasil e em diversas áreas do mundo, exceto os perfis SB1064 e SB0852 que foram descritos pela primeira vez no país. Os resultados obtidos demonstram que a associação dos métodos diagnósticos utilizados foram a base do primeiro estudo de identificação das micobactérias encontradas no estado do Ceará, o que pode contribuir para a criação de um banco de dados para o estudo epidemiológico da TB no estado.(AU)

Mycobacterium tuberculosis spoligotypes and drug-resistant characterization from Beira compared to genotypes circulating in Mozambique

Abstract INTRODUCTION Mozambique is one of three countries with high prevalence of tuberculosis (TB), TB/human immunodeficiency virus coinfection, and multidrug-resistant TB. We aimed to describe Mycobacterium tuberculosis spoligotypes circulating among drug resistant (DR) strains from Beira, Mozambique comparing them with genotypes in the country. METHODS: We performed spoligotyping of 79 M. tuberculosis suspected of DR-TB compared all spoligotype patterns published on the international database and PubMed. RESULTS: Both in Beira and Mozambique (n=578), the main clades were Latin-American-Mediterranean, East-African-Indian, Beijing and T, with no extensively DR TB cases. CONCLUSIONS: Beira and Mozambique share the same population genetic structure of M. tuberculosis.

Contaminación cruzada por Mycobacterium tuberculosis en el Laboratorio Nacional de Referencia de Tuberculosis de Cuba / Cross-contamination of Mycobacterium tuberculosis at the National Rference Laboratory of Tuberculosis of Cuba

Introducción: la contaminación cruzada por Mycobacterium tuberculosis (Mtb) en el laboratorio es más común de lo que se piensa. Objetivo: confirmar un posible episodio de contaminación cruzada por Mtb en el Laboratorio Nacional de Referencia de Tuberculosis, Lepra y Micobacterias del Instituto Pedro Kouri. Métodos: se realizó un estudio descriptivo y retrospectivo de los indicadores de calidad del cultivo del 3er trimestre de 2014. Se observó un incremento de cultivos positivos a Mtb con codificaciones bajas, durante un día de trabajo. Se encontraron 10 aislamientos de 19 muestras procesadas: ocho con sospecha de contaminación cruzada y dos aislamientos pertenecientes a uno de los pacientes involucrados y que tuvo un cultivo positivo procesado en una fecha diferente. Las muestras se procesaron según lo establecido por el Programa Nacional de Control de la Tuberculosis de Cuba. A los cultivos positivos se les realizó la tipificación con oligonucleótidos espaciadores Spoligotyping. Resultados: el aislamiento de la muestra 1 435 (paciente 1) fue un patrón único al no aparecer en la base de datos internacional SITVIT2. En los cultivos de las muestras 1 438 y 1 439 (paciente 2) se identificó el linaje Beijing (tipo 1). En los siete cultivos restantes (pacientes del 3 al 7) se identificó el linaje S tipo 71; los cultivos posteriores de estos pacientes fueron negativos a excepción del paciente 5 (muestra 1 561), en el que se aisló Mtb con el mismo patrón genético. Conclusiones: los resultados de la genotipificación permitieron confirmar los cultivos positivos contaminados e inferir una posible fuente de contaminación durante ese día de trabajo. También resalta la importancia de identificar eventos de contaminación cruzada, pues puede implicar un mal manejo clínico de los pacientes, así como el uso innecesario de un tratamiento largo y costoso, además de influir en el análisis e interpretación de los resultados desde el punto de vista epidemiológico(AU)

Introduction: cross contamination of Mycobacterium tuberculosis in the laboratory is more common than thought. Objective: to confirm a possible event of cross-contamination of Mycobacterium tuberculosis in the National Reference Laboratory of Tuberculosis, Leprosy and Mycobacteria of Pedro Kouri Institute. Methods: a descriptive and retrospective study of quality indicators of culture for the third quarter of 2014 was conducted. An increased Mycobacterium tuberculosis-positive culture with low encodings was observed during a working day. Ten isolates of 19 processed samples were found: eight suspected cross-contamination and two isolates from one of the patients involved and had a positive culture processed on a different date. The samples were processed as established by the National Program for Tuberculosis Control in Cuba .Positive cultures were typed by using oligonucleotide spacers Spoligotyping. Results: the isolation of the sample 1 435 (patient 1) showed a unique pattern that does not appear in the international database SITVIT2. In the culturing of 1 438 and 1 439 samples (patient 2), the Beijing lineage (type 1) was identified. In the remaining seven cultures (patients 3 through 7), the lineage S type 71 was identified. The subsequent cultures of the samples taken from these patients were negative except for patient 5 (sample 1 561) in whom Mtb was isolated with the same genetic pattern. Conclusions: the results of genotyping allowed confirming the contaminated positive cultures and inferring a possible source of contamination during that workday. It also highlights the importance of identifying cross-contamination events, because it may involve poor clinical management of patients and thus the unnecessary use of a long and costly treatment, in addition to influencing the analysis and interpretation of the results from the epidemiological viewpoint(AU)

Molecular characterization of mycobacterium tuberculosis isolates from Tehran, Iran by restriction fragment length polymorphism analysis and spoligotyping

Abstract: INTRODUCTION Characterization of Mycobacterium tuberculosis (MTB) isolates by DNA fingerprinting has contributed to tuberculosis (TB) control. The aim of this study was to determine the genetic diversity of MTB isolates from Tehran province in Iran. METHODS MTB isolates from 60 Iranian and 10 Afghan TB patients were fingerprinted by standard IS6110-restriction fragment length polymorphism (RFLP) analysis and spoligotyping. RESULTS The copy number of IS6110 ranged from 10-24 per isolate. The isolates were classified into 22 clusters showing ≥ 80% similarity by RFLP analysis. Fourteen multidrug-resistant (MDR) isolates were grouped into 4 IS6110-RFLP clusters, with 10 isolates [71% (95% CI: 45-89%)] in 1 cluster, suggesting a possible epidemiological linkage. Eighteen Iranian isolates showed ≥ 80% similarity with Afghan isolates. There were no strains with identical fingerprints. Spoligotyping of 70 isolates produced 23 distinct patterns. Sixty (85.7%) isolates were grouped into 13 clusters, while the remaining 10 isolates (14.2%) were not clustered. Ural (formerly Haarlem4) (n = 22, 31.4%) was the most common family followed by Central Asian strain (CAS) (n = 18, 25.7%) and T (n = 9, 12.8%) families. Only 1strain was characterized as having the Beijing genotype. Among 60 Iranian and 10 Afghan MTB isolates, 25% (95% CI: 16-37) and 70% (95% CI: 39-89) were categorized as Ural lineage, respectively. CONCLUSIONS A higher prevalence of Ural family MTB isolates among Afghan patients than among Iranian patients suggests the possible transmission of this lineage following the immigration of Afghans to Iran.

Evaluación del poder discriminatorio de técnicas de epidemiología molecular en aislados Venezolanos de Mycobacterium tuberculosis / Evaluation of discriminatory power of molecular epidemiology techniques in Mycobacterium tuberculosis Venezuelan isolates

La técnica de espoligotipaje y el método de unidades repetitivas micobacterianas interdiseminadas y número variable de repetidos tándem (MIRU-VNTR) 24 loci se emplean para estudiar la epidemiología molecular de tuberculosis. En el presente trabajo evaluamos la discriminación del método MIRU-VNTR 24 loci solo y en asociación con espoligotipaje en aislados clínicos de M. tuberculosis en Venezuela, la diversidad alélica de los 24 loci, y el poder discriminativo para la combinación de 24 y 15 loci, 12 loci tradicionales (12t), aquellos con más alta diversidad alélica y una nueva combinación que denominamos 12 inv. Se estudiaron 104 cepas de diferentes linajes y 431 cepas de la familia Latin-America y Mediterráneos (LAM). Los loci 4052, 2163b, 424 y 2996 presentaron la más alta diversidad alélica. Las tasas de agrupamiento de MIRU-VNTR 24 loci, espoligotipaje y MIRU-VNTR combinado con espoligotipaje para 104 aislados fueron de 18,27%, 71,15% y 14,4%, respectivamente, mientras que para cepas LAM fue de 43,2%, 95,8% y 37,4%. Las combinaciones de 15, 12inv y 4 loci MIRU-VNTR mas discriminativos, fueron más discriminatorios que 12t. Las tasas de agrupamiento para 15 y 12inv MIRU VNTR acoplados a espoligotipaje en los 104 aislados fueron de 21% y 23%, mientras que para cepas LAM fue de 52% y 46% respectivamente. El número de patrones diferentes fue similar para 12inv y 15 loci. Se propone el uso de un número reducido de loci MIRU-VNTR informativos acoplado a espoligotipaje para estudios de la transmisión de la tuberculosis en Venezuela.

The techniques of spoligotyping and mycobacterial interspersed repetitive unit and variable-number tandem repeat typing with 24 loci (MIRU-VNTR-24), have been used to study the molecular epidemiology of tuberculosis. The aim of this study was: to evaluate the discriminative power of MIRU-VNTR 24 loci alone and in association with spoligotyping in clinical isolates of M. tuberculosis in Venezuela; the allelic diversity of the 24 loci; and the discriminative power for the combination of 24 and 15 loci, 12 traditional loci (12t), those with higher allelic diversity and a new combination named 12inv. We analyzed one set of 104 strains of different lineages and a second set of 431 strains belonging to the Latin-America and Mediterranean lineage (LAM) that is predominant in Venezuela. The determination of allelic diversity showed that 4052, 2163b, 424 y 2996 are highly discriminative. Clustering rates of MIRU-VNTR 24 loci, spoligotyping and MIRU-VNTR combined with spoligotyping for 104 isolates were 18.27%, 71.15% and 14.4%, respectively, whereas with the 431 LAM strains the values were 43.2 %, 95.8% and 37.4%. MIRU-VNTR combinations of 15, 12inv and 4 loci were more discriminatory than 12t. Clustering rates for MIRU-VNTR 15 and 12inv loci coupled with spoligotyping in the 104 isolated was 21% and 23%, while for LAM strains was 52% and 46% respectively. The number of different genetics patterns for 15 and 12inv loci were similar. In conclusion, we propose the use of a small number of informative loci MIRU-VNTR coupled to spoligotyping to investigate the transmission of tuberculosis in Venezuela.

Molecular profiling of drug resistant isolates ofMycobacterium tuberculosis in the state of Santa Catarina, southern Brazil

Drug resistance is a global threat and one of the main contributing factors to tuberculosis (TB) outbreaks. The goal of this study was to analyse the molecular profile of multidrug-resistant TB (MDR-TB) in the state of Santa Catarina in southern Brazil. Fifty-three MDR Mycobacterium tuberculosisclinical isolates were analysed by spoligotyping and a partial region of therpoB gene, which is associated with rifampicin resistance (RMP-R), was sequenced. Some isolates were also distinguished by their mycobacterial interspersed repetitive units (MIRU). S531L was the most prevalent mutation found within rpoBin RMP-R isolates (58.5%), followed by S531W (20.8%). Only two MDR isolates showed no mutations withinrpoB. Isolates of the Latin American Mediterranean (LAM) family were the most prevalent (45.3%) found by spoligotyping, followed by Haarlem (9.4%) and T (7.5%) families. SIT106 was found in 26.4% of isolates and all SIT106 isolates typed by MIRU-12 (5 out of 14) belong to MIT251. There was a high correlation between the S531W mutation and the LAM family mainly because all SIT2263 (LAM9) isolates carry this mutation. Among isolates with the S531W mutation in rpoB MIRU demonstrates a cluster formed by four isolates (SIT2263 and MIT163) and very similar profiles were observed between eight of the nine isolates. Better characterisation of TB isolates may lead to new ways in which to control and treat TB in this region of Brazil.

Identificação e genotipagem de mycobacterium bovis em bovinos positivos no teste intradérmico para tuberculose em Mato Grosso do Sul / Identification and genotyping of mycobacterium bovis from positive cattle in skin test for tuberculosis in the state of Mato Grosso do Sul, Brazil

Neste estudo, realizou-se genotipagem de isolados de Mycobacterium bovis, provenientes de amostras de tecidos de bovinos positivos no teste cervical comparativo (TCC) para tuberculose em Mato Grosso do Sul, por meio da técnica de spoligotyping. Tecidos de 13 bovinos positivos, oriundos de diferentes municípios do estado, foram cultivados em meio de Stonebrink. As colônias resultantes foram submetidas à coloração de Ziehl-Neelsen e todos os isolados apresentaram características tintoriais de BAAR. Os 13 isolados de BAAR foram identificados por PCR multiplex (mPCR). O gene hsp65 foi alvo para identificação de Mycobacterium spp, a sequência de inserção IS6110 foi alvo para identificação de complexo Mycobacterium tuberculosis (CMT) e a região rvd1rv2031c foi explorada para detecção de M. bovis. Os isolados micobacterianos foram genotipados pela técnica de spoligotyping. Dos 13 bovinos, sete tinham pelo menos uma lesão sugestiva de tuberculose em linfonodos retrofaríngeos, parotídeos e pulmonares ou no pulmão, e em seis não foram encontradas lesões visíveis sugestivas da doença. Na mPCR, 11/13 (84,6%) isolados foram positivos para Mycobacterium spp, 8/13 (61,5%) positivos para CMT e 7/13 (53,8%) positivos para M. bovis. Com base no spoligotyping, oito isolados de BAAR foram agrupados dentro de três diferentes agrupamentos de genótipos e uma amostra remanescente apresentou perfil único, sendo quatro isolados com padrão de espoligotipo SB0121, dois SB1145, dois SB0881 e um SB0140. A técnica de spoligotyping demonstrou que há diversidade genética entre os espoligotipos presentes no estado de Mato Grosso do Sul, embora predomine o perfil SB0121.

Spoligotyping was performed in the present study to genotype Mycobacterium bovis isolates obtained from tissues of cattle that were positive in the comparative intradermal tuberculin test (CITT) in the state of Mato Grosso do Sul (Brazil). Tissue samples from 13 positive cattle from different municipalities of the state were cultured using a Stonebrink medium. The resulting colonies were subjected to Ziehl-Neelsen staining and all isolates exhibited the staining characteristics of AFB. The 13 isolates of AFB were identified by means of a multiplex PCR (mPCR) assay. The hsp65 gene was targeted for the identification of Mycobacterium spp., whereas the IS6110 insertion sequence was targeted for the identification of the Mycobacterium tuberculosis complex (MTC) and the rvd1rv2031c region was explored for the detection of Mycobacterium bovis. The spoligotyping assay was performed to genotype mycobacterial isolates. Of the 13 cattle, seven had at least one lesion suggestive of tuberculosis in the retropharyngeal, parotid and lung lymph nodes or lung. The remaining six exhibited no lesions suggestive of the disease. In the mPCR, 11 of the 13 isolates (84.6%) were positive for Mycobacterium spp., 8/13 (61.5%) were positive for the MTC and 7/13 (53.8%) were positive for M. bovis. Based on the spoligotyping, eight isolates were grouped into three different groups of genotypes and one isolate exhibited an orphan type. Four isolates exhibited spoligotype pattern SB0121, while two isolates were associated with the pattern SB1145, another two were associated with pattern SB0881 and one was associated with pattern SB0140. Spoligotyping confirmed the genetic diversity present among isolates found in the state of Mato Grosso do Sul. In addition, SB0121 was confirmed as the predominant profile.