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.

ABSTRACT 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.

Tuberculosis (TB) is an infectious disease in which the molecular typing methods allow to have important information about the dynamics of transmission and to assist properly in disease control. Although the ERIC-PCR (Enterobacterial repetitive intergenic consensus-PCR) assay is fast and easy to perform, scarce studies have reported its use in epidemiological studies in TB outbreaks. In this study, we aimed to genotype Mycobacterium tuberculosis and M. bovis isolates by ERIC-PCR and compare its discriminatory power with two other classically used methods: 12 loci-MIRU (Mycobacterial Interspersed Repetitive Units) and Spoligotyping. The M. tuberculosis isolates studied were from northwestern and southwestern and M. bovis from northwestern Parana, Brazil. ERIC-PCR rendered banding patterns with great diversity (1 to 12 bands) of molecular sizes, ranging from 100 to 1600 bp. ERIC-PCR showed to be fast, simple and affordable to differentiate isolates. ERIC-PCR would be an important tool in the epidemiology of TB as screening in case of outbreak, which demands rapid intervention. However if any doubt persist, as it may occur with the application of only one genotypic method, other genotyping methods should be applied and carefully interpreted, always with additional epidemiological information.

ABSTRACT The present study evaluated the antimicrobial susceptibility profile, β-lactamase production, and genetic diversity of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter spp. using phenotypic identification, antimicrobial susceptibility testing, and β-lactamase phenotypic detection. Isolates were obtained from patients in an intensive care unit in a hospital in southern Brazil. Bacterial genomic DNA was extracted, followed by the genotypic detection of carbapenemases and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). Fifty-six isolates (26 Klebsiella pneumoniae, five Escherichia coli, three Enterobacter aerogenes, nine P. aeruginosa, and 13 Acinetobacter spp.) were evaluated. The phenotypic extended spectrum β-lactamase (ESBL) test was positive in 53.8% of the K. pneumoniae isolates, 100.0% of the E. coli isolates, and 100.0% of the E. aerogenes isolates. Phenotypic and genotypic testing of K. pneumoniae carbapenemase (KPC) was positive in 50.0% of the K. pneumoniae isolates. Phenotypic and genotypic testing showed that none of the P. aeruginosa or Acinetobacter spp. isolates were positive for metallo- β-lactamase (MBL). The bla OXA gene was detected only in Acinetobacter spp. The lowest genetic diversity, determined by ERIC-PCR, was observed among the KPC-producing K. pneumoniae isolates and OXA-producing Acinetobacter spp. isolates, indicating the inadequate dissemination control of multidrug-resistant bacteria in this hospital environment.

RESUMO 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.

ABSTRACT 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.

SUMMARY Introduction: Group B streptococcus (GBS) or Streptococcus agalactiae can colonize the gastrointestinal and genitourinary tracts and has been considered one of the most important risk factors for the development of neonatal disease. The present study evaluated the antimicrobial susceptibility of GBS isolates from pregnant women who were attended at a public health service in Northern Paraná, Brazil. Methods: A descriptive analytical cross-sectional study was performed with 544 pregnant women, at ≥ 35 weeks of gestation. One hundred and thirty-six GBS isolates from pregnant women were tested for antimicrobial susceptibility. Results: All of the GBS isolates showed susceptibility to the drug that is most frequently used for intrapartum prophylaxis: penicillin. Resistance to clindamycin and erythromycin was detected, thus decreasing the options of prophylaxis in women who are allergic to penicillin. Conclusions: Additional studies should be conducted to increase the knowledge of GBS sensitivity profile to antimicrobials in other health centers.

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.

We evaluated the in vitro anti-Mycobacterium tuberculosis activity and the cytotoxicity of dichloromethane extract and pure compounds from the leaves of Calophyllum brasiliense. Purification of the dichloromethane extract yielded the pure compounds (-) mammea A/BB (1), (-) mammea B/BB (2) and amentoflavone (3). The compound structures were elucidated on the basis of spectroscopic and spectrometric data. The contents of bioactive compounds in the extracts were quantified using high performance liquid chromatography coupled to an ultraviolet detector. The anti-M. tuberculosis activity of the extracts and the pure compounds was evaluated using a resazurin microtitre assay plate. The cytotoxicity assay was performed in J774G.8 macrophages using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colourimetric method. The quantification of the dichloromethane extract showed (1) and (2) at concentrations of 31.86 ± 2.6 and 8.24 ± 1.1 µg/mg of extract, respectively. The dichloromethane and aqueous extracts showed anti-M. tuberculosis H37Rv activity of 62.5 and 125 µg/mL, respectively. Coumarins (1) and (2) showed minimal inhibitory concentration ranges of 31.2 and 62.5 µg/mL against M. tuberculosis H37Rv and clinical isolates. Compound (3) showed no activity against M. tuberculosis H37Rv. The selectivity index ranged from 0.59-1.06. We report the activity of the extracts and coumarins from the leaves of C. brasiliense against M. tuberculosis.

A febre tifóide é doença bacteriana aguda causada por Salmonella enterica sorotipo typhi, que é adquirida pela ingestão de água ou alimento contaminado. O objetivo do presente trabalho é descrever um caso de febre tifóide ocorrido em Maringá, após três anos sem notificação da doença no Estado do Paraná.

Typhoid fever is an acute bacterial disease caused by Salmonella enterica serotype typhi, which is acquired by consumption of contaminated food or water. This paper had the aim of describing a case of typhoid fever that occurred in Maringá, State of Paraná, after three years without any notifications of the disease.

Procurou-se verificar a ocorrência de flebotomíneos naturalmente infectados por Leishmania. Estes insetos foram coletados com armadilhas luminosas de Falcão, Shannon e HP, nos municípios de Doutor Camargo e Maringá, de novembro de 2004 a outubro de 2005. Coletaram-se 11.033 flebotomíneos em Doutor Camargo e destes 2.133 fêmeas foram dissecadas, especialmente de Nyssomyia neivai (86,87%). Em Maringá coletaram-se 136 flebotomíneos, dos quais foram dissecadas 79 fêmeas de N. whitmani e 1 Migonemyia migonei. Parte das fêmeas dissecadas foi armazenada em pools de 10 espécimes. A PCR, utilizando os iniciadores MP1L/MP3H, foi realizada em 1.190 fêmeas de N. neivai e 190 de N. whitmani de Doutor Camargo e 30 N. whitmani de Maringá. Não se constatou a infecção natural por Leishmania pelos métodos utilizados. Estes resultados indicam que é baixa a taxa de infecção natural de flebotomíneos por Leishmania, corroborando outros trabalhos realizados em áreas endêmicas de LTA, inclusive no Paraná.

The purpose of this work was to verify the occurrence of Leishmania in naturally infected sandflies. The insects were collected with Falcão, Shannon and HP light-traps, in Doutor Camargo and Maringá municipalities between November 2004 and October 2005. Of the 11,033 sandflies collected in Doutor Camargo, 2,133 surviving females were dissected, particularly those of the Nyssomyia neivai species (86.87%). In Maringá, 136 sandflies were collected, of which 79 N. whitmani females and 1 Migonemyia migonei female were dissected. The dissected insects were identified and stored in the pools of 10 specimens. The PCR was carried out on 1,190 females of N. neivai and 190 of N. whitmani from Doutor Camargo, and on 30 of N. whitmani from Maringá, using the primers MP1L/MP3H. The natural infection by Leishmania in sandflies was not confirmed by either of the methods used. The results suggested the low natural infection rate of sandflies by Leishmania in these areas, corroborating other studies carried out in endemic areas of ACL.

Realizou-se coletas de flebotomíneos de maio de 2005 a abril de 2006. Os resultados foram comparados com os das coletas realizadas entre abril de 2001 e setembro de 2002, para avaliar as medidas empregadas para diminuir a densidade destes insetos, no Recanto Marista, município de Doutor Camargo, Estado do Paraná. As coletas foram feitas com armadilhas de Falcão, em domicílios e galinheiros, das 22 às 2 horas, semanalmente, quatro vezes ao mês. Em 2005 e 2006, coletaram-se 213.195 flebotomíneos, 1.113,8 em média por hora (MH) e em 2001 e 2002, 199.821 (MH=1.653,5). Nyssomyia neivai prevaleceu (75,4%) em todos os ecótopos que, juntamente com Nyssomyia whitmani (23,4%), Migonemyia migonei (0,8%) e Pintomyia fischeri (0,4%), representaram 99,7% do total coletado. Nos ecótopos representados por galinheiros foram coletados 88,7% dos flebotomíneos. Constatou-se que houve queda na densidade de flebotomíneos no período de coletas 2005 e 2006 em relação à de 2001 e 2002, especialmente no domicílio.

Collections of sandflies were made between May 2005 and April 2006. The results were compared with those from collections undertaken between April 2001 and September 2002, in order to evaluate the measures used to decrease the density of these insects in Recanto Marista, municipality of Doutor Camargo, State of Paraná. The collections were carried out by Falcão traps inside domiciles and hen sheds, from 10 p.m. to 2 a.m. once a week, four times a month. In 2005 and 2006, 213,195 sandflies were collected (average of 1,113.8 per hour), compared with 199,821 (average of 1,653.5 per hour) in 2001 and 2002. Nyssomyia neivai predominated (75.4%) in all the ecotopes. Nyssomyia neivai, Nyssomyia whitmani, Migonemyia migonei and Pintomyia fischeri accounted for 99.7% of all the sandflies collected. The ecotope of hencoops accounted for 88.7% of the sandflies collected. It was observed that the sandfly density had decreased between the 2001-2002 and 2005-2006 collections, especially in homes.

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.