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BACKGROUND: The genetic diversity of M. bovis in Tunisia is still underestimated despite the implementation of an eradication program. The lack of data about spatial distribution of the M. bovis population hinders the control of bovine tuberculosis (bTB) progress. This study represents the largest molecular analysis of M. bovis isolates in Tunisia. It is aimed to upgrade the understanding of bTB epidemiology and the geographical distribution of the infection. Tuberculosis research was performed in cattle (n = 149) with TB-compatible lesions collected over 5 months from a slaughterhouse located in Sfax, Tunisia. RESULTS: Ninety-four animals were found to be infected by M. bovis and two others by M. caprae. Spoligotyping revealed twenty-five patterns, SB0120, SB0134, and SB0121 being the most prevalent profiles (36.4%, 11.4%, and 7.2%, respectively). Three new spoligotypes were detected: SB2345, SB2344 and SB2343. MIRU-VNTR analysis classified the isolates in seventy-three profiles and showed a large genotypic variety observed within the main spoligotype which was split into several MIRU-VNTR types: 29 in SB0120 (h = 0.983), 10 in SB0134 (h = 0.981) and 7 in SB0121 (h = 1). Genotyping revealed a common pattern in different geographic regions. It also showed that Sfax, located in southern-Tunisia, represents a high-risk area with an elevated genetic diversity. CONCLUSIONS: Spatial analysis may provide insights into disease transmission, which affects the effectiveness of eradication campaigns in cattle.
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Mycobacterium bovis/genética , Tuberculose Bovina/microbiologia , Animais , Bovinos , Feminino , Variação Genética/genética , Técnicas de Genotipagem/veterinária , Masculino , Repetições Minissatélites/genética , Reação em Cadeia da Polimerase/veterinária , Tuberculose Bovina/epidemiologia , Tunísia/epidemiologiaRESUMO
INTRODUCTION: In Tunisia, almost 77% of clinically and bacteriologically diagnosed cases of extrapulmonary tuberculosis (EPTB) are zoonotic TB, caused by M. bovis. Although several studies have analyzed bovine TB in cattle in Tunisia, no study has evaluated the risk of transmission to humans in such an endemic country. We aimed to study the genetic diversity of M. bovis human isolates, to ascertain the causes of human EPTB infection by M. bovis and to investigate the distribution and population structure of this species in Tunisia. MATERIALS AND METHODS: A total of 110 M. bovis isolates taken from patients with confirmed EPTB were characterized by spoligotyping and MIRU-VNTR typing methods. RESULTS: Among the 15 spoligotypes detected in our study, 6 (SB0120, SB0121, SB2025, SB1200, SB1003 and SB0134) were the most prevalent (83.5%) of which SB0120, SB0121 and SB2025 were the most prevailing. MIRU-VNTR typing method showed a high genotypic and genetic diversity. The genetic differentiation based on MIRU-VNTR was significant between populations from South East (Tataouine, Medenine) and Central West (Gafsa, Sidi Bouzid, Kasserine) regions. Of note, 13/15 (86.7%) spoligotypes detected in our study were previously identified in cattle in Tunisia with different frequencies suggesting a peculiar ability of some genotypes to infect humans. Using combined spoligotyping and MIRU-VNTR method, a high clustering rate of 43.9% was obtained. Our results underlined that human EPTB due to M. bovis was more commonly found in female gender and in young patients. Most of our patients, 66.4% (73/110) were raw milk or derivatives consumers, whereas 30.9% (34/110) patients would have contracted EPTB through contact with livestock. The findings suggest that the transmission of Zoonotic TB caused by M. bovis to humans mainly occurred by oral route through raw milk or derivatives. CONCLUSION: Our study showed the urgent need of a better veterinary control with the implementation of effective and comprehensive strategies in order to reach a good protection of animals as well as human health.
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Mycobacterium bovis/genética , Tuberculose/microbiologia , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Feminino , Variação Genética , Genótipo , Técnicas de Genotipagem , Humanos , Lactente , Líbia/etnologia , Gado , Masculino , Pessoa de Meia-Idade , Leite , Mycobacterium bovis/isolamento & purificação , Estudos Prospectivos , Tuberculose/epidemiologia , Tunísia/epidemiologia , ZoonosesRESUMO
A combined enrichment/ newly developed invA TaqMan® real-time PCR (qPCR) method as a screening assay to detect Salmonella spp. in 500 naturally food matrices is evaluated. DNA template for qPCR was extracted from an overnight pre-enriched sample in buffered peptone water using lysis-guanidine isothiocyanate method. Heterologous internal amplification control (IAC) was incorporated during qPCR assays and co-amplified with the invA gene of the target pathogen. InvA qPCR exhibited 100% specificity when testing 94 Salmonella strains (inclusivity) and 32 non-Salmonella strains (exclusivity). The qPCR showed a consistent detection of two copies of the invA gene/PCR reaction, a good intra- and inter-run reproducibility with a good PCR efficiency (89.6%). QPCR was sensitive and showed Salmonella detection at 8.5 × 100 CFU mL-1 of artificially spiked poultry meat -BWP solution in less than 40 cycles. When analyzing 500 different food matrices and comparing the results with the ISO 6579:2002 conventional culture method, the sensitivity and specificity were 100 and 76.6%, respectively. QPCR showed Salmonella spp. DNA in raw poultry meat 27/45 (60%), milk 31/93 (33.3%), raw red meat 5/13 (38.5%), and fish 11/46 (23.9%) samples. The prevalence of Salmonella spp. in cakes, dairy, cooked meals, charcuterie products using qPCR was 11/14 (26.8%), 5/22 (22.7%), 32/150 (21.3%), and 5/20 (25%), respectively, compared to 0% as demonstrated by culture. S. Anatum was the most common serovar found associated with red meat compared to S. kentucky isolated from fish and poultry meat. In conclusion, our study is the first to use a combined enrichment/invA qPCR method as a screening assay to detect Salmonella DNA in different types of commercialized food in Southern Tunisia. QPCR results indicate that Salmonella contamination is common in milk and in other types of food samples.
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INTRODUCTION: Tunisia has one of the highest burdens of extrapulmonary tuberculosis (EPTB) among tuberculosis (TB) cases but the contribution of MTBC-mediated human EPTB is unknown. EPTB diagnosis is challenging due to the paucibacillary nature of clinical samples. Therefore, a need of a simplified molecular method for sensitive and specific TB detection and differentiation of MTBC members caused EPTB remains a priority to an early diagnosis, optimize successful anti-TB treatment and minimize transmission. We evaluated the performance of a single tube tetraplex Taq Man real time PCR for EPTB detection and differentiation between MTBC members directly on extrapulmonary samples. MATERIALS AND METHODS: Extrapulmonary samples obtained from clinically suspected EPTB patients from 2013 to April 2015 were tested by Ziehl Neelsen Staining, mycobacterial culture and qPCR assay for RD1, RD9, RD12 and ext-RD9 targets (MTBC-RD qPCR). The performance of qPCR was compared to a reference standard based on MTBC culture and/or at least two criteria of a composite reference standard (CRS) including clinical, radiological, histopathological and therapeutic findings. RESULTS: EPTB was identified in 157/170 (92.4%) of included patients of whom 99 (63%) were confirmed by culture and 58 (36.9%) by CRS criteria. The sensitivity and specificity of qPCR, in comparison to the reference standard were 100% (157/157) and 92.3% (12/13), respectively. The sensitivity of qPCR was statistically significant as compared to culture and smear microscopy (P< 0.001). QPCR results showed M. bovis identification in 77.1% of extrapulmonary samples in occurrence to lymphadenitis infection. M. tuberculosis and M.bovis BCG were detected in 21.6% and 1.3% of cases, respectively. CONCLUSIONS: MTBC-RD qPCR proved to be a rapid and sensitive assay for simultaneously TB detection and MTBC members identification on extrapulmonary samples within 1.5 days after sample receipt. Its high sensitivity could make this method a useful tool in diagnosing TB in addition to routine conventional methods and TB clinical parameters.
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Reação em Cadeia da Polimerase Multiplex/métodos , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Tuberculose/diagnóstico , Tuberculose/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Técnicas Bacteriológicas , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Microscopia , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Sensibilidade e Especificidade , Fatores de Tempo , Tunísia , Adulto JovemRESUMO
OBJECTIVE/BACKGROUND: Tuberculosis is a major public health problem and the emergence of drug resistance complicates the situation even more. It is therefore crucial to implement all conclusions from the studies that aim at a better understanding of the molecular mechanisms which govern the emergence and the evolution of drug resistance. The aim of this study is to assess the degree of involvement of the inhA and katG genes in the acquisition of isoniazid resistance in clinical strains of Mycobacterium tuberculosis. METHODS: The inhA and katG genes were sequenced in 21 strains of M. tuberculosis with different resistance profiles and from different regions. RESULTS: Analysis of the sequences obtained by comparison to those of the reference strain H37Rv showed that 95.2% had mutations. KatG S315T was the most common mutation (85.7%). The mutation katG T275A was revealed in two strains (9.5%). Two different point mutations in the inhA gene and its promoter region were identified as C-15T and G56A at a frequency equal to 14% and 10%, respectively. The G56A mutation is a new silent mutation. Our study showed no correlation between found mutations and multidrug resistance. Among the 21 strains studied, only one strain showed no mutations. CONCLUSION: In terms of this study, we characterized the mutations involved in resistance to isoniazid. katG S315T was by far the most frequent mutation, followed by C-15T. The frequency of these mutations was concordant with those reported in literature including those in intermediate tuberculosis endemic countries.
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AIMS AND OBJECTIVES: Current methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB) are either costly or slow. As the prevalence of multidrug-resistant (MDR) strains increases, the need for fast, reliable, and inexpensive methods is obvious. This study evaluated a rapid colorimetric nitrate reductase assay (NRA) for direct DST of MTB directly from clinical sputum samples. METHODS: A total of 111 sputa with positive microscopy results for acid-fast bacilli (AFB) with more than 10 AFB per high-power field were used in the study. The samples were decontaminated using the modified Petroff method. The NRA results were compared with the reference indirect proportion method. RESULTS: The sensitivity and the specificity of the direct NRA were 90% and 97.3%, 92.6% and 98.2%, 52.9% and 100%, and 28.6% and 100% for rifampin, isoniazid, streptomycin, and ethambutol, respectively. The results were in most cases available in 28days (84.3%). CONCLUSIONS: The direct NRA could be used as a rapid, inexpensive, and accurate method to determine rifampin and isoniazid susceptibility directly from sputum. The technique might become a valid alternative to traditional methods, especially in low-income countries.
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Antituberculosos/farmacologia , Ensaios Enzimáticos , Testes de Sensibilidade Microbiana/métodos , Microscopia , Mycobacterium tuberculosis/efeitos dos fármacos , Nitrato Redutase/metabolismo , Escarro/microbiologia , Colorimetria , Análise Custo-Benefício , Humanos , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana/economia , Rifampina/farmacologia , Sensibilidade e Especificidade , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
INTRODUCTION: GeneXpert MTB/RIF is a fully-automated diagnostic molecular test which simultaneously detects tuberculosis (TB) and rifampicin (RIF) drug resistance. The purpose of this study is to evaluate the performance of the GeneXpert MTB/RIF test for the detection of Mycobacterium tuberculosis complex (MTBC) in lymph node specimens and to show the place of Mycobacterium bovis as a major cause of TB lymphadenitis. MATERIAL AND METHODS: This study was conducted simultaneously in the National Reference Laboratory for Mycobacteria of Ariana and the Central Laboratory of Sfax, from January to December 2013. In total, 174 lymph node specimens were processed simultaneously for Ziehl-Neelsen, auramine and immuno-histochemical staining. Conventional culture on both Lowenstein-Jensen and liquid medium (Bactec MGIT 960 BD system) and the new molecular-based GeneXpert MTB/RIF assay system were performed. Positive cultures were confirmed using molecular identification (Genotype MTBC Hain Lifescience). RESULTS: Among the 174 samples tested, the GeneXpert detected the DNA of MTBC in 134 samples (77%). Standard bacteriological assays, including AFB microscopy and culture, were positive, respectively, in 41 (23.6%) and 79 (45.4%) specimens. M. bovis was isolated in 76% of positive cultures. GeneXpert sensitivity and specificity results were assessed according to smear and culture results, clinical and histological findings. The sensitivity and specificity of the Xpert assay were 87.5% (126/144) and 73.3%, respectively. CONCLUSION: The implementation of the GeneXpert MTB/RIF assay may dramatically improve the rapid diagnosis of lymph node TB.
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Tipagem Molecular/métodos , Mycobacterium bovis/isolamento & purificação , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose dos Linfonodos/microbiologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Linfonodos/microbiologia , Masculino , Pessoa de Meia-Idade , Tipagem Molecular/instrumentação , Mycobacterium bovis/classificação , Mycobacterium bovis/genética , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Tuberculose dos Linfonodos/diagnóstico , Tunísia , Adulto JovemRESUMO
AIM: To evaluate patients' profiles, demographics, clinical and therapeutic approaches and strategies in patients with tuberculous lymphadenitis (TBG). PATIENTS AND METHODS: A retrospective study of all TBG-confirmed cases admitted in a tuberculosis-specific health care facility between 1 January 2009 and 16 June 2013. RESULTS: A total of 181 clinical files were examined. Mean age was 32years old; the female/male ratio was 1.78 to 1. Raw milk consumption was noted in 1/3 of patients. Most cases involved the head and neck region (83.4%), nodes involvement, including axillary (12 cases), and mediastinal (9 cases). Clinical symptoms were present in only 55.2%. Tuberculin skin test (TST) was conducted with 82.6% positive responses. Diagnostics confirmation was done with anatomical pathology in most of the patients; only 56 of them had any microbiology analysis done. Demonstration of acid-fast bacilli in microscopy from either fine-needle aspirates or biopsies was done in 17.5% of cases, and cultures yielded positive results in 27%. Treatment duration was varied. Paradoxical reactions were noted in 12% and persistent lymphadenopathy after treatment completion was noted in 10% of cases. CONCLUSIONS: TBG remains a disease of interest. Today, its diagnosis and management is still a problem despite its increasing worldwide incidence, and especially in this study area. Disease control should be strengthened in this country.