Comparative evaluation of pncA gene, IS6110 and 12.7-Kb fragment based PCR assays for simultaneous detection of Mycobacterium tuberculosis complex (M. tuberculosis and M. bovis) in cultured strains and clinical specimens

PCR based molecular techniques help in discrimination of two closely related Mycobacterium tuberculosis and M.bovis. Here, we analyzed 24 M. bovis, 39 M. tuberculosis, 21 fresh acid-fast positive sputum samples and standardmycobacterial strains with pncA, 12.7 Kb and IS6100 based PCR assays. DNA from cultures and sputum yielded a positiveamplification of 185 bp with M. tuberculosis specific reverse primer pncAMT-2 but not with M. bovis specific reverseprimer pncAMB-2 and all M. bovis strains showed a positive amplification of 185 bp with M. bovis specific reverse primerpncAMB-2 but not with M. tuberculosis specific reverse primer pncAMT-2. The 12.7 Kb fragment based PCR performed onDNA extracted from cultures of M. tuberculosis and sputum yielded product of 168 bp while M. bovis showed 262 bpproducts. M. tuberculosis complex specific IS6110 PCR assay performed on DNA extracted from M. tuberculosis, M. boviscultures and sputum samples yielded M. tuberculosis complex specific 123-bp amplified products. The sequence analysis ofrepresentative PCR products of IS6110 and 12.7 Kb fragment showed 99-100% and 100% identity in amplicon products,respectively. To test reliability of primers, M. tuberculosis and M. bovis cultures were mixed and subjected to IS6110, pncAand 12.7 Kb PCR assay. pncA primers could not successfully and reliably discriminate the mixed culture, however, 12.7 Kbfragment primers successfully discriminated the mixed culture of M. tuberculosis and M. Bovis.

Tuberculosis en menores de 15 años / Tuberculosis in children younger than 15 years

Introducción: La Habana es la provincia con mayor incidencia de tuberculosis infantil en Cuba. Objetivo: caracterizar la tuberculosis infantil en La Habana. Métodos: se realizó un estudio descriptivo, transversal, en el universo de enfermos menores de 15 años, en el período 2001-2010. De las encuestas epidemiológicas se revisaron: variables demográficas, localización, forma clínica y datos que condujeron al diagnóstico. Resultados: se reportó una incidencia de 44 casos, y tasas al inicio y final de 2,2 y 2,1 x 100 mil habitantes de 0-14 años respectivamente. Exceptuando los años 2003 y 2006, las tasas de La Habana estuvieron muy por encima de la media nacional, al igual que el porcentaje de casos infantiles en relación con el total de enfermos. Predominaron los menores de 5 años; no se reportaron lactantes. El sexo femenino (54,5 por ciento) fue el más afectado en general, aunque predominó el sexo masculino en los menores de 5 años. Los municipios más afectados fueron: Cerro y Arroyo Naranjo (7 y 5 casos respectivamente), seguidos por Centro Habana, Plaza, Marianao y 10 de Octubre con 4. Predominó la localización pulmonar (38-86,4 por ciento), fundamentalmente formas primarias, con 15,8 por ciento de baciloscopias positivas, todas en formas pulmonares tipo adulto. El resultado de la prueba de Mantoux fue ³ 10 mm en 68,2 por ciento. Se identificó el contacto infectante en el 88 por ciento, sobre todo, los menores de 5 años, que se infectaron con más frecuencia en el hogar. Conclusiones: aunque La Habana tiene condiciones demográficas que favorecen la transmisión, hay que profundizar en la pesquisa activa en los controles de foco, y a otros grupos más vulnerables para contribuir a la eliminación de la TB

Introduction: Havana is the province with the highest t incidence of infant tuberculosis in Cuba. Objective: to characterize infant tuberculosis in Havana. Methods: a cross-sectional descriptive study was performed in patients younger than 5 years in the period of 2001 through 2010. Some variables were checked in the epidemiological surveys such as demographic variables, location, clinical form and data, which led to the diagnosis. Results: there were reported an incidence of 44 cases, and initial and final rates of 2.2 and 2.1 per 100 000 inhabitants aged 0-14 years, respectively. Except for the years 2003 and 2006, the rates of Havana province were well over the domestic mean as it was the case of the percentage of infant cases with respect to the total number of patients. Children younger than 5 years prevailed; no nurslings were reported. Females (54.5 percent) were the most affected in general, although males predominated in children under-5 years of age. The most affected municipalities were Cerro and Arroyo Naranjo (7 and 5 cases, respectively) followed by Centro Habana, Plaza, Marianao and 10 de Octubre with 4 each. Pulmonary location predominated (38-86.4 percent), mainly the primary form, with 15.8 percent positive bacilloscopies, all in adult-type pulmonary forms. The result of Mantoux's test was ³ 10 mm in 68.2 percent. The infective contact was identified in 88 percent of cases, mainly in children aged less than 5 years, who got infected more frequently at home. Conclusions: although Havana province has demographic conditions that favor disease transmission, one must perform more thorough active screening to control foci, and for other more vulnerable groups to contribute to eliminate tuberculosis

A rapid and low-cost microscopic observation drug susceptibility assay for detecting TB and MDR-TB among individuals infected by HIV in South India.

Background: The converging epidemics of HIV and tuberculosis (TB) pose one of the greatest public health challenges of our time. Rapid diagnosis of TB is essential in view of its infectious nature, high burden of cases, and emergence of drug resistance. Objective: The purpose of this present study was to evaluate the feasibility of implementing the microscopic observation drug susceptibility (MODS) assay, a novel assay for the diagnosis of TB and multi-drug-resistant tuberculosis (MDR-TB) directly from sputum specimens, in the Indian setting. Materials and Methods: This study involved a cross-sectional, blinded assessment of the MODS assay on 1036 suspected cases of pulmonary TB in HIV-positive and HIV-negative patients against the radiometric method, BD-BACTEC TB 460 system. Results: Overall, the sensitivity, specificity, positive predictive value, and negative predictive value of the MODS assay in detecting MTB among TB suspected patients were 89.1%, 99.1%, 94.2%, 95.8%, respectively. In addition, in the diagnosis of drug-resistant TB, the MODS assay was 84.2% sensitive for those specimens reporting MDR, 87% sensitivity for those specimens reporting INH mono-resistance, and 100% sensitive for specimens reporting RIF mono-resistance. The median time to detection of TB in the MODS assay versus BACTEC was 9 versus 21 days (P < 0.001). Conclusion: Costing 5 to 10 times lesser than the automated culture methods, the MODS assay has the potential clinical utility as a simple and rapid method. It could be effectively used as an alternative method for diagnosing TB and detection of MDR-TB in a timely and affordable way in resource-limited settings.