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The state of Ceará, in the Northeast Region of Brazil, presents the simultaneous circulation of Zika (ZIKV), dengue (DENV) and chikungunya (CHIKV) viruses. In 2017 there were a high number of cases of these three arboviruses, especially CHIKV. Here, we detected the presence of arboviruses ZIKV, DENV and CHIKV and their coinfections in women in endemic regions of the city of Fortaleza, Ceará in a post-Zika epidemic year. Sociodemographic and environmental characteristics associated with arbovirus positivity were also analyzed. Women (n = 1289) between 15 and 39 years old were included. RT-qPCR was performed for virus detection and IgM antibody positivity was also analyzed. One hundred and six (8.3%) participants were positive for one or more arboviruses. Monoinfections (76; 5.9%) were distributed between 22 (1.7%) for ZIKV, 39 (3.1%) for DENV and 15 (1.2%) for CHIKV. Co-infections were detected in 30 (2.3%) of the positive participants and one case with triple infection was found. IgM positivity was found in 2.4% of ZIKV RT-qPCR, 9.6% of DENV and 16.3% of CHIKV. RT-qPCR positivity for arboviruses was associated with low socioeconomic class and presence of a water box sealing in the household. A higher positivity to the three viruses occurred in the month with the lowest wind velocity, which was also preceded by the highest peak of rain and humidity. We identified the simultaneous circulation and co-infection of ZIKV, DENV and CHIKV in Fortaleza in a post-Zika epidemic year. We also highlight the need for continuous epidemiological surveillance combined with molecular diagnostic tools.

Background Mycobacterium leprae was the first microorganism directly associated with a disease, however, there are still important gaps in our understanding of transmission. Although household contacts are prioritized, there is evidence of the importance of extrahousehold contacts. The goal of this article is to contribute to our understanding of the transmission of leprosy ex-household. Methods We compare co-location data of 397 leprosy cases and 211 controls drawn from the Centro de Dermatologia Sanitária D. Libânia in Fortaleza, Brazil. We collected lifetime geolocation data related to residence, school attendance and workplace and developed novel methods to establish a critical distance (Rc) for exposure and evaluated the potential for transmission for residence, school and workplace. Results Our methods provide different threshold values of distance for residence, school and workplace. Residence networks demonstrate an Rc of about 500 m. Cases cluster in workplaces as well. Schools do not cluster cases. Conclusions Our novel network approach offers a promising opportunity to explore leprosy transmission. Our networks confirm the importance of coresidence, provide a boundary and suggest a role for transmission in workplaces. Schools, on the other hand, do not demonstrate a clustering of cases. Our findings may have programmatic relevance.

Triatomine insects, native to northeastern Brazil, have been found in the urban peridomicile. The city of Sobral has a high number of tuberculosis cases and several triatomine species. This study investigates the presence of mycobacteria, particularly Mycobacterium tuberculosis complex (MTBC) species, in triatomines captured in the urban perimeter of Sobral.We analyzed 167 triatomines captured in urban households and peridomiciles of Sobral. Mycobacteria were identified by the PRA-hsp65 method followed by partial sequencing of the hsp65 and rpoB genes. The sequences confirmed as MTBC were also typed by mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) and spoligotyping.Triatoma brasiliensis (38.6%), Triatoma pseudomaculata (32.9%), Panstrongylus lutzi (24.3%) were the most frequently identified. In 51.1% (70/167) of them, species of the Mycobacteriaceae family were detected by PRA-hsp65; of these, 31.4% (22/70) were identified as belonging to MTBC species. Nine (12.9%) of the triatomine samples were confirmed by sequencing as belonging to MTBC species. MIRU-VNTR genotyping suggests that the presence of different MTBC sublines in the triatomines should be investigated.This is the first report of MTBC lineages in triatomine insects. These results indicate the migration and adaptation of these insects in an urban setting.

Tuberculosis (TB) affects around 10 million people worldwide in 2019. Approximately 3.4 % of new TB cases are multidrug-resistant. The gold standard method for detecting Mycobacterium tuberculosis , which is the aetiological agent of TB, is still based on microbiological culture procedures, followed by species identification and drug sensitivity testing. Sputum is the most commonly obtained clinical specimen from patients with pulmonary TB. Although smear microscopy is a low-cost and widely used method, its sensitivity is 50–60 %. Thus, owing to the need to improve the performance of current microbiological tests to provide prompt treatment, different methods with varied sensitivity and specificity for TB diagnosis have been developed. Here we discuss the existing methods developed over the past 20 years, including their strengths and weaknesses. In-house and commercial methods have been shown to be promising to achieve rapid diagnosis. Combining methods for mycobacterial detection systems demonstrates a correlation of 100 %. Other assays are useful for the simultaneous detection of M. tuberculosis species and drug-related mutations. Novel approaches have also been employed to rapidly identify and quantify total mycobacteria RNA, including assessments of global gene expression measured in whole blood to identify the risk of TB. Spoligotyping, mass spectrometry and next-generation sequencing are also promising technologies; however, their cost needs to be reduced so that low- and middle-income countries can access them. Because of the large impact of M. tuberculosis infection on public health, the development of new methods in the context of well-designed and -controlled clinical trials might contribute to the improvement of TB infection control.

The dried blood spot (DBS) samples are a useful resource for viral DNA isolation and important in increasing access to HBV diagnosis. However, the choice of the DNA extraction method is crucial for reliable results. We compared the reliability of four DNA extraction methods using DBS samples for the qualitative and quantitative detection of HBV. A panel of serially diluted HBV DNA in whole blood was spotted onto filter paper (Whatman 903 paper and Whatman FTA cards). Four methods were used to extract DNA: QIAamp® DNA Blood Mini Kit (Qiagen); High Pure Viral Nucleic Acid Kit (Roche); Invisorb Spin Blood Midi Kit (Invitek), and DBS Genomic DNA Isolation Kit (Norgen Biotek). Two qualitative PCRs for the core and surface gene regions of HBV were used, and in‐house real‐time PCR was also evaluated. It was possible to detect HBV DNA using all extraction and PCR protocols. The lowest limit of detection was found using Whatman 903 paper, Roche extraction, and qualitative PCR (20 copies of HBV DNA per ml) for the surface/polymerase HBV region. In the case of in‐house real‐time PCR, the lowest limit of detection was found using both Roche and Qiagen assays (estimated 2 × 103 copies per ml). These results suggest the importance of both the extraction method and PCR protocol in detecting HBV DNA in DBS. This study provides insights into the utility of DBS samples in HBV molecular diagnosis and their feasibility in low resource areas where cold storage and transportation may be difficult., Dried blood spots (DBS) could be used for hepatitis B virus (HBV) DNA detection with qualitative PCR for the surface/polymerase region. Here, using in‐house real‐time PCR, the lowest limit of detection was 2000 copies/ml. DBS testing might be feasible in low resource areas.

CD44 and CD133 have been considered as cancer stem cell (CSC) markers. Stem cell markers are rarely described in healthy stomach tissues. However, the clinicopathological and prognostic value of CD44 and CD133 in gastric cancer remains controversial. This study investigated the expression of CD44 and CD133 in gastric cancer and non-neoplastic gastric mucosa. We used samples of primary gastric adenocarcinomas (n = 69), metastatic lymph nodes (n = 30), intestinal metaplasia (n = 17), and histologically normal gastric tissues of surgical margins (n = 54). The expression of CD44 and CD133 were studied in samples by immunohistochemistry. Fisher’s exact test and a logistic regression model were used in this study. CD44 expression was observed in 12% of samples with intestinal metaplasia, 20% with lymph node metastases, 22% with normal mucosa, to 30% of samples with primary tumors. Most of these positive tumors showed immunostaining in less than 4% of cancerous cells, mainly in the diffuse type. CD133 expression was observed in 7% (intestinal metaplasia) to 46% (normal mucosa). In the positive cases of cancer (24%), in most of them, less than 3% of cells were marked. CD44 and CD133 expression in the histologically normal gastric mucosa was restricted to the deeper regions of the gastric crypts at the level where stem cells and progenitor cells are usually found. CD44 and CD133 expression occurs in few gastric cancer cells, mainly in diffuse carcinomas, and are expressed in histologically normal gastric mucosae. None of the markers are specific for cancer and are also present in intestinal metaplasia and the normal mucosa.

Background Outbreaks of Chikungunya virus (CHIKV) occurred in Brazil during 2015–2017. Fortaleza was the city that reported the most cases. Methods The first round of a cohort study was conducted among women aged 15–39 y in Fortaleza, Brazil, in 2018 (Zika in Fortaleza). We collected sera to detect CHIKV IgG and IgM antibodies. Factors for CHIKV infection were identified using a Poisson regression model. Results We evaluated 1466 serum samples and 13.8% and 37.2% of women were found positive for CHIKV IgM and IgG antibodies, respectively. Living with more than four others in the same house and having an abandoned house nearby were associated with CHIKV infection. Being currently pregnant was associated with a decreased probability of CHIKV infection, which was also associated with pregnant women reporting using more repellent, both inside and outside the house, than non-pregnant women. Conclusions Crowding in households and abandoned houses nearby can increase potential transmission. Policies providing better living conditions and regulation of abandoned sites and buildings are necessary to control the mosquito population. Programmes providing repellant at low or no cost to pregnant women should be implemented in the neighbourhoods where arbovirus infections are endemic.

Federal University of Cear?. School of Medicine. Department of Pathology and Legal Medicine. Fortaleza, CE, Brazil. Federal University of Cear?. School of Medicine. Department of Pathology and Legal Medicine. Fortaleza, CE, Brazil. Federal University of Cear?. School of Medicine. Department of Pathology and Legal Medicine. Fortaleza, CE, Brazil. Cear? State University. Center for Health Sciences. Fortaleza,CE, Brazil. Federal University of Cear?. School of Medicine. Department of Pathology and Legal Medicine. Fortaleza, CE, Brazil. Dona Libania National Reference Center in Sanitary Dermatology. Fortaleza, CE, Brazil Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Federal University of Cear?. School of Medicine. Department of Pathology and Legal Medicine. Fortaleza, CE, Brazil. Objectives: To investigate the subclinical infection of leprosy in contacts younger than 15 years old of new leprosy cases diagnosed at the Dona Libania Dermatology Center in Fortaleza, Ceara. Methods: This was a cross-sectional study involving 69 leprosy cases and 101 household contacts under 15 years old. Nasal secretion samples were collected in order to investigate the Bacilloscopic Index (BI) for M. leprae and to detect the DNA of the bacilli; we also investigated the seropositivity of the IgM antibody to the PGL1 antigen through the ML-Flow technique. Results: Evaluating the positivity of all three techniques used in the study, we obtained a frequency of 33.7% for ML-Flow, 16.1% for PCR of DNA and 1.98% for BI in nasal secretion. The positivity for RLEP PCR and in the anti-PGL-1 indicates stimulation of the immune response and consequent subclinical infection. In our study, all positive BI cases were also RLEP PCR positive, which confirms the presence of the bacillus in the nasal mucosa of the studied contacts. The seropositivity to anti-PGL-1 was higher among the female sex, the ones between 11 and 15 years old and among the contacts of paucibacillary (PB) cases. Conclusions: Such techniques when used together help the monitoring of contacts with greater risk of developing the disease, especially in childhood.

The essential oil (EO) extracted from the bark of Cinnamomum zeylanicum (Czey; also known as cinnamon), mostly derives its properties from its major compound trans-cinnamaldehyde (TCin). The present study evaluated the antimycobacterial activity of the essential oil from Czey (CzeyEO) and TCin against sensitive and resistant clinical isolates of Mycobacterium tuberculosis, as well as the combinatorial effects of CzeyEO and TCin with the anti-tuberculosis (TB) drugs rifampicin (RIF) and isoniazid (INH). The resazurin microtiter assay method was used to determine the minimum inhibitory concentration (MIC) of the components tested on the clinical isolates of M. tuberculosis. The effects of the CzeyEO/RIF, CzeyEO/INH, TCin/RIF, and TCin/INH combinations on the M. tuberculosis H37Rv reference strain were evaluated using the checkerboard method to determine the fractional inhibitory concentration index (FICI). CzeyEO and TCin inhibited all bacterial clinical isolates. In the interactive experiment, CzeyEO and TCin were found to be highly effective in reducing the resistance of resistant M. tuberculosis to RIF and INH. All four tested combinations demonstrated synergistic and additive effects, with no antagonistic effects. The synergistic combinations of CzeyEO/RIF and CzeyEO/INH exhibited FICI values of 0.375 and 0.5, respectively, while the TCin/RIF and TCin/INH combinations exhibited FICI values of 0.31 and 0.5, respectively. These results indicate that CzeyEO and TCin are potential candidates for the treatment of drug-resistant tuberculosis in combination therapy with INH and RIF. O óleo essencial (EO) extraído da casca do Cinnamomum zeylanicum (CzeyEO), conhecido como canela, tem como seu principal composto o trans-cinamaldeído (TCin). O presente estudo avaliou a atividade antimicobacteriana de CzeyEO e do TCin contra isolados clínicos sensíveis e resistentes de Mycobacterium tuberculosis, bem como os efeitos das associações de CzeyEO e do TCin com os fármacos anti-TB, rifampicina (RIF) e isoniazida (INH). A técnica de ensaio de microtitulação da resazurina foi utilizada para determinar a concentração inibitória mínima (CIM) dos componentes testados nos isolados clínicos de M. tuberculosis. Os efeitos das associações CzeyEO/RIF, CzeyEO/INH, TCin/RIF e TCin/INH contra a cepa de referência H37Rv de M. tuberculosis foram avaliados pelo método Checkerboard, determinando o índice de concentração inibitória fracionária (ICIF). Todos os isolados clínicos bacterianos foram inibidos por CzeyEO e TCin. As interações de CzeyEO e TCin foram altamente eficazes na redução da resistência do M. tuberculosis resistente a RIF e INH. Todas as quatro combinações testadas resultaram em efeitos sinérgicos e aditivos, sem efeito antagônico. Ambas as associações de sinergismo de CzeyEO/RIF e CzeyEO/INH mostraram valores de ICIF de 0,375 e 0,5, enquanto as associações de TCin/RIF e TCin/INH apresentaram valores de ICIF de 0,31 e 0,5. CzeyEO e TCin são potenciais candidatos em terapia combinada com INH e RIF para o tratamento da tuberculose resistente.

Background: This study compares the strains of genotypes of M. leprae from nasal secretions (NS) and skin biopsy (SB) in the same patient, supplementing conventional epidemiology to gain insight into the infection of leprosy in Fortaleza, Brazil. Methods: The sample consisted of 38 newly diagnosed leprosy patients attending the National Reference Center of Dermatology Dona Libania (CDERM), in Fortaleza, who tested positive for M. leprae by PCR in DNA extracts of nasal secretions. DNA was also extracted from skin biopsy (SB) scrapings of each patient and used for multiplex PCR amplification of M. leprae VNTR loci. The number of repeats at 15 loci were determined by the fragment length analysis method. Results: locus VNTR genotypes were achieved in 38 NS, and in 38 SB specimens. M. leprae strains differed in their genotypes in paired specimens in all but two of 38 patients. The genotype similarity in the remainder ranged from 53% to 87%. Conclusion: M. leprae 15 VNTR loci genotypes of paired nasal and biopsy skin samples from five patients were identical, while as many as seven loci differed in the 33 other patients. When the NS and biopsy genotypes were pooled and compared, it was found that there was a great variability among different VNTR markers. It is important to investigate other molecular markers suitable for typing genetic variations of the bacilli.

Universidade Federal Cear?. Faculdade de Medicina. Departamento de Cirurgia e Maternidade Escola Assis Chateaubriand. Fortaleza, CE, Brasil. Universidade Federal Cear?. Faculdade de Medicina. Departamento de Cirurgia e Maternidade Escola Assis Chateaubriand. Fortaleza, CE, Brasil. Universidade Federal Cear?. Faculdade de Medicina. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brasil. Universidade Federal Cear?. Faculdade de Medicina. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brasil. Universidade Federal Cear?. Faculdade de Medicina. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brasil. Universidade Federal Cear?. Faculdade de Medicina. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brasil. Universidade Federal Cear?. Faculdade de Medicina. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Universidade Federal Cear?. Faculdade de Medicina. Fortaleza, CE, Brasil. Universidade Federal Cear?. Faculdade de Medicina. Fortaleza, CE, Brasil. Universidade Federal Cear?. Faculdade de Medicina. Fortaleza, CE, Brasil. Universidade Federal Cear?. Faculdade de Medicina. Fortaleza, CE, Brasil. Universidade Federal Cear?. Faculdade de Medicina. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brasil. OBJECTIVE: The aim of this study is to investigate the presence of human papillomavirus DNA and genotypes in breast cancer and normal breast tissue samples obtained from women from the northeast region of Brazil. METHOD: One hundred three breast cancer samples and 95 normal breast samples, as the non-malignant controls, were studied. DNA extraction was verified by human beta-globin gene amplification, and polymerase chain reaction was conducted based on HPV L1-specific consensus primers MY09/MY11 and GP5+/GP6+, followed by nested multiplex polymerase chain reaction with type-specific primers for the E6/E7 consensus region. RESULTS: Human papillomavirus DNA was detected in 51 (49.5%) breast carcinoma samples and 15 (15.8%) normal breast samples (p

This work was funded by Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES), Institut d'Estudis Catalans (IEC) and the Swiss National Science Foundation (grants 310030_166687 and IZRJZ3_164171). Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Go?s. Rio de Janeiro, RJ, Brazil. Universidade do Estado do Par?. Programa de P?s-gradua??o em Biologia Parasit?ria na Amaz?nia. Bel?m, PA, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Universidade do Estado do Par?. Programa de P?s-gradua??o em Biologia Parasit?ria na Amaz?nia. Bel?m, PA, Brazil. Universidade do Estado do Par?. Programa de P?s-gradua??o em Biologia Parasit?ria na Amaz?nia. Bel?m, PA, Brazil. Universidade do Estado do Par?. Programa de P?s-gradua??o em Biologia Parasit?ria na Amaz?nia. Bel?m, PA, Brazil. Universidade Federal do Par?. Hospital Universit?rio Jo?o de Barros Barreto. Bel?m, PA, Brazil. Universidade Federal do Par?. Hospital Universit?rio Jo?o de Barros Barreto. Bel?m, PA, Brazil. University of Delhi. Delhi College of Engineering. New Delhi, India. Universidade do Estado do Par?. Programa de P?s-gradua??o em Biologia Parasit?ria na Amaz?nia. Bel?m, PA, Brazil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Biologia Molecular Aplicada as Micobact?rias, Rio de Janeiro, RJ, Brazil. Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Go?s. Rio de Janeiro, RJ, Brazil. Universidade Federal do Cear?. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brazil. Swiss Tropical & Public Health Institute. Basel, Switzerland / University of Basel. Basel, Switzerland. Swiss Tropical & Public Health Institute. Basel, Switzerland / University of Basel. Basel, Switzerland. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Biologia Molecular Aplicada as Micobact?rias, Rio de Janeiro, RJ, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Tuberculosis (TB) is an infectious disease with a higher risk for infection and disease among household contacts (HHC). Here, we report a molecular epidemiology-based approach to study disease transmission and the genetic characteristics of Mycobacterium tuberculosis (Mtb) strains among HHC in the city of Belem, the capital of the state of Para in north Brazil. The study included 63 TB patients belonging to 26 HHC groups (HHC1 to HHC26). Spoligotyping and 24-loci Mycobacterial Interspersed Repetitive Unit - Variable Number of Tandem Repeat (MIRU-VNTR) revealed indistinguishable bacterial genotypes among 26 patients in 14 (53.8%) HHC groups. Drug susceptibility testing (DST) revealed that 45 (71.4%) of the Mtb isolates were multidrug resistant. The major cluster composed of isolates from five HHCs and on three of these, whole genome sequencing (WGS) was performed confirming their high genetic similarity. These results pinpoint the need for improved vigilance for TB control in households in the city of Bel?m. When comparing WGS versus phenotypic resistance detection methods as DST and Minimum Inhibitory Concentration (MIC) our data suggest that depending on the colonies selection, results may present variation.

Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico, Brazil under [grant number CNPq?410573/2006-0]. Federal University of Cear?. Faculdade de Medicina. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brazil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Federal University of Cear?. Faculdade de Medicina. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brazil. Funda??o Osvaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Biologia Molecular Aplicada a Micobact?rias. Rio de Janeiro, RJ, Brasil. Funda??o Osvaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Biologia Molecular Aplicada a Micobact?rias. Rio de Janeiro, RJ, Brasil. Federal University of Cear?. Departamento de Estat?stica e Matem?tica Aplicada. Fortaleza, CE, Brazil. Universidade de Fortaleza. Programa de P?s-Gradua??o em Sa?de Coletiva. Fortaleza, CE, Brazil. Centro de Dermatologia Dona Lib?nia. Department of the State of Cear?. Fortaleza, CE, Brazil. Centro de Dermatologia Dona Lib?nia. Department of the State of Cear?. Fortaleza, CE, Brazil. Tulane University. School of Public Health and Tropical Medicine. Department of Global Community Health and Behavioral Sciences. New Orleans , LA , USA. Federal University of Cear?. Faculdade de Medicina. Departamento de Sa?de Comunit?ria. Fortaleza, CE, Brazil. This study analyzed the genetic diversity by MIRU-VNTR of Mycobacterium leprae isolates from nasal cavities and related to epidemiological and clinical data. The sample consisted of 48 newly diagnosed leprosy cases that tested positive for M. leprae PCR in nasal secretion (NS) attending to the National Reference Center of Dermatology Dona Libania (CDERM), Fortaleza, Brazil. Total DNA was extracted from NS of each patient and used for amplification of four M. leprae VNTR loci. Four clusters of M. leprae isolates were formed with identical genotypes. In the spatial analysis, 12 leprosy cases presented similar genotypes organized into 4 clusters. The most common genotypes in the current study was AC8b: 8, AC9: 7, AC8a: 8, GTA9: 10, which may represent a genotype of circulating strains most often in Cear?. A minimum set of four MIRU-VNTR loci was demonstrated to study the genetic diversity of M. leprae isolates from NS.

EnglishThe rise in cases of antibiotic-resistant Mycobacterium tuberculosis has become a major obstacle to the effective control of tuberculosis (TB) worldwide. Essential oils (EO) are complex mixtures that may contain between 20 and 60 components, with two or three major compounds at relatively high concentrations (20-70%) that are responsible for their pharmacological properties. The objective of this study was to assess the antimicrobial activity of the EOs, bushy lippia (Lippia alba), rosemary pepper (Lippia sidoides), lemon grass (Cymbopogon citratus), Mexican mint or Indian borage (Plectranthus amboinicus), and true cinnamon (Cinnamomum zeylanicum), against Mycobacterium tuberculosis H37Rv. Chemical characterization of the EOs was performed by gas chromatography coupled to mass spectrometry. The minimum inhibitory concentration (MIC) was determined by the microdilution-based resazurin microtiter assay. Four EOs were able to inhibit the growth of M. tuberculosis, with MICs of 286.5±130.2μg/mL (C. zeylanicum), 299.5±117.2μg/mL (L. sidoides), 351.6±39.06μg/mL (P. amboinicus), and 1,250μg/mL (C. citratus). Only the EO of L. alba showed no antimycobacterial activity at the tested concentrations, with an MIC greater than 1,250µg/mL. Results of this study suggested that C. zeylanicum, L. sidoides, and P. amboinicus could be important sources of bactericidal compounds against M. tuberculosis and require further investigation. The activity against M. tuberculosis of these three EOs has not been reported previously. The results show the high potential of the tested antimycobacterial EOs, making them a promising alternative for TB treatment. This data also confirms the importance of bioprospecting studies for active substances with antimycobacterial activity, which are still scarce. Key words: anti-bacterial agent; essential oils; Mycobacterium tuberculosis; Lippia alba; Lippia sidoides; Cymbopogon citrates; Plectranthus amboinicus; Cinnamomum zeylanicum. portuguesO aumento no numero de casos de Mycobacterium tuberculosis resistentes tem se tornado um grande obstaculo no controle efetivo da tuberculose (TB) mundialmente. Os oleos essenciais (OE), que sao misturas complexas que podem conter entre 20 a 60 componentes, apresentam dois ou tres compostos principais, em concentracoes relativamente elevadas, 20 a 70%, que sao responsaveis pelas suas propriedades farmacologicas. O objetivo deste estudo foi avaliar a atividade antimicobacteriana dos seguintes oleos essenciais (OEs) em Mycobacterium tuberculosis H37Rv: erva-cidreira (Lippia alba), alecrim-pimenta (Lippia sidoides), capim-limao (Cymbopogon citratus), oregano (Plectranthus amboinicus) e canela (Cinnamomum zeylanicum). A caracterizacao quimica dos OEs foi realizada por cromatografia gasosa acoplada a espectrometria de massa. A Concentracao Inibitoria Minima (CIM) foi determinada pela tecnica de microdiluicao da resazurina. Quatro OEs foram capazes de inibir o crescimento de M. tuberculosis, com CIM de 286,5±130,2μg/mL (C. zeylanicum), 299,5±117,2μg/mL (L. sidoides), 351,6±39,06μg/mL (P. amboinicus) e 1250μg/mL (C. citratus). Somente o OE de L. alba nao mostrou atividade antimicobacteriana nas conscentracoes testadas, considerando CIM maiores que 1250µg/mL. Os resultados deste estudo sugerem que L. sidoides, C. zeylanicum e P. amboinicus podem ser fontes importantes de compostos bactericidas contra M. tuberculosis e provaveis candidatos a serem investigados. A atividade contra M. tuberculosis desses tres OEs nao foi relatada em estudos anteriores. Os resultados mostram o elevado potencial antimicobacteriano dos OEs analisados, fazendo deles uma alternativa promissora para o tratamento da TB. Os resultados obtidos demonstraram a importância de pesquisas para bioprospeccao de substâncias ativas com acao antimicobacteriana, que ainda sao escassas. Palavras-chave: agente antibacteriano; oleos essenciais; Mycobacterium tuberculosis; Lippia alba; Lippia sidoides; Cymbopogon citrates; Plectranthus amboinicus; Cinnamomum zeylanicum.

Mycobacterium leprae bacilli are mainly transmitted by the dissemination of nasal aerosols from multibacillary (MB) patients to susceptible individuals through inhalation. The upper respiratory tract represents the main entry and exit routes of M. leprae. Therefore, this study aimed to evaluate the sensitivity and specificity of real-time quantitative polymerase chain reaction (qPCR) in detecting M. leprae in nasal secretion (NS) and skin biopsy (SB) samples from MB and paucibacillary (PB) cases. Fifty-four NS samples were obtained from leprosy patients at the Dona Libânia National Reference Centre for Sanitary Dermatology in Ceará, Brazil. Among them, 19 MB cases provided both NS and SB samples. Bacilloscopy index assays were conducted and qPCR amplification was performed using specific primers for M. leprae 16S rRNA gene, generating a 124-bp fragment. Primer specificity was verified by determining the amplicon melting temperature (T(m) = 79.5 °C) and detection limit of qPCR was 20 fg of M. leprae DNA. Results were positive for 89.7 and 73.3% of NS samples from MB and PB cases, respectively. SB samples from MB patients were 100% positive. The number of bacilli detected in NS samples were 1.39 × 10(3)–8.02 × 10(5), and in SB samples from MB patients were 1.87 × 10(3)–1.50 × 10(6). Therefore, qPCR assays using SYBR Green targeting M. leprae 16S rRNA region can be employed in detecting M. leprae in nasal swabs from leprosy patients, validating this method for epidemiological studies aiming to identify healthy carriers among household contacts or within populations of an endemic area.

Universidade Federal do Cear?. Faculdade de Medicina. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brasil. Universidade Federal do Cear?. Faculdade de Medicina. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Universidade Federal do Cear?. Departamento de Geologia. Fortaleza, CE, Brasil. Universidade Federal do Cear?. Departamento de Geologia. Fortaleza, CE, Brasil. Universidade de Fortaleza. Programa de P?s-Gradua??o em Sa?de Coletiva. Fortaleza, CE, Brasil. Tulane University. School of Public Health and Tropical Medicine. Department of Global Community Health and Behavioral Sciences. New Orleans, LA, USA Universidade Federal do Cear?. Faculdade de Medicina. Departamento de Sa?de Comunit?ria. Fortaleza, CE, Brasil. Universidade Federal do Cear?. Faculdade de Medicina. Departamento de Patologia e Medicina Legal. Fortaleza, CE, Brasil. BACKGROUND: The detection of live Mycobacterium leprae in soil and animals other than humans suggests that the environment plays a role in the transmission of leprosy. OBJECTIVE: The objective of this study was to investigate the presence of viable M. leprae in natural water sources used by the local population in five municipalities in the state of Cear?, northeastern Brazil. METHODS: Samples were collected from 30 different sources. Viable bacilli were identified by reverse transcriptase polymerase chain reaction (PCR) of the M. leprae gyrA gene and sequencing of the PCR products. Physicochemical properties of each water source were also assessed. FINDINGS: M. leprae gyrA mRNA was found in 23 (76.7%) of the water sources. No association was found between depth of the water and sample positivity, nor was there any association between the type of water used by the population and sample positivity. An association between viable M. leprae and temperature and pH was found. Georeferencing showed a relation between the residences of leprosy cases and water source containing the bacterium. MAIN CONCLUSIONS: The finding of viable M. leprae in natural water sources associated with human contact suggests that the environment plays an important role in maintaining endemic leprosy in the study region.

Mycobacterium leprae bacilli are mainly transmitted by the dissemination of nasal aerosols from multibacillary (MB) patients to susceptible individuals through inhalation. The upper respiratory tract represents the main entry and exit routes of M. leprae. Therefore, this study aimed to evaluate the sensitivity and specificity of real-time quantitative polymerase chain reaction (qPCR) in detecting M. leprae in nasal secretion (NS) and skin biopsy (SB) samples from MB and paucibacillary (PB) cases. Fifty-four NS samples were obtained from leprosy patients at the Dona Libânia National Reference Centre for Sanitary Dermatology in Ceará, Brazil. Among them, 19 MB cases provided both NS and SB samples. Bacilloscopy index assays were conducted and qPCR amplification was performed using specific primers for M. leprae 16S rRNA gene, generating a 124-bp fragment. Primer specificity was verified by determining the amplicon melting temperature (Tm = 79.5 °C) and detection limit of qPCR was 20 fg of M. leprae DNA. Results were positive for 89.7 and 73.3% of NS samples from MB and PB cases, respectively. SB samples from MB patients were 100% positive. The number of bacilli detected in NS samples were 1.39 × 103–8.02 × 105, and in SB samples from MB patients were 1.87 × 103–1.50 × 106. Therefore, qPCR assays using SYBR Green targeting M. leprae 16S rRNA region can be employed in detecting M. leprae in nasal swabs from leprosy patients, validating this method for epidemiological studies aiming to identify healthy carriers among household contacts or within populations of an endemic area.

INTRODUCTION: It is estimated that over two billion individuals are infected by Mycobacterium tuberculosis worldwide. Interleukin-6 (IL-6) is an important cytokine whose serum levels are commonly high in active pulmonary tuberculosis (TB). IL-6 screening in contacts of patients with TB may be useful to monitor the progress of the infectious process and to infer the risk of progression to active disease. OBJECTIVE: To evaluate the serum levels of interleukin-6 in contacts of patients with active pulmonary tuberculosis and to compare them with two other groups: a) patients affected by active pulmonary tuberculosis; b) non-contacts of tuberculosis. METHODS: Cross-sectional study with 15 contacts of patients with active pulmonary tuberculosis, selected according to the protocol recommended by the Ministry of Health. The serum levels of interleukin-6 were measured by Enzyme-linked immunosorbent assay (ELISA). The same test was also applied in the two comparison groups: 38 patients with active pulmonary tuberculosis (confirmed by clinical examination, X-rays of the chest and baciloscopy) and 63 non-contacts (healthy blood donors). RESULTS: In the contact group, the median IL-6 concentration was 1.7 pg/ml (0.96-4.8 pg/ml). For those affected by active pulmonary tuberculosis and non-contact individuals, these values corresponded to 4.3 pg/ml (0.5-24 pg/ml) and 0.5 pg/ml (0-2.8 pg/ml), respectively (p < 0.0001). CONCLUSION: Contacts of patients with active pulmonary tuberculosis had significantly higher IL-6 serum levels (3.4 times higher) in relation to non-contact individuals, but on a lower level (2.5 times lower) when compared to those affected by active disease. INTRODUÇÃO: Estima-se em mais de dois bilhões o número de infectados pelo Mycobacterium tuberculosis no mundo. A interleucina-6 (IL-6) é uma importante citocina, cujos níveis séricos estão comumente elevados na tuberculose pulmonar ativa. A dosagem dessa citocina em contatos de pacientes com tuberculose pode ser útil para se acompanhar a evolução do processo infeccioso, bem como para inferir o risco de progressão da doença ativa. OBJETIVO: Avaliar os níveis séricos de IL-6 em contatos de pacientes com tuberculose pulmonar ativa e compará-los com os de dois outros grupos: a) pacientes acometidos por tuberculose pulmonar ativa; b) não contatos de tuberculose. MÉTODOS: Estudo transversal com 15 contatos de pacientes com tuberculose pulmonar ativa, selecionados de acordo com protocolo preconizado pelo Ministério da Saúde. Os níveis séricos de IL-6 foram mensurados por meio de ensaio imunoenzimático (ELISA). O mesmo ensaio também foi empregado nos dois grupos de comparação: 38 pacientes com tuberculose pulmonar ativa (confirmada por exame clínico, raios X de tórax e baciloscopia) e 63 não contatos, doadores saudáveis de sangue. RESULTADOS: Para o grupo de contatos, a mediana de concentração de IL-6 foi de 1,7 pg/ml (0,96-4,8 pg/ml); para os acometidos por tuberculose pulmonar ativa e indivíduos não contatos, esses valores foram 4,3 pg/ml (0-12 pg/ml) e 0,5 pg/ml (0-2,8 pg/ml), respectivamente (p < 0,0001). CONCLUSÃO: Contatos de pacientes com tuberculose pulmonar ativa apresentaram níveis séricos de IL-6 significativamente aumentados (3,4 vezes mais), em relação aos indivíduos não contatos, mas em um patamar inferior (2,5 vezes menos), quando comparados com os acometidos pela doença ativa.

Leprosy is endemic in large part of Brazil with 28,761 new patients in 2015, the second largest number worldwide and reaches 9/10.000 in highly endemic regions and 2.7/10.000 in the city of Fortaleza, Ceará, Northeast Brazil. For better understanding of risk factors for leprosy transmission, we conducted an epidemiologic study supplemented by 17 locus VNTR and SNP 1–4 typing of Mycobacterium leprae in skin biopsy samples from new multibacillary (MB) patients diagnosed at a reference center in 2009 and 2010. Among the 1,519 new patients detected during the study period, 998 (65.7%) were MB and we performed DNA extraction and genotyping on 160 skin biopsy samples, resulting in 159 (16%) good multilocus VNTR types. Thirty-eight of these patients also provided VNTR types from M. leprae in nasal swabs. The SNP-Type was obtained for 157 patients and 87% were of type 4. Upon consideration all VNTR markers, 156 different genotypes and three pairs with identical genotypes were observed; no epidemiologic relation could be observed between individuals in these pairs. Considerable variability in differentiating index (DI) was observed between the different markers and the four with highest DI [(AT)15, (TA)18, (AT)17 and (GAA)21] frequently demonstrated differences in copy number when comparing genotypes from both type of samples. Excluding these markers from analysis resulted in 83 genotypes, 20 of which included 96 of the patients (60.3%). These clusters were composed of two (n = 8), three (n = 6), four (n = 1), five (n = 2), six (n = 1), 19 (n = 1) and 23 (n = 23) individuals and suggests that recent transmission is contributing to the maintenance of leprosy in Fortaleza. When comparing epidemiological and clinical variables among patients within clustered or with unique M. leprae genotypes, a positive bacterial index in skin biopsies and knowledge of working with someone with the disease were significantly associated with clustering. A tendency to belong to a cluster was observed with later notification of disease (mean value of 3.4 months) and having disability grade 2. A tendency for lack of clustering was observed for patients who reported to have lived with another leprosy case but this might be due to lack of inclusion of household contacts in the study. Although clusters were spread over the city, kernel analysis revealed that some of the patients belonging to the two major clusters were spatially related to some neighborhoods that report poverty and high disease incidence in children. Finally, inclusion of genotypes from nasal swabs might be warranted. A major limitation of the study is that sample size of 160 patients from a two year period represents only 15% of the new patients and this could have weakened statistical outcomes. This is the first molecular epidemiology study of leprosy in Brazil and although the high clustering level suggests that recent transmission is the major cause of disease in Fortaleza; the existence of two large clusters needs further investigation., Author summary Leprosy is a transmissible disease that is still endemic in several countries including in Brazil, a country with highly variable region associated incidence of disease. Fortaleza is a city in Northeast Brazil with high incidence and conventional epidemiology studies are suggestive for high levels of recent transmission. Genotyping of M. leprae allows the recognition of individuals that have been infected with the same strain (called a cluster) and therefore being suggestive for belonging to the same transmission network. In the present work, by analyzing genotypes of M. leprae in the skin lesion of multibacillary patients, we made observations that improve our knowledge on interpretation of genotypes and clusters and confirm the high levels of recent transmission in the city of Fortaleza. This is one of the few studies that used molecular epidemiology to look for risk factors for recent transmission of leprosy and to our knowledge, the first in Brazil. Our data support further investigation of the workplace as a source of infection, preferentially by a study designed on a larger number of patients and including analysis of M. leprae present in the nose.

Burkholderia pseudomallei, the causative agent of melioidosis was found in a small cluster of cases in Tejucuoca, Ceara, Brazil. Tests were carried out to determine its phenotypic characteristics: colony morphology on Ashdown agar and MacConkey agar, biochemical profile in conventional biochemical tests and API 20NE, arabinose assimilation and susceptibility testing by disk diffusion, comparing with data in the literature. This study confirms the presence of B. pseudomallei in Brazil and describes its characteristics.

The seroprevalence rates of IgM anti-phenolic glycolipid-I (PGL-I) antibodies in four study groups with differing exposure to Mycobacterium leprae in Ceara, Brazil were investigated between March 2005 and August 2006. The first three groups in a high prevalence area included 144 cases of leprosy, their 380 contacts and 317 participants with no known leprosy contact. The fourth group in a low prevalence area consisted of 87 participants with no known leprosy contact living in an area in which no cases of leprosy had been reported in the previous 6 months. Seropositivity and levels of IgM antibodies to PGL-I were investigated using ELISA. The seropositivity levels of anti-PGL-I among the different clinical forms of leprosy cases were 61% for lepromatous, 25% for tuberculoid and 27% indeterminate. The levels of anti-PGL-I antibodies in the endemic area differentiated leprosy cases from non-cases. However, the seropositivity was similar among contact cases (15.8%) and no known leprosy contact cases from high (15.1%) and low (13.8%) prevalence areas. The seropositivity of both contacts and no known contacts was much higher than previously reported among no known contacts in other endemic areas. The study indicates that anti-PGL-I antibodies are not useful as immunological markers of household leprosy contacts and no known leprosy contacts in endemic areas.

In this study, we have identified a secreted 13 kDa lectin from Mtb (Mtb, Mycobacterium tuberculosis; sMTL-13) by homology search of a non-redundant lectin database. Bioinformatic analysis revealed that sMTL-13 belongs to the ricin-type beta-trefoil family of proteins containing a Sec-type signal peptide present in Mtb complex species, but not in non-tuberculous mycobacteria. Following heterologous expression of sMTL-13 and generation of an mAb (clone 276.B7/IgG1kappa), we confirmed that this lectin is present in culture filtrate proteins from Mtb H37Rv, but not in non-tuberculous mycobacteria-derived culture filtrate proteins. In addition, sMTL-13 leads to an increased IFN-gamma production by PBMC from active tuberculosis (ATB) patients. Furthermore, sera from ATB patients displayed high titers of IgG Ab against sMTL-13, a response found to be decreased following successful anti-tuberculosis therapy. Together, our findings reveal a secreted 13 kDa ricin-like lectin from Mtb, which is immunologically recognized during ATB and could serve as a biomarker of disease treatment.

The hepatitis C virus (HCV) is classified into six different genotypes and their distribution is different throughout the world. Epidemiologic studies are important to determine several characteristics of the virus, as well as the disease. This study analysed the prevalence of HCV and its genotypes among patients from a leading hospital in Ceará, which is located in Northeast Brazil. A total of 119 anti-HCV-seropositive patients, each having previously completed a questionnaire about risk behaviours related to HCV infection were tested for HCV infection using a qualitative HCV polymerase chain reaction (PCR) assay and genotyping by restriction fragment length polymorphism (RFLP). The detection was based on amplifying of the non-coding 5’ region. Of the 119 patients, 95 showed positive results in the qualitative HCV test. History of surgery was the most reported risk factor, followed by the use of drugs, having tattoos, undergoing haemodialysis and occupational exposure. Genotype 1 was the most prevalent (46.9%), followed by genotype 3 (34.4%) and 2 (8.3%). The genotype distribution was similar for all of the various risk behaviours.

INTRODUCTION: This study quantified Mycobacterium leprae bacilli in environmental water samples from five municipalities in the State of Ceará by quantitative polymerase chain reaction (qPCR) and compared the identified genotypes with those obtained from leprosy patient biopsies. METHODS: We collected five replicas from each of the 30 selected reservoirs and skin lesion biopsies from 25 new leprosy cases treated at a reference center in Fortaleza, Ceará from 2010 to 2013. The 16S rRNA gene region of M. leprae was amplified by qPCR and a standard curve was created with the pIDTBlue 16SrRNAMlep plasmid. The Juazeiro do Norte water samples and the biopsies were genotyped (single nucleotide polymorphism [SNP] 1 to 4) and the SNP 4 genotypes were subtyped. RESULTS: Of the 149 water samples analyzed, 54.4% were positive for the M. leprae DNA. The M. leprae bacilli copy number ranged from 1.42 × 10 -1 to 1.44 × 10 + 2 . Most biopsies showed SNP type 4 (64%), while all samples from Juazeiro do Norte were SNP type 4, with subtype 4-N appearing at the highest frequency. CONCLUSIONS: We suggest that environmental waters containing M. leprae bacilli play an important role in disease transmission, justifying PGL-1 seropositivity in individuals living in areas where there is no reported case, and in leprosy cases individuals who report no previous contact with other case. Therefore, further investigation is needed to clarify disease transmission in this region and to explore the role of the environment. We also suggest that in this area surveillance for leprosy cases should be intensified.

Objectives: This study has used a combination of clinical information, spoligotyping, and georeferencing system to elucidate the genetic diversity of the Mycobacterium tuberculosis isolates circulating in a TB-prevalent municipality of Northeast Brazil. Methods: A total of 115 M. tuberculosis strains were isolated from pulmonary tuberculosis patients from January 2007 to March 2008 in Fortaleza. Drug susceptibility and spoligotyping assays were performed and place of residence of the patients were georeferenced. Results: Of the M. tuberculosis strains studied, 51 (44.3%) isolates were resistant to at least one drug (R-TB) and 64 (55.7%) were sensitive to all the drugs tested (S-TB). A high frequency of resistance was found in previously treated cases (84%) and among new cases (16%; p

Infection with both Human Immunodeficiency Virus (HIV) and Mycobacterium tuberculosis is currently the world's leading cause of death due to infectious agents. We evaluated factors related to the development of tuberculosis (TB) in HIV-infected patients who were being treated at an infectious diseases hospital in Fortaleza, Ceara, Brazil. From January 2004 to December 2005, we made an epidemiological study through the analysis of the medical records of 171 patients, who were diagnosed as having both HIV and tuberculosis. Among these co-infected patients, most (81%, p=0.0006) were male. Co-infection was more frequent (87.8%) among patients over 40 years of age and those with lower educational levels (less than eight years of schooling). Forty-one percent of the patients in the study had not had a smear culture test for acid-fast bacilli (AFB). CD4 cell counts were lower than 200 cells/[micro]L in 71.9% of the patients, the mean being 169 cells/[micro]L. This type of data is important for establishing strategies to improve the control of tuberculosis in HIV-infected patients.