Fast detection of Mycobacterium tuberculosis in culture-positive sputum samples by nitrate reductase activity

Microscopy and bacterial culture are the main tools in the diagnosis of tuberculosis. Since the slow growth of Mycobacterium tuberculosis impairs rapid diagnosis strategies, especially in countries where the latter are the only available resources, the ongoing development of new and inexpensive tools based on mycobacterial metabolism optimizing growth detection with preliminary identification is greatly welcome. When compared to the other species from the M. tuberculosis complex, M. tuberculosis is a strong nitrate reducer. Current assay compares the nitrate reductase activity of M. tuberculosis from pulmonary specimens cultivated in nitrate-supplemented media. Fifty-five sputum samples were decontaminated and inoculated in conventional (Middlebrook 7H9, Ogawa Kudoh-OK) and in nitrate-supplemented media (Middlebrook 7H9-N, Ogawa Kudoh-N). An aliquot from the media directly reacted with Griess reagent (7H9-N and OK-N) every five days, or transferred to a nitrate substrate solution (7H9, OK). Nitrate to nitrite reduction was considered positive, revealed by the pink color, indicating bacterial growth. As reference method, the Mycobacteria Growth Indicator Tube (MGIT) was used for sensitivity calculations and statistical analysis. 7H9-N and OK-N assays proved to perform better in detecting M. tuberculosis than conventional assays (7H9 and OK). Indeed, broth nitrate-supplemented medium (7H9-N) was comparable to MGIT to detect M. tuberculosis, except in growth detection time. Results show that 7H9-N may be used as an alternative tool particularly in low-income countries since it is a simple and cheap technique, and does not restrict diagnosis to single-source products

Critical analysis: use of polymerase chain reaction to diagnose leprosy

Leprosy is a neglected tropical disease and an important public health problem, especially in developing countries. It is a chronic infectious disease that is caused by Mycobacterium leprae, which has a predilection for the skin and peripheral nerves. Although it has low sensitivity, slit-skin smear (SSS) remains the conventional auxiliary laboratory technique for the clinical diagnosis of leprosy. Polymerase chain reaction (PCR) is a molecular biology technique that holds promise as a simple and sensitive diagnostic tool. In the present study, the performance of two PCR methods, using different targets, PCR-LP and PCR-P, were compared with SSS with regard to leprosy diagnosis in a reference laboratory. M. leprae DNA was extracted from 106 lymph samples of 40 patients who had clinical suspicion of leprosy. The samples were subjected to both PCR techniques and SSS. Amplification of the human b-globin gene was used as PCR inhibitor control. The specificity of both PCR techniques was 100%, and sensitivity was 0.007 and 0.015 µg/ml for PCR-LP and PCR-P, respectively. No significant difference was found between either the PCR-LP or PCR-P results and SSS results (p >; 0.05). Although PCR is not yet a replacement for SSS in the diagnosis of leprosy, this technique may be used as an efficient auxiliary tool for early detection of the disease, especially in endemic regions. This strategy may also be useful in cases in which SSS results are negative (e.g., in paucibacillary patients) and cases in which skin biopsy cannot be performed.A hanseníase é uma doença tropical negligenciada e ainda um importante problema de saúde pública, especialmente nos países em desenvolvimento. É uma doença infecciosa crônica causada pelo Mycobacterium leprae, que tem predileção pela pele e nervos periféricos. Embora com baixa sensibilidade, o esfregaço de linfa (SSS) continua sendo o método laboratorial convencional auxiliar no diagnóstico clínico da hanseníase. A biologia molecular representada pela Reação em Cadeia da Polimerase (PCR) trouxe a expectativa de ser uma ferramenta diagnóstica simples e sensível. No presente estudo, o desempenho de dois métodos de PCR usando alvos diferentes, PCR-P e PCR-LP, foi comparado com SSS no diagnóstico da hanseníase em um laboratório de referência. DNA de M. leprae foi extraído de 106 amostras de linfa de 40 pacientes que apresentavam suspeita clínica de hanseníase. As amostras foram submetidas tanto a PCR como SSS. A amplificação do gene humano β-globina foi usada como controle de inibição da PCR. A especificidade de ambas as técnicas de PCR foi de 100% e a sensibilidade foi de 0,007 μg/mL e 0,015 μg/mL para a PCR-P e PCR-LP, respectivamente. Não se observou diferença estatística entre os resultados da PCR-LP e PCR-P, quando comparado com SSS (p >; 0,05). Apesar de a PCR ainda não substituir o SSS no diagnóstico da hanseníase, esta técnica pode ser usada como ferramenta auxiliar eficiente para a detecção precoce da doença, especialmente em regiões endêmicas. Esta estratégia pode também ser útil nos casos em que os resultados de SSS forem negativos (ex. em pacientes paucibacilares) e em casos onde a biópsia da pele não pode ser realizada

Is the efflux pump inhibitor Verapamil a potential booster for isoniazid against Mycobacterium tuberculosis?

The membrane-based efflux pump systems are recognized to have an important role in pathogenicity and drug resistance in Mycobacterium tuberculosis by the extrusion of toxic substrates and drugs from the inner bacillus. This study aimed to investigate the in vitro interaction of Verapamil (VP), an efflux pump inhibitor, with the classical first-line anti-tuberculosis drug isoniazid (INH) in resistant and susceptible M. tuberculosis clinical isolates. Seven multidrug-resistant (MDR), three INH monoresistant and four susceptible M. tuberculosis clinical isolates were tested for the INH and VP combination by modified Resazurin Microtiter Assay Plate (REMA). Fractional Inhibitory Concentration (FIC) and Modulation Factor (MF) were determined. The INH plus VP combination showed no significant change in the Minimum inhibitory concentration (MIC) values of INH (FIC≥ 0.5; MF=1 or 2).The use of VP in tuberculosis therapy should be managed carefully, considering the resistance caused by specific mutation in katG and inhA genes, in which the use of these EPIs may have no success. The use of EPIs as an adjunctive drug in the anti-tuberculosis therapy should be further investigated on a larger number of M. tuberculosis clinical isolates with different resistant profile

DETECTION OF Leishmania (Viannia) IN Nyssomyia neivai AND Nyssomyia whitmani BY MULTIPLEX POLYMERASE CHAIN REACTION, IN SOUTHERN BRAZIL

Flebotomíneos transmitem os patógenos das leishmanioses. Foi avaliada a infecção natural de flebotomíneos por Leishmania (Viannia) em municípios do Estado do Paraná, sul do Brasil. Os flebotomíneos foram coletados com armadilhas de Falcão e Shannon. Após dissecação para pesquisa de flagelados no tubo digestório e identificação das espécies, as fêmeas de flebotomíneos foram submetidas a Multiplex Reação em Cadeia da Polimerase (multiplex PCR) para a detecção do fragmento do kDNA de Leishmania (Viannia) e do fragmento do gene IVS6 da cacofonia de flebotomíneos. A análise foi realizada em pools contendo sete a 12 tubos digestórios de fêmeas da mesma espécie. Um total de 510 fêmeas foram analisadas, incluindo nove Migonemyia migonei, 17 Pintomyia fischeri, 216 Nyssomyia neivai e 268 Nyssomyia whitmani. Embora nenhuma fêmea tenha sido encontrada naturalmente infectada com flagelados pela dissecação, o fragmento de DNA de Leishmania (Viannia) foi mostrado por multiplex PCR em uma amostra de Ny. neivai (0,46%) e três amostras de Ny. whitmani (1,12%). Conclui-se que Ny. neivai e Ny. whitmani são suscetíveis à infecção por Leishmania, e que multiplex PCR, devido à sua sensibilidade, especificidade e viabilidade, pode ser utilizada em estudos epidemiológicos para a detecção da infecção natural do inseto vetor.Sandflies transmit pathogens of leishmaniasis. The natural infection of sandflies by Leishmania (Viannia) was assessed in municipalities, in the state of Paraná, in Southern Brazil. Sandflies were collected with Falcão and Shannon traps. After dissection in search of flagellates in digestive tubes and identification of the species, female sandflies were submitted to the Multiplex Polymerase Chain Reaction (multiplex PCR) for detection of the fragment of the kDNA of Leishmania (Viannia) and the fragment from the IVS6 cacophony gene region of the phlebotomine insects. The analysis was performed in pools containing seven to 12 guts from females of the same species. A total of 510 female sandflies were analyzed, including nine Migonemyia migonei, 17 Pintomyia fischeri, 216 Nyssomyia neivai, and 268 Nyssomyia whitmani. Although none of the females was found naturally infected by flagellates through dissection, the fragment of DNA from Leishmania (Viannia) was shown by multiplex PCR in one sample of Ny. neivai (0.46%) and three samples of Ny. whitmani (1.12%). It was concluded that Ny. neivai and Ny. whitmani are susceptible to Leishmania infection, and that multiplex PCR can be used in epidemiological studies to detect the natural infection of the sandfly vector, because of its sensitivity, specificity and feasibility

Pesquisa de infecção natural de flebotomíneos por Leishmania, no Estado do Paraná Research of natural infection of phlebotomines for Leishmania, in the State of Paraná

A leishmaniose tegumentar americana tem sido notificada em todos os estados do Brasil e no Paraná essa doença é endêmica. O objetivo deste trabalho foi detectar a infecção natural de flebotomíneos para verificar a competência vetorial destes insetos e a identificação da espécie parasitária. Os flebotomíneos foram coletados com armadilhas de Falcão e Shannon, nos municípios de Doutor Camargo, Fênix e Mandaguari, de novembro de 2005 a agosto de 2006. Coletaram-se 12.930 flebotomíneos, dos quais 2.487 fêmeas foram dissecadas e destes 1.230 fêmeas foram submetidas à reação em cadeia da polimerase. Pelo método da dissecação, foi detectada uma fêmea de Nyssomyia whitmani com infecção natural por flagelados e pela reação em cadeia da polimerase não se detectou a presença de DNA de Leishmania em nenhuma das fêmeas. Apesar de não ter sido detectada a infecção natural de Nyssomyia neivai nas localidades em apreço e ainda que os requisitos de incriminação vetorial não tenham sido atendidos, não se deve negligenciar o potencial vetorial desta espécie.American cutaneous leishmaniasis has been reported in all Brazilian states and in the Paraná this disease is endemic. The objective of this work was to detect natural infections in phlebotomines to verify the vector competence of these insects and the identification of the parasite species. Phlebotomines were collected using Falcão and Shannon traps, in the municipalities of Doutor Camargo, Fênix and Mandaguari, between November 2005 and August 2006. from 12,930 phlebotomines were collected, 2,487 females were dissected and 1,230 dissected females had been submitted to polymerase chain reaction. Flagellates were detected in a female Nyssomyia whitmani that had been dissected and for polymerase chain reaction failed to detect Leishmania DNA in any females. Even though flagellates were not detected in Nyssomyia neivai it should still be considered as a potencial vector

Influence of the identification of contacts on the adherence of index tuberculosis cases to treatment in a high incidence country

Background: Health professionals must interview index tuberculosis (TB) cases to identify and examine their contacts, because human interaction favors disease transmission. Revealing their contacts implies the disclosure of their health condition to close friends and family. The aim of this study was to evaluate the influence of the identification of contacts of TB index cases on the outcomes of TB treatment. Methods: This observational, cross-sectional, epidemiological study was conducted using data provided by SINAN-Net on subjects diagnosed with TB between 2008 and 2012 in Paraná, Brazil. The inclusion criteria were new cases of pulmonary TB in individuals older than 15 years. Results: A total of 9867 new cases of TB were identified. In total, 29% of adult cases did not have their contacts examined, and of these, 61.8% were smear-positive. The adults whose contacts were not examined underwent fewer tests and presented a lower cure rate and higher rates of treatment dropout and death. Conclusions: The detection of the contacts of index cases constitutes an epidemiological and public health strategy for the surveillance and control of TB. The health professionals who promote patient adherence to treatment and the involvement of their families in the fight against TB achieve better results regarding the identification of contacts of index cases, adherence to treatment, and cure. Keywords: Tuberculosis, Contact tracing, Infectious diseases, Epidemiological surveillanc