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.

Abstract INTRODUCTION The incidence of syphilis has increased since the 1970s. METHODS This was a descriptive and analytical cross-sectional study with a non-probabilistic sample. RESULTS: Of 973 patients with human immunodeficiency virus, 179 (18.4%) tested positive for both human immunodeficiency virus and syphilis, 84.8% were men, 50.9% were aged between 36 and 50 years, 47.8% with syphilis were diagnosed with human immunodeficiency virus for 10-20 years, and 40.3% received antiretroviral therapy for 10-20 years. CONCLUSIONS The prevalence of syphilis in patients with human immunodeficiency virus is higher than expected, making it urgent to adopt efficient public health measures.

ABSTRACT Visceral leishmaniasis, associated with HIV/AIDS coinfection, is becoming a more aggressive disease, complicating an accurate prognosis. A 21-year-old HIV-positive female presenting with clinical features of visceral leishmaniasis was enrolled in this study. Bone marrow cytology, Novy-MacNeal-Nicolle culture and kDNA PCR of peripheral blood were all positive. Typing methods, multilocus enzyme electrophoresis and ITS1-RFLP PCR of peripheral blood confirmed infection by Leishmania (L.) infantum chagasi . PCR has proved to be safer and more affordable than other characterization methods; ITS1-RFLP PCR can diagnose and type Leishmania spp. in both endemic and non-endemic areas, favoring the prognosis and allowing the appropriate treatment of patients.

Vários flebotomíneos têm sido considerados como possíveis vetores suspeitos ou comprovados de parasitas do gênero Leishmania no Brasil, mas os dados para região nordeste continuam incipientes. Neste estudo, 600 flebotomíneos foram agrupados em grupos de 10 espécimes e testados por uma PCR específica para o gênero Leishmania e por outra PCR para Leishmania (Leishmania) infantum. Quatorze dos 60 grupos foram positivos por PCR gênero-específica, sendo cinco de L. migonei, sete L. complexa, um de L. sordellii e um de L. naftalekatzi, o que corresponde a uma taxa mínima de infecção de 2,3% (14/600). Nossos resultados, associados à antropofilia e abundância dessas espécies, sugerem a participação de L. migonei e L. complexa como vetores de Leishmania no nordeste do Brasil. Notavelmente, a detecção de DNA de Leishmania em L. sordellii e L. naftalekatzi é relatada pela primeira vez no Brasil, mas futuros estudos são necessários para compreender melhor o significado desses achados.

Several phlebotomine sand fly species have been regarded as putative or proven vectors of parasites of the genus Leishmania in Brazil, but data for the northeastern region remains incipient. In this study, a total of 600 phlebotomine sand flies were grouped in pools of 10 specimens each and tested by a Leishmania genus-specific PCR and by a PCR targeting Leishmania (Leishmania) infantum. Fourteen out of 60 pools were positive by the genus-specific PCR, being five pools of L. migonei, seven of L. complexa, one of L. sordellii and one of L. naftalekatzi, which correspond to a minimal infection rate of 2.3% (14/600). Our results, associated with their known anthropophily and their abundance, suggest the participation of L. migonei and L. complexa as vectors of Leishmania in northeastern Brazil. Remarkably, this is the first time in this country that the detection of Leishmania DNA in L. sordellii and L. naftalekatzi has been reported, but future studies are necessary to better understand the significance of these findings.

Introduction Polymerase chain reaction (PCR) may offer an alternative diagnostic option when clinical signs and symptoms suggest visceral leishmaniasis (VL) but microscopic scanning and serological tests provide negative results. PCR using urine is sensitive enough to diagnose human visceral leishmaniasis (VL). However, DNA quality is a crucial factor for successful amplification. Methods A comparative performance evaluation of DNA extraction methods from the urine of patients with VL using two commercially available extraction kits and two phenol-chloroform protocols was conducted to determine which method produces the highest quality DNA suitable for PCR amplification, as well as the most sensitive, fast and inexpensive method. All commercially available kits were able to shorten the duration of DNA extraction. Results With regard to detection limits, both phenol: chloroform extraction and the QIAamp DNA Mini Kit provided good results (0.1 pg of DNA) for the extraction of DNA from a parasite smaller than Leishmania (Leishmania) infantum (< 100fg of DNA). However, among 11 urine samples from subjects with VL, better performance was achieved with the phenol:chloroform method (8/11) relative to the QIAamp DNA Mini Kit (4/11), with a greater number of positive samples detected at a lower cost using PCR. Conclusion Our results demonstrate that phenol:chloroform with an ethanol precipitation prior to extraction is the most efficient method in terms of yield and cost, using urine as a non-invasive source of DNA and providing an alternative diagnostic method at a low cost.

INTRODUÇÃO: Staphylococcus spp. é um importante patógeno associado aos cuidados em saúde, e a identificação de isolados resistentes à meticilina em amostras de colonização pode fornecer dados para auxiliar no sucesso da terapia antimicrobiana. OBJETIVOS: Determinar a ocorrência de colonização por Staphylococcus spp. resistentes à meticilina (MRS) por meio da detecção do gene mecA e avaliar diferentes métodos fenotípicos para a detecção presuntiva da resistência à meticilina em amostras da cavidade nasal anterior e das mãos de profissionais de saúde de um hospital universitário no Estado de Pernambuco, Brasil. MÉTODOS: Foram selecionados 28 isolados de Staphylococcus spp. que mostraram perfil intermediário ou resistente à oxacilina, detectado pela técnica de Kirby Bauer. Os métodos utilizados foram o teste de disco difusão de cefoxitina, concentração inibitória mínima pelo E-test de oxacilina, screening para avaliação da resistência à oxacilina e reação em cadeia da polimerase (PCR) para detecção do gene mecA. RESULTADOS: Dos métodos fenotípicos utilizados, apenas o E-test de oxacilina não mostrou diferença estatística significante em relação à PCR para a detecção do gene mecA, considerado o método padrão-ouro. CONCLUSÃO: O E-test de oxacilina foi o melhor método fenotípico utilizado. É necessário detectar corretamente o MRS em indivíduos saudáveis, pois eles podem atuar como portadores, sendo uma fonte potencial de microrganismos envolvidos em infecções hospitalares.

INTRODUCTION: Staphylococcus spp. is an important healthcare-associated pathogen and the identification of methicillin-resistant strains in samples of colonization may provide data to assist in the antimicrobial therapy success. OBJECTIVES: To determine the occurrence of colonization by methicillin-resistant Staphylococcus spp. (MRS), through the detection of the mecA gene and to evaluate different phenotypic methods for the presumptive detection of methicillin resistance in samples of the anterior nasal cavity and hands of the health care personnel of a university hospital in the state of Pernambuco, Brazil. METHODS: We selected the 28 isolates of Staphylococcus spp., which showed an intermediate or resistant phenotypic profile for oxacillin, detected by the Kirby Bauer technique. The methods used were disk-diffusion tests for cefoxitin, minimal inhibitory concentration by E-test for oxacillin, screening for oxacillin resistance and mecA gene detection by polymerase chain reaction (PCR). RESULTS: About the phenotypic methods utilized, only the E-test of oxacillin did not show a statistically significant difference in relation to PCR for the mecA gene detection, considered the gold standard. CONCLUSION: The E-test of oxacillin was the best of the phenotypic methods utilized. It is necessary to correctly detect MRS in healthy individuals, because they can act as carriers and can therefore be a potential source of microorganisms involved in hospital infections.

This study was performed with the objective to identify the prevalence of colonization by Staphylococcus aureus in nursing professionals from a teaching hospital in Pernambuco, and evaluate the resistance profile of these isolates. To do this, we performed a cross-sectional study where biological samples were collected from the hands and nasal cavities of the subjects. S. aureus was identified using agar (blood agar and mannitol salt) via catalase and coagulase tests. The sensitivity profile was determined by Kirby Bauer technique and determination of methicillin resistance was performed with oxacillin screening with sodium chloride (NaCl) addition. Of the 151 professionals evaluated, 39 were colonized which showed a prevalence of 25.8%. Among the variables studied, age and use of PPE were associated with colonization by the organism. Of all the isolates, only five were resistant to methicillin.

Estudio realizado para identificar prevalencia de colonización por Staphylococcus aureus en profesionales de enfermería de hospital universitario de Pernambuco, así como evaluar el perfil de resistencia de la bacteria aislada. Se realizó un estudio transversal en el que se recolectaron muestras biológicas de manos y cavidad nasal. La identificación del S. aureus se realizó mediante cultivo en agar-sangre, agar-manitol salado y mediante pruebas de catalasa y coagulasa. El perfil de sensibilidad se determinó por técnica de Kirby Bauer y para la determinación de resistencia a meticilina se realizó screening en placa con oxalacina, con adición de 4% de NaCl. De 150 profesionales evaluados, 39 estaban colonizados, lo que demostró prevalencia de 25,8%. Entre las variables estudiadas, faja etaria y cantidad de EPI se presentaron asociadas con la colonización por la bacteria. De todas las cepas aisladas, apenas cinco presentaron resistencia a meticilina.

O presente estudo foi realizado com o objetivo de identificar a prevalência de colonização pelo Staphylococcus aureus em profissionais de enfermagem de um hospital universitário de Pernambuco, bem como avaliar o perfil de resistência deles isoladamente. Para isso, foi realizado um estudo transversal, no qual foram coletadas amostras biológicas das mãos e da cavidade nasal. A identificação do S. aureus foi realizada por meio do semeio em agar-sangue, agar manitol-salgado e através dos testes de catalase e coagulase. O perfil de sensibilidade foi determinado pela técnica de Kirby Bauer e para determinação da resistência à meticilina foi realizado o screening em placa com oxacilina com adição de 4% de NaCl. Dos 151 profissionais avaliados, 39 se encontravam colonizados, o que demonstrou uma prevalência de 25,8%. Dentre as variáveis estudadas, a faixa etária e a quantidade de EPI apresentaram-se associadas à colonização pelo microrganismo. De todas as linhagens isoladas, apenas cinco apresentaram resistência à meticilina.

O presente estudo foi realizado com o objetivo de identificar a prevalência de colonização pelo Staphylococcus aureus em profissionais de enfermagem de um hospital universitário de Pernambuco, bem como avaliar o perfil de resistência deles isoladamente. Para isso, foi realizado um estudo transversal, no qual foram coletadas amostras biológicas das mãos e da cavidade nasal. A identificação do S. aureus foi realizada por meio do semeio em agar-sangue, agar manitol-salgado e através dos testes de catalase e coagulase. O perfil de sensibilidade foi determinado pela técnica de Kirby Bauer e para determinação da resistência à meticilina foi realizado o screening em placa com oxacilina com adição de 4% de NaCl. Dos 151 profissionais avaliados, 39 se encontravam colonizados, o que demonstrou uma prevalência de 25,8%. Dentre as variáveis estudadas, a faixa etária e a quantidade de EPI apresentaram-se associadas à colonização pelo microrganismo. De todas as linhagens isoladas, apenas cinco apresentaram resistência à meticilina.

This study was performed with the objective to identify the prevalence of colonization by Staphylococcus aureus in nursing professionals from a teaching hospital in Pernambuco, and evaluate the resistance profile of these isolates. To do this, we performed a cross-sectional study where biological samples were collected from the hands and nasal cavities of the subjects. S. aureus was identified using agar (blood agar and mannitol salt) via catalase and coagulase tests. The sensitivity profile was determined by Kirby Bauer technique and determination of methicillin resistance was performed with oxacillin screening with sodium chloride (NaCl) addition. Of the 151 professionals evaluated, 39 were colonized which showed a prevalence of 25.8%. Among the variables studied, age and use of PPE were associated with colonization by the organism. Of all the isolates, only five were resistant to methicillin.

Estudio realizado para identificar prevalencia de colonización por Staphylococcus aureus en profesionales de enfermería de hospital universitario de Pernambuco, así como evaluar el perfil de resistencia de la bacteria aislada. Se realizó un estudio transversal en el que se recolectaron muestras biológicas de manos y cavidad nasal. La identificación del S. aureus se realizó mediante cultivo en agar-sangre, agar-manitol salado y mediante pruebas de catalasa y coagulasa. El perfil de sensibilidad se determinó por técnica de Kirby Bauer y para la determinación de resistencia a meticilina se realizó screening en placa con oxalacina, con adición de 4% de NaCl. De 150 profesionales evaluados, 39 estaban colonizados, lo que demostró prevalencia de 25,8%. Entre las variables estudiadas, faja etaria y cantidad de EPI se presentaron asociadas con la colonización por la bacteria. De todas las cepas aisladas, apenas cinco presentaron resistencia a meticilina.

Liver biopsy is the gold-standard method to stage fibrosis; however, it is an invasive procedure and is potentially dangerous. The main objective of this study was to evaluate biological markers, such as cytokines IL-13, IFN-γ, TNF-α and TGF-β, platelets, bilirubins (Bil), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), total proteins, γ-glutamil transferase (γ-GT) and alkaline phosphatase (AP), that could be used to predict the severity of hepatic fibrosis in schistosomiasis and hepatitis C (HC) as isolated diseases or co-infections. The following patient groups were selected: HC (n = 39), HC/hepatosplenic schistosomiasis (HSS) (n = 19), HSS (n = 22) and a control group (n = 13). ANOVA and ROC curves were used for statistical analysis. P < 0.05 was considered significant. With HC patients we showed that TNF-α (p = 0.020) and AP (p = 0.005) could differentiate mild and severe fibrosis. With regard to necroinflammatory activity, AST (p = 0.002), γ-GT (p = 0.034) and AP (p = 0.001) were the best markers to differentiate mild and severe activity. In HC + HSS patients, total Bil (p = 0.008) was capable of differentiating between mild and severe fibrosis. In conclusion, our study was able to suggest biological markers that are non-invasive candidates to evaluate fibrosis and necroinflammatory activity in HC and HC + HSS.

O trabalho visou à otimização de um método baseado na reação em cadeia da polimerase multiplex - para diferenciação de micobactérias de interesse para a saúde pública. A PCR Multiplex baseou-se na amplificação simultânea do genehsp65, presente em todo gênero Mycobacterium, do gene dnaJ, presente apenas em Mycobacterium tuberculosis e Mycobacterium avium e da sequência de inserção IS6110 presente no complexo Mycobacterium tuberculosis, gerando amplicons de 165pb, 365pb e 541pb, respectivamente. O limite de detecção foi de 1fg para o alvo hsp65, 100pg para o dnaJ e 0,1fg para o IS6110. A PCR multiplex detectou até 100pg de DNA de Mycobacterium tuberculosis. O sistema demonstrou ser específico e sensível na detecção de Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium e Mycobacterium smegmatis. Os resultados obtidos utilizando cepas de referência demonstraram que a PCR multiplex pode ser uma ferramenta rápida, sensível e específica na diferenciação de micobactérias.

This study aimed to optimize a method based on the polymerase chain reaction - multiplex PCR - for differentiation of mycobacteria species of interest for public health. The multiplex PCR was based on simultaneous amplification of the hsp65 gene, which is present in all species of the Mycobacterium genus, the dnaJ gene, which is present only in Mycobacterium tuberculosis and Mycobacterium avium and the IS6110 insertion sequence, which is present in the Mycobacterium tuberculosis complex, generating amplicons of 165 bp, 365 bp and 541 bp, respectively. The detection limit was 1 fg for the hsp65 target, 100 pg for dnaJ and 0.1 fg for IS6110. The multiplex PCR detected down to 100 pg of DNA of Mycobacterium tuberculosis. The system was shown to be specific and sensitive for detection of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. The results obtained using reference strains of mycobacteria showed that multiplex PCR may be a fast, sensitive and specific tool for differentiation of mycobacteria.

Staphylococcus aureus is the main human pathogen that colonizes individuals in general population. The objective of the study was evaluate the epidemiological and sensitivity profile of S. aureus lineage, isolated in health care workers (HCW) of a University Hospital in Pernambuco state, Brazil. Biological samples of hands and nasal cavities were sown in agar sheep blood. Colonies under suspicion of being S. aureus were identified using Gram staining, catalase test and coagulase, mannitol-salty agar fermentation and DNAse agar. The resistance to mupirocin was analyzed through the Kirby Bauer technique. In relation to methicillin and vancomycin the determination was by the minimum inhibitory concentration method (E-test). From the 202 HCW evaluated, 52 were colonized by S. aureus (25,7%). The factors associated to the colonization by S. aureus were: age-group, professional category, use of individual protection equipments (frequency and numbers). All S. aureus isolate lineages were sensitive to mupirocin and vancomycin, and three of them were identified as methicillin-resistant. The prevalence of MSSA and MRSA among HCW was considered low and was below the results described in the literature. The isolate S. aureus lineages have shown low resistance profile.