Development and evaluation of a single-tube nested PCR with colorimetric assay for Mycobacterium tuberculosis detection.

Molecular techniques have revolutionized tuberculosis (TB) diagnosis by offering a faster and more sensitive approach, detecting Mycobacterium tuberculosis (Mtb) DNA directly from samples. Single-tube nested PCR (STNPCR) combines two PCR reactions with separate oligonucleotide sets in a single tube. Moreover, colorimetric methods in PCR products have been studied for pathogen detection. Thus, this study aimed to establish a novel system based on colorimetric STNPCR for Mtb detection using microtiter plates with IS6110-amplified fragments. The results showed a general colorimetric STNPCR detection limit of 1 pg/µl. Its general sensitivity and specificity were 76.62 and 60.53%, respectively, with kappa index agreement of 0.166.

In vitro bioevaluation and docking study of dihydrosphingosine and ethambutol analogues against sensitive and multi-drug resistant Mycobacterium tuberculosis.

Tuberculosis remains a major public health problem and one of the top ten causes of death worldwide. The alarming increase in multidrug-resistant and extensively resistant variants (MDR, pre-XDR, and XDR) makes the disease more difficult to treat and control. New drugs that act against MDR/XDR strains are needed for programs to contain this major epidemic. The present study aimed to evaluate new compounds related to dihydro-sphingosine and ethambutol against sensitive and pre-XDR Mycobacterium strains, as well as to characterize the pharmacological activity through in vitro and in silico approaches in mmpL3 protein. Of the 48 compounds analyzed, 11 demonstrated good to moderate activity on sensitive and MDR Mycobacterium tuberculosis (Mtb), with a Minimum Inhibitory Concentration (MIC) ranging from 1.5 to 8 µM. They presented 2 to 14 times greater potency of activity when compared to ethambutol in pre-XDR strain, and demonstrated a selectivity index varying between 2.21 and 82.17. The substance 12b when combined with rifampicin, showed a synergistic effect (FICI = 0.5) on sensitive and MDR Mtb. It has also been shown to have a concentration-dependent intracellular bactericidal effect, and a time-dependent bactericidal effect in M. smegmatis and pre-XDR M. tuberculosis. The binding mode of the compounds in its cavity was identified through molecular docking and using a predicted structural model of mmpL3. Finally, we observed by transmission electron microscopy the induction of damage to the cell wall integrity of M. tuberculosis treated with the substance 12b. With these findings, we demonstrate the potential of a 2-aminoalkanol derivative to be a prototype substance and candidate for further optimization of molecular structure and anti-tubercular activity in preclinical studies.

Evaluation of completeness, consistency and non-duplication of leprosy notification data on the Notifiable Health Conditions Information System, João Pessoa, Paraíba, Brazil: a descriptive study, 2001-2019.

OBJECTIVE: to analyze the completeness, consistency and non-duplication of leprosy notification data in João Pessoa, Paraíba, Brazil, 2001-2019. METHODS: this was a descriptive study, conducted with data from the Notifiable Health Conditions Information System, which checked for "duplication" (acceptable: < 5%), "completeness" (excellent = incompleteness ≤ 5%) and "consistency" (excellent: ≥ 90.0%), based on the proportion of complete and consistent fields. RESULTS: the sample consisted of 2,410 notifications. Duplication was acceptable (0.3%). The completeness of the "bacilloscopy", "affected nerves", "examined contacts" and "reactive episode" fields was very poor (more than 50% incomplete). Consistency between the "operational classification" and "initial treatment regimen" fields was excellent (99.6%), while consistency between "operational classification" and "clinical form" was low (50.7%). CONCLUSION: although duplication was acceptable, poor completeness of diagnosis and follow-up fields hinders epidemiological analysis, recognition of the status of the disease and adoption of measures to control it.

Performance of IS6110-LAMP assay for detection of Mycobacterium tuberculosis complex in blood and urine samples from patients with extrapulmonary tuberculosis.

BACKGROUND: The diagnosis of extrapulmonary tuberculosis (EPTB) shows numerous difficulties because of non-specific symptomatology and low sensitivity of conventional methods. Loop-mediated isothermal amplification (LAMP) is a fast and low-cost technique, which can amplify under isothermal conditions an amount of target DNA copies into approximately a billion copies. OBJECTIVE: The present study aimed to evaluate a IS6110-LAMP system for Mycobacterium tuberculosis detection in blood and urine samples from patients with EPTB. METHODS: The collected samples (n = 122) were stratified in two groups: Group EPTB - patient samples with confirmed EPTB (n = 61); Group non-TB - patient samples without TB (n = 61). The urine samples underwent decontamination, and the components of blood samples were separated (plasma and PBMC). DNA extractions were performed in all biological samples followed by IS6110-LAMP assay technique. The detection limit was evaluated through dilution curves (1:10) using Mtb reference strain (H37Rv) genomic DNA. FINDINGS: The detection limit of IS6110-LAMP was 10 fg/µL (∼10-20 bacilli/µL). The IS6110-LAMP technique sensitivity and specificity were 95.65 % and 79.25 %, respectively, with a general kappa agreement index of 0.762. MAIN CONCLUSIONS: Based on these results, IS6110-LAMP test showed considerable diagnostic parameters, being able to aid in the speed and accuracy of the final EPTB diagnosis.

Avaliação da completitude, consistência e não duplicidade dos dados de notificação da hanseníase no Sistema de Informação de Agravos de Notificação, João Pessoa, Paraíba: estudo descritivo, 2001-2019 / Evaluation of completeness, consistency and non-duplication of leprosy notification data on the Notifiable Health Conditions Information System, João Pessoa, Paraíba, Brazil: a descriptive study, 2001-2019 / Evaluación de la completitud, consistencia y no duplicidad de notificación de lepra en el Sistema de Información e Enfermidades de Declaración Obligatoria, en João Pessoa, Paraíba: estudio descriptivo, 2001-2019

Objetivo: analisar a completitude, consistência e não duplicidade dos dados da notificação da hanseníase em João Pessoa, Paraíba, Brasil, 2001-2019. Métodos: estudo descritivo, com dados do Sistema de Informação de Agravos de Notificação; verificou-se a duplicidade (aceitável quando < 5%), completitude (grau excelente = incompletitude ≤ 5%) e consistência (excelente quando ≥ 90,0%) dos dados, utilizando-se a proporção de campos preenchidos e coerentes. Resultados: a amostra de 2.410 notificações apresentou duplicidade aceitável (0,3%); a completitude dos campos "baciloscopia", "no de nervos afetados", "contatos examinados" e "episódio reacional" foi muito ruim (mais de 50% incompletos); e a consistência entre os campos "classificação operacional" e "esquema terapêutico inicial", excelente (99,6%), enquanto entre "classificação operacional" e "forma clínica" foi baixa (50,7%). Conclusão: embora a duplicidade observada fosse aceitável, a completitude de campos sobre diagnóstico e acompanhamento foi ruim, dificultando a análise epidemiológica, o reconhecimento da situação do agravo e a adoção de medidas de controle da doença.

Objective: to analyze the completeness, consistency and non-duplication of leprosy notification data in João Pessoa, Paraíba, Brazil, 2001-2019. Methods: this was a descriptive study, conducted with data from the Notifiable Health Conditions Information System, which checked for "duplication" (acceptable: ≤5%), "completeness" (excellent = incompleteness ≤ 5%) and "consistency" (excellent: ≤ 90.0%), based on the proportion of complete and consistent fields. Results: the sample consisted of 2,410 notifications. Duplication was acceptable (0.3%). The completeness of the "bacilloscopy", "affected nerves", "examined contacts" and "reactive episode" fields was very poor (more than 50% incomplete). Consistency between the "operational classification" and "initial treatment regimen" fields was excellent (99.6%), while consistency between "operational classification" and "clinical form" was low (50.7%). Conclusion: although duplication was acceptable, poor completeness of diagnosis and follow-up fields hinders epidemiological analysis, recognition of the status of the disease and adoption of measures to control it.

Objetivo: analizar la completitud, consistencia y no duplicidad de los datos de notificación de lepra en João Pessoa, Paraíba, 2001 - 2019. Métodos: estudio descriptivo, realizado con datos del Sistema de Información de Enfermedades de Declaración Obligatoria, que verificó "duplicidad" (aceptable: < 5%). "completitud" (grado excelente: incompletitud ≤ 5%) y "consistencia" (excelente: ≥ 90,0%), utilizando la proporción de campos completos y coherentes. Resultados: se obtuvo una muestra de 2.410 notificaciones. La duplicidad se consideró aceptable (0,3%). La completitud de los campos "baciloscopía", "nervios afectados", "contactos examinados" y "episodio reaccional" fue muy pobre (más del 50% incompleta). La consistencia entre los campos "clasificación operativa" y "régimen terapéutico inicial" fue excelente (99,6%), mientras que "clasificación operativa" y "forma clínica" fue baja (50,7%). Conclusión: aunque la duplicidad es aceptable, la completitud de los campos de diagnóstico y seguimiento fue deficiente, lo que dificulta el análisis epidemiológico, el reconocimiento de la situación de la enfermedad y la adopción de medidas de control de la enfermedad.

Tendência temporal da tuberculose drogarresistente (TBDR) e dos tipos de resistência no estado de Pernambuco, Brasil / Temporal trend of Drug-Resistant Tuberculosis (DRTB) and resistance types in the state of Pernambuco, Brazil

Resumo Introdução A tuberculose drogarresistente (TBDR) é um dos grandes desafios para a saúde pública. Objetivo Este estudo analisou a tendência temporal da TBDR e a razão da proporção dos tipos de resistência no estado de Pernambuco, no período de 2002 a 2014. Método Trata-se de um estudo observacional do tipo tendência temporal de série histórica utilizando dados do Sistema de Informação de Tratamentos Especiais da Tuberculose e análise pelo modelo autorregressivo de médias móveis de ordem 2. Resultados Foram notificados 298 novos casos de TBDR, com incidência variando entre 0,06 e 0,71/100 mil habitantes. Pelo menos 1 caso foi notificado em 40 dos 185 municípios do estado. A tendência temporal da TBDR e da proporção entre os tipos de resistência não apresentou comportamento linear. Nos últimos cinco anos, houve uma tendência ascendente, concentrando 71,1% dos casos da série histórica. Em 2014, foi observado um crescimento expressivo tanto da TBDR primária como da secundária, quando comparado ao ano de 2002. Conclusão Apesar da baixa incidência e de a tendência da TBDR não ter comportamento linear, o aumento de casos nos últimos anos da série histórica pode ser considerado um sinal de alerta para os programas de controle da doença em Pernambuco.

Abstract Background Drug-Resistant Tuberculosis (DRTB) is one of the most relevant challenge to public health. Objective This study analyzed the temporal trend of the DRTB and resistance types in the state of Pernambuco, from 2002 to 2014. Method This was an observational study of the temporal trend type of a historical series using data from the Special Treatment Information System of Tuberculosis (SITE-TB) and also analysis by the autoregressive moving average models of order 2. Results A total of 298 new DRTB cases were reported, ranging from 0.06 to 0.71 per 100,000 inhabitants. At least one case was reported in 40 of the 185 municipalities in the state. The temporal trend of DRTB and the proportion between resistance types did not present a linear behavior. In the last 5 years, there has been an upward trend, concentrating 71.1% of the historical series. In 2014, there was an expressive growth of both primary and secondary DRTB, when compared to the year 2002. Conclusion Despite the low incidence and tendency of DRTB not to be linear, the increase in cases in the last years of the historical series can be considered as a warning sign for the disease control programs of Pernambuco.

Spatial analysis of tuberculosis in children under 15 years of age and socioeconomic risk: an ecological study in Paraíba, Brazil, 2007-2016. / Análise espacial da tuberculose em menores de 15 anos de idade e risco socioeconômico: um estudo ecológico na Paraíba, 2007-2016.

OBJECTIVE: To analyze spatial distribution of tuberculosis in individuals under 15 years old and socioeconomic factors in Paraíba, Brazil, 2007-2016. METHODS: This was an ecological study based on data from the Notifiable Health Conditions Information System (SINAN), taking each municipality to be a unit of analysis. Spatial distribution of incidence was performed, the local empirical Bayesian method and Moran's I were applied. Socioeconomic data were crossed-checked to identify areas of social prosperity. RESULTS: 426 cases were notified, with average incidence of 4.5/100,000 inhabitants. Moran's I was 0.59 (p=0.010). The Moran Map revealed concentration of cases in children under 15 in 38 high priority municipalities, in clusters with high-high and low-low patterns, in the east and northwest of the state, coinciding with areas of low social prosperity. CONCLUSION: There were clusters with greater tuberculosis transmission, indicating priority areas for addressing tuberculosis.

Treatment of mycobacteriosis in a patient with compatible symptoms after a cesarean delivery.

This study aims to report the diagnostic course and treatment of a fast-growing mycobacteria infection after cesarean delivery. We report the case of a 37-year-old woman admitted to the Infectious Diseases' Clinic of the Hospital das Clinicas da Universidade Federal de Pernambuco, Pernambuco State, Brazil, four months after a cesarean section, presenting with healing difficulties and located pain outside the surgical site. The first diagnosis was a probable rejection of the sutures that were not absorbed, but based on the clinical signs, reported history, complementary laboratory tests and no response to treatment with the conventional antibiotic therapy (cephalosporins/quinolones) prescribed, the ultimate diagnosis was a mycobacteriosis caused by Micobacterium fortuitum. Since fast-growing mycobacteria do not easily penetrate host tissues, they is mainly related to post-trauma or post-surgical procedures. It is extremely important to call attention to these occurrences in the gynecological-obsthetric field. Treatment for mycobacteriosis is often complicated because of the side effects of antibiotics, especially the ototoxicity of amikacin.

Análise espacial da tuberculose em menores de 15 anos de idade e risco socioeconômico: um estudo ecológico na Paraíba, 2007-2016 / Análisis espacial de la tuberculosis en menores de 15 años y riesgo socioeconómico: un estudio ecológico en Paraíba, Brasil, 2007-2016 / Spatial analysis of tuberculosis in children under 15 years of age and socioeconomic risk: an ecological study in Paraíba, Brazil, 2007-2016

Objetivo: Analisar a distribuição espacial da tuberculose em indivíduos menores de 15 anos de idade e fatores socioeconômicos na Paraíba, Brasil, 2007-2016. Métodos: Estudo ecológico, com dados do Sistema de Informação de Agravos de Notificação, sendo o município a unidade de análise. Realizou-se distribuição espacial da incidência, aplicou-se o método bayesiano empírico local e a estatística de Moran. Dados socioeconômicos foram cruzados, para identificação das áreas de prosperidade social. Resultados: Foram notificados 426 casos, com incidência média de 4,5/100 mil habitantes. O índice de Moran foi de 0,59 (p-valor=0,010). O Moran Map revelou concentração de casos em menores de 15 anos em 38 municípios com alta prioridade da atenção, em conglomerados nos padrões alto-alto e baixo-baixo, nas regiões leste e noroeste do estado, coincidindo com áreas de baixa prosperidade social. Conclusão: Houve clusters com maior transmissão da tuberculose, apontando áreas prioritárias para abordagem da tuberculose.

Objetivo: Analizar la distribución espacial de la tuberculosis en menores de 15 años y los factores socioeconómicos en Paraíba, Brasil, 2007-2016. Métodos: Estudio ecológico, con datos de Sistema de Información de Agravamientos de Notificación, considerando el municipio como unidad de análisis. Se realizó la distribución espacial de la incidencia, se aplicó el método empírico local Bayesiano y la estadística de Moran. Se cruzaron datos socioeconómicos para identificar áreas de vulnerabilidad social. Resultados: Se notificaron 426 casos, con una incidencia promedio de 4,5/100 mil habitantes. El índice de Moran = 0,59 (p=0,010). El Mapa de Moran reveló una concentración de casos en menores de 15 años en 38 municipios con alta prioridad de atención, en conglomerados en los patrones alto-alto y bajo-bajo, en las regiones este y noroeste del estado, coincidiendo con áreas de baja prosperidad social. Conclusión: Existían clusters con mayor transmisión de tuberculosis, lo que indica áreas prioritarias para abordar la tuberculosis.

Objective: To analyze spatial distribution of tuberculosis in individuals under 15 years old and socioeconomic factors in Paraíba, Brazil, 2007-2016. Methods: This was an ecological study based on data from the Notifiable Health Conditions Information System (SINAN), taking each municipality to be a unit of analysis. Spatial distribution of incidence was performed, the local empirical Bayesian method and Moran's I were applied. Socioeconomic data were crossed-checked to identify areas of social prosperity. Results: 426 cases were notified, with average incidence of 4.5/100,000 inhabitants. Moran's I was 0.59 (p=0.010). The Moran Map revealed concentration of cases in children under 15 in 38 high priority municipalities, in clusters with high-high and low-low patterns, in the east and northwest of the state, coinciding with areas of low social prosperity. Conclusion: There were clusters with greater tuberculosis transmission, indicating priority areas for addressing tuberculosis.

Identification of nontuberculous mycobacteria species by multiplex real-time PCR with high-resolution melting.

INTRODUCTION: Nontuberculous mycobacteria (NTM) species, as human pathogens, are increasing in the world, as is the difficulty of accurately identifying them. Differential diagnosis, especially between the M. tuberculosis complex and NTM species, and the characterization of NTM species is important. This study aimed to evaluate the performance of a molecular system based on multiplex real-time PCR with high-resolution melting (HRM) for the identification and differentiation of NTM species of clinical importance of an endemic area for tuberculosis in northeastern Brazil. METHODS: The technical protocol of the molecular system was based on multiplex real-time PCR-HRM, and evaluated the sensitivity and specificity of the detection of NTM species in mycobacterial clinical isolates from the studied region. The gold standard method was specific gene sequencing. RESULTS: The sensitivity and specificity of multiplex real-time PCR-HRM modified for differentiation between NTM and M. tuberculosis were 90% and 100%, respectively. The PCR-HRM sensitivities for the characterization of NTM species (M. kansasii, M. abscesses, M. avium, and M. fortuitum) were 94.59%, 80%, 57.14%, and 54%, respectively. CONCLUSIONS: The multiplex real-time PCR-HRM modified assay has the potential to rapidly and efficiently identify nontuberculous mycobacteria of clinical importance, which is crucial for immediate implementation of the appropriate therapy and thus avoiding complications and sequelae in patients.

Differential distribution in vitamin D receptor gene variants and expression profile in Northeast Brazil influences upon active pulmonary tuberculosis.

Tuberculosis is an infectious disease with variable outcomes. This variability is due to host immune capacity in containing the infection process initiated by the Mycobacterium tuberculosis (MTB). Vitamin D is able to modulate a very specific immune response against MTB infection, and its action relies on vitamin D receptor (VDR) binding. Altered VDR forms may compromise vitamin D pathway and proper immune response after MTB infection. Herein we assessed the relationship of five potentially functional polymorphisms from VDR: rs2228570 FokI, rs11568820 Cdx-2, rs2248098, rs1540339 and rs4760648, with tuberculosis susceptibility. The SNP rs4760648 T/T was associated with differential susceptibility to tuberculosis (OR = 2.50, 95%CI = 1.20-5.36, p = 0.01). The SNP rs1540339 presented association to both T allele (OR = 0.55, 95%CI = 0.35-0.88, p = 0.01) and the T/T genotype (OR = 0.404, 95%CI = 0.20 - 0.78, p = 0.005). The FokI T allele was identified as associated to diminished susceptibility (OR = 0.67, 95% CI = 0.45-0.99, p = 0.04) to active TB, as well as T/T genotype (OR = 0.15, 95%CI = 0.04-0.45, p = 9.58 × 10-5). We also performed the expression analyses and observed a down-regulation of VDR in patients (-10.717 FC, p = 8.42e-12), and according to the presence of associated FokI SNP, we observed that the C/T and T/T genotypes presence increases VDR expression (+ 1.25 and + 2.35 FC, p = 0.425 and p = 0.506, respectively). This study shows that vitamin D receptor variants can influence upon pulmonary tuberculosis susceptibility and VDR mRNA levels are decreased in those patients.

Rapid detection of Mycobacterium tuberculosis in children using blood and urine specimens.

INTRODUCTION: Laboratory and clinical features of childhood tuberculosis (TB) are non-specific and establishing an accurate diagnosis remains a challenge. This study evaluated a Single tube nested-PCR (STNPCR) to detect genomic DNA of Mycobacterium tuberculosis complex in blood and urine. METHODS: Biological samples were obtained from children (<15 years old) with clinical suspicion of pulmonary and extrapulmonary TB at public hospitals in Recife-Pernambuco, Brazil. Cultures yielded negative results in a majority of childhood TB cases, which are generally paucibacillary. A set of clinical, epidemiological, radiological, and laboratory criteria with evident clinical improvement after anti-TB treatment were frequently used to define childhood TB cases. RESULTS: Ninety children with clinical suspicion were enrolled in this study (44 with TB and 46 without TB). The pulmonary TB group had 20 confirmed cases and 46 negative controls, while the extrapulmonary TB group had 24 confirmed cases. The STNPCR showed sensitivities to pulmonary and extrapulmonary TB of 47.4% and 52.2% (blood) and 38.8% and 20% (urine), respectively. Considering the low performance of STNPCR on separate samples, we decided to perform a combined analysis (parallel sensitivity analysis) of the results from blood and urine samples. The parallel sensitivity increased to 65% in blood and 62.5% in urine. The specificity in both samples ranged from 93.5-97.8%. CONCLUSIONS: Although STNPCR showed moderate sensitivity, the specificity is high; therefore, the test can be used as an auxiliary tool to diagnose TB in children. It is a rapid test that demonstrated better performance than other diagnostic tests in paucibacillary samples as it does in childhood tuberculosis.

Tuberculose: conhecimento e adesão às medidas profiláticas em indivíduos contatos da cidade do Recife, Pernambuco, Brasil / Tuberculosis: knowledge and adherence to prophylactic measures in contact individuals of the city of Recife, Pernambuco, Brazil

Resumo Introdução Para o controle da tuberculose, é fundamental interromper a cadeia de transmissão da doença. O Ministério da Saúde preconiza que 100% dos contatos sejam examinados e iniciem tratamento da Infecção Latente por Mycobacterium tuberculosis. Nesse sentido, o conhecimento sobre a doença e a adesão à profilaxia por parte desses contatos são fatores que podem interferir no adequado controle da tuberculose. Objetivo Descrever o conhecimento dos contatos de portadores de tuberculose sobre a doença e sua adesão às medidas profiláticas no Distrito Sanitário II em Recife/PE. Método Estudo quantitativo, descritivo, utilizando questionários padronizados, aplicados a 140 contatos de tuberculose notificados de janeiro a dezembro de 2015. Análise dos dados realizada por meio de frequências simples. Resultados Dentre os entrevistados, 75,7% eram do sexo feminino, 55% pardos, com baixos níveis de escolaridade e renda familiar. Destes, 84,3% acreditam que a tuberculose é grave, 48,6% consideram que a transmissão se faz compartilhando utensílios. Apenas 55% foram convidados para serem examinados e 76% referiram não saber que deveriam ir à consulta ou a importância desta. Conclusão Os contatos de tuberculose possuem precário conhecimento sobre a doença, baixa adesão à atenção primária à saúde e a busca ativa dos contatos ainda é ineficiente.

Abstract Background For the control of tuberculosis, it is essential to interrupt its chain of transmission. The Ministry of Health recommends 100% of contacts being examined and initiated treatment of the Latent Mycobacterium tuberculosis infection. In this sense, the knowledge about the disease and adherence to prophylaxis by these contacts are factors that can interfere in the adequate control of tuberculosis. Objective To describe the knowledge of the contacts of tuberculosis patients on the disease and their adherence to prophylactic measures in the Sanitary District II in Recife / PE. Method A quantitative and descriptive study was carried out using standardized questionnaires, applied to 140 contacts of tuberculosis notified from January to December 2015. Data analysis carried out through simple frequencies. Results Among the interviewees, 75.7% were female, 55% brown, with low levels of schooling and family income; of these 84.3% believe that tuberculosis is serious, 48.6% consider that transmission is done by sharing utensils. Only 55% were invited to be examined and 76% reported not knowing they should go to the consultation or the importance of these. Conclusion The contacts of tuberculosis have poor knowledge about the disease, low adherence to primary health care and the active search for contacts is still inefficient.

Rapid detection of Mycobacterium tuberculosis in children using blood and urine specimens

Abstract INTRODUCTION: Laboratory and clinical features of childhood tuberculosis (TB) are non-specific and establishing an accurate diagnosis remains a challenge. This study evaluated a Single tube nested-PCR (STNPCR) to detect genomic DNA of Mycobacterium tuberculosis complex in blood and urine. METHODS: Biological samples were obtained from children (<15 years old) with clinical suspicion of pulmonary and extrapulmonary TB at public hospitals in Recife-Pernambuco, Brazil. Cultures yielded negative results in a majority of childhood TB cases, which are generally paucibacillary. A set of clinical, epidemiological, radiological, and laboratory criteria with evident clinical improvement after anti-TB treatment were frequently used to define childhood TB cases. RESULTS: Ninety children with clinical suspicion were enrolled in this study (44 with TB and 46 without TB). The pulmonary TB group had 20 confirmed cases and 46 negative controls, while the extrapulmonary TB group had 24 confirmed cases. The STNPCR showed sensitivities to pulmonary and extrapulmonary TB of 47.4% and 52.2% (blood) and 38.8% and 20% (urine), respectively. Considering the low performance of STNPCR on separate samples, we decided to perform a combined analysis (parallel sensitivity analysis) of the results from blood and urine samples. The parallel sensitivity increased to 65% in blood and 62.5% in urine. The specificity in both samples ranged from 93.5-97.8%. CONCLUSIONS: Although STNPCR showed moderate sensitivity, the specificity is high; therefore, the test can be used as an auxiliary tool to diagnose TB in children. It is a rapid test that demonstrated better performance than other diagnostic tests in paucibacillary samples as it does in childhood tuberculosis.

Identification of nontuberculous mycobacteria species by multiplex real-time PCR with high-resolution melting

Abstract INTRODUCTION: Nontuberculous mycobacteria (NTM) species, as human pathogens, are increasing in the world, as is the difficulty of accurately identifying them. Differential diagnosis, especially between the M. tuberculosis complex and NTM species, and the characterization of NTM species is important. This study aimed to evaluate the performance of a molecular system based on multiplex real-time PCR with high-resolution melting (HRM) for the identification and differentiation of NTM species of clinical importance of an endemic area for tuberculosis in northeastern Brazil. METHODS: The technical protocol of the molecular system was based on multiplex real-time PCR-HRM, and evaluated the sensitivity and specificity of the detection of NTM species in mycobacterial clinical isolates from the studied region. The gold standard method was specific gene sequencing. RESULTS: The sensitivity and specificity of multiplex real-time PCR-HRM modified for differentiation between NTM and M. tuberculosis were 90% and 100%, respectively. The PCR-HRM sensitivities for the characterization of NTM species (M. kansasii, M. abscesses, M. avium, and M. fortuitum) were 94.59%, 80%, 57.14%, and 54%, respectively. CONCLUSIONS The multiplex real-time PCR-HRM modified assay has the potential to rapidly and efficiently identify nontuberculous mycobacteria of clinical importance, which is crucial for immediate implementation of the appropriate therapy and thus avoiding complications and sequelae in patients.

Performance of the IS6110-TaqMan® assay in the diagnosis of extrapulmonary tuberculosis from different biological samples.

INTRODUCTION: This study evaluated the performance of the IS6110-TaqMan® assay in different types of biological samples and tissues for laboratory diagnosis of extrapulmonary tuberculosis. METHODS: 143 biological samples and tissues from patients with suspected extrapulmonary tuberculosis from the health services of Recife/Pernambuco/Brazil were evaluated with the IS6110-TaqMan® assay. RESULTS: The sensitivities of the IS6110-TaqMan® assay calculated for blood, urine, both blood and urine samples, tissue biopsies, extrapulmonary body fluid samples, and all samples from patients calculated together were 55.9%, 33.3%, 68.8%, 43.8%, 29.6%, and 73.7%, respectively, and the specificities were 80%, 100%, 78.6%, 100%, 100%, and 84.2%, respectively. CONCLUSIONS: The accuracy of qPCR was high in various clinical sample types. The analysis of more than one type of clinical sample collected from the same patient with extrapulmonary tuberculosis enhances the diagnostic power of the IS6110-TaqMan® assay when compared with the use of only one clinical sample.

Performance of the IS6110-TaqMan® assay in the diagnosis of extrapulmonary tuberculosis from different biological samples

Abstract INTRODUCTION: This study evaluated the performance of the IS6110-TaqMan® assay in different types of biological samples and tissues for laboratory diagnosis of extrapulmonary tuberculosis. METHODS: 143 biological samples and tissues from patients with suspected extrapulmonary tuberculosis from the health services of Recife/Pernambuco/Brazil were evaluated with the IS6110-TaqMan® assay. RESULTS: The sensitivities of the IS6110-TaqMan® assay calculated for blood, urine, both blood and urine samples, tissue biopsies, extrapulmonary body fluid samples, and all samples from patients calculated together were 55.9%, 33.3%, 68.8%, 43.8%, 29.6%, and 73.7%, respectively, and the specificities were 80%, 100%, 78.6%, 100%, 100%, and 84.2%, respectively. CONCLUSIONS The accuracy of qPCR was high in various clinical sample types. The analysis of more than one type of clinical sample collected from the same patient with extrapulmonary tuberculosis enhances the diagnostic power of the IS6110-TaqMan® assay when compared with the use of only one clinical sample.

DEFB1 gene polymorphisms and tuberculosis in a Northeastern Brazilian population

Abstract β-Defensin-1, an antimicrobial peptide encoded by the DEFB1 gene, is known to play an important role in lung mucosal immunity. In our association study we analyzed three DEFB1 functional polymorphisms -52G>A (rs1799946), -44C>G (rs1800972) and -20G>A (rs11362) in 92 tuberculosis patients and 286 healthy controls, both from Northeast Brazil: no association was found between the studied DEFB1 polymorphisms and the disease. However we cannot exclude that this lack of association could be due to the low number of subjects analyzed, as suggested by the low statistical power achieved for the three analyzed SNPs (values between 0.16 and 0.50).

DEFB1 gene polymorphisms and tuberculosis in a Northeastern Brazilian population.

ß-Defensin-1, an antimicrobial peptide encoded by the DEFB1 gene, is known to play an important role in lung mucosal immunity. In our association study we analyzed three DEFB1 functional polymorphisms -52G>A (rs1799946), -44C>G (rs1800972) and -20G>A (rs11362) in 92 tuberculosis patients and 286 healthy controls, both from Northeast Brazil: no association was found between the studied DEFB1 polymorphisms and the disease. However we cannot exclude that this lack of association could be due to the low number of subjects analyzed, as suggested by the low statistical power achieved for the three analyzed SNPs (values between 0.16 and 0.50).

Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine.

INTRODUCTION: Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.