Lack of evidence for human infection with Xenotropic murine leukemia virus-related virus in the Brazilian Amazon basin

Introduction This study confirmed the absence of natural infection with Xenotropic murine leukemia virus-related virus (XMRV) or XMRV-related disease in human populations of the Brazilian Amazon basin. We demonstrated that 803 individuals of both sexes, who were residents of Belem in the Brazilian State of Pará, were not infected with XMRV. Methods Individuals were divided into 4 subgroups: healthy individuals, individuals infected with human immunodeficiency virus, type 1 (HIV-1), individuals infected with human T-lymphotrophic virus, types 1 or 2 (HTLV-1/2), and individuals with prostate cancer. XMRV infection was investigated by nested PCR to detect the viral gag gene and by quantitative PCR to detect pol. Results There was no amplification of either gag or pol segments from XRMV in any of the samples examined. Conclusions This study supports the conclusions of the studies that eventually led to the retraction of the original study reporting the association between XMRV and human diseases.

The occurrence and dissemination of methicillin and vancomycin-resistant Staphylococcus in samples from patients and health professionals of a university hospital in Recife, State of Pernambuco, Brazil

Introduction Methicillin-resistant Staphylococcus aureus (MRSA) strains have been responsible for many nosocomial outbreaks. Within hospitals, colonized employees often act as reservoirs for the spread of this organism. This study collected clinical samples of 91 patients admitted to the intensive care unit (ICU), hemodialysis/nephrology service and surgical clinic, and biological samples from the nasal cavities of 120 professionals working in those environments, of a University Hospital in Recife, in the State of Pernambuco, Brazil. The main objective of this study was to determine the occurrence and dissemination of methicillin- and vancomycin-resistant Staphylococcus spp. Methods The isolates obtained were tested for susceptibility to oxacillin and vancomycin and detection of the mecA gene. In addition, the isolates were evaluated for the presence of clones by ribotyping-polymerase chain reaction (PCR). Results MRSA occurrence, as detected by the presence of the mecA gene, was more prevalent among nursing technicians; 48.1% (13/27) and 40.7% (11/27) of the isolates were from health professionals of the surgical clinic. In patients, the most frequent occurrence of mecA-positive isolates was among the samples from catheter tips (33.3%; 3/9), obtained mostly from the hemodialysis/nephrology service. Eight vancomycin-resistant strains were found among the MRSA isolates through vancomycin screening. Based on the amplification patterns, 17 ribotypes were identified, with some distributed between patients and professionals. Conclusions Despite the great diversity of clones, which makes it difficult to trace the source of the infection, knowledge of the molecular and phenotypic profiles of Staphylococcus samples can contribute towards guiding therapeutic approaches in the treatment and control of nosocomial infections.

Evaluation of three enzyme immunoassays and a nucleic acid amplification test for the diagnosis of Clostridium difficile-associated diarrhea at a university hospital in Brazil

Introduction Despite the known importance of Clostridium difficile as a nosocomial pathogen, few studies regarding Clostridium difficile infection (CDI) in Brazil have been conducted. To date, the diagnostic tests that are available on the Brazilian market for the diagnosis of CDI have not been evaluated. The aim of this study was to compare the performances of four commercial methods for the diagnosis of CDI in patients from a university hospital in Brazil. Methods Three enzyme immunoassays (EIAs) and one nucleic acid amplification test (NAAT) were evaluated against a cytotoxicity assay (CTA) and toxigenic culture (TC). Stool samples from 92 patients with suspected CDI were used in this study. Results Twenty-five (27.2%) of 92 samples were positive according to the CTA, and 23 (25%) were positive according to the TC. All EIAs and the NAAT test demonstrated sensitivities between 59 and 68% and specificities greater than 91%. Conclusions All four methods exhibited low sensitivities for the diagnosis of CDI, which could lead to a large number of false-negative results, an increased risk of cross-infection to other patients, and overtreatment with empirical antibiotics.

The existence of only one haplotype of Leishmania major in the main and potential reservoir hosts of zoonotic cutaneous leishmaniasis using different molecular markers in a focal area in Iran

IntroductionLeishmania major is the causative agent of zoonotic cutaneous leishmaniasis (ZCL), and great gerbils are the main reservoir hosts in Iran. Abarkouh in central Iran is an emerging focal point for which the reservoir hosts of ZCL are unclear. This research project was designed to detect any Leishmania parasites in different wild rodent species.MethodsAll rodents captured in 2011 and 2012 from Abarkouh district were identified based on morphological characteristics and by amplification of the rodent cytochrome b (Cyt b) gene. To detect Leishmania infection in rodents, deoxyribonucleic acid (DNA) of each ear was extracted. Internal transcribed spacer-ribosomal deoxyribonucleic acid (ITS-rDNA), microsatellites, kinetoplast deoxyribonucleic acid (kDNA) and cytochrome b genes of Leishmania parasites were amplified by polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) and sequencing were employed to confirm the Leishmania identification.ResultsOf 68 captured rodents in the region, 55 Rhombomys opimus were identified and nine Leishmaniainfections (9/55) were found. In addition, eight Meriones libycus and two Tatera indicawere sampled, and one of each was confirmed to be infected. Two Meriones persicus and one Mus musculuswere sampled with no infection.ConclusionsThe results showed that all 11 unambiguously positive Leishmania infections were Leishmania major. Only one haplotype of L. major(GenBank access No. EF413075) was found and at least three rodents R. opimus, M. libycus and T. indica—appear to be the main and potential reservoir hosts in this ZCL focus. The reservoir hosts are variable and versatile in small ZCL focal locations.

High similarity of Trypanosoma cruzikDNA genetic profiles detected by LSSP-PCR within family groups in an endemic area of Chagas disease in Brazil

IntroductionDetermining the genetic similarities among Trypanosoma cruzi populations isolated from different hosts and vectors is very important to clarify the epidemiology of Chagas disease.MethodsAn epidemiological study was conducted in a Brazilian endemic area for Chagas disease, including 76 chronic chagasic individuals (96.1% with an indeterminate form; 46.1% with positive hemoculture).ResultsT. cruzi I (TcI) was isolated from one child and TcII was found in the remaining (97.1%) subjects. Low-stringency single-specific-primer-polymerase chain reaction (LSSP-PCR) showed high heterogeneity among TcII populations (46% of shared bands); however, high similarities (80-100%) among pairs of mothers/children, siblings, or cousins were detected.ConclusionsLSSP-PCR showed potential for identifying similar parasite populations among individuals with close kinship in epidemiological studies of Chagas disease.

Polymerase chain reaction-based method for the identification of Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in mucosal tissues conserved in paraffin

ABSTRACTINTRODUCTION: In the Americas, mucosal leishmaniasis is primarily associated with infection by Leishmania (Viannia) braziliensis. However, Leishmania (Viannia) guyanensis is another important cause of this disease in the Brazilian Amazon. In this study, we aimed at detecting Leishmaniadeoxyribonucleic acid (DNA) within paraffin-embedded fragments of mucosal tissues, and characterizing the infecting parasite species.METHODS: We evaluated samples collected from 114 patients treated at a reference center in the Brazilian Amazon by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses.RESULTS: Direct examination of biopsy imprints detected parasites in 10 of the 114 samples, while evaluation of hematoxylin and eosin-stained slides detected amastigotes in an additional 17 samples. Meanwhile, 31/114 samples (27.2%) were positive for Leishmania spp. kinetoplast deoxyribonucleic acid (kDNA) by PCR analysis. Of these, 17 (54.8%) yielded amplification of the mini-exon PCR target, thereby allowing for PCR-RFLP-based identification. Six of the samples were identified as L. (V.) braziliensis, while the remaining 11 were identified as L. (V.) guyanensis.CONCLUSIONS: The results of this study demonstrate the feasibility of applying molecular techniques for the diagnosis of human parasites within paraffin-embedded tissues. Moreover, our findings confirm that L. (V.) guyanensisis a relevant causative agent of mucosal leishmaniasis in the Brazilian Amazon.

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

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

Detection of canine visceral leishmaniasis by conjunctival swab PCR

Abstract: INTRODUCTION: Conjunctival swab PCR was evaluated as a tool to diagnose visceral leishmaniasis in dogs. METHODS: Conjunctival swab PCR was compared to indirect immunofluorescence antibody test and blood PCR. RESULTS: Indirect immunofluorescence was significantly correlated with conjunctival swab PCR (p < 0.05), but not with blood PCR (p > 0.05). In addition, conjunctival swab PCR was significantly associated with presence of clinical symptoms (p < 0.05), whereas blood PCR was associated with absence of clinical symptoms (p < 0.05). CONCLUSIONS: Results indicate that conjunctival swab PCR is useful in epidemiological surveys of canine visceral leishmaniasis.

Extraction of Trypanosoma cruzi DNA from food: a contribution to the elucidation of acute Chagas disease outbreaks

Abstract: INTRODUCTION: Before 2004, the occurrence of acute Chagas disease (ACD) by oral transmission associated with food was scarcely known or investigated. Originally sporadic and circumstantial, ACD occurrences have now become frequent in the Amazon region, with recently related outbreaks spreading to several Brazilian states. These cases are associated with the consumption of açai juice by waste reservoir animals or insect vectors infected with Trypanosoma cruzi in endemic areas. Although guidelines for processing the fruit to minimize contamination through microorganisms and parasites exist, açai-based products must be assessed for quality, for which the demand for appropriate methodologies must be met. METHODS: Dilutions ranging from 5 to 1,000 T. cruzi CL Brener cells were mixed with 2mL of acai juice. Four Extraction of T. cruzi DNA methods were used on the fruit, and the cetyltrimethyl ammonium bromide (CTAB) method was selected according to JRC, 2005. RESULTS: DNA extraction by the CTAB method yielded satisfactory results with regard to purity and concentration for use in PCR. Overall, the methods employed proved that not only extraction efficiency but also high sensitivity in amplification was important. CONCLUSIONS: The method for T. cruzi detection in food is a powerful tool in the epidemiological investigation of outbreaks as it turns epidemiological evidence into supporting data that serve to confirm T. cruzi infection in the foods. It also facilitates food quality control and assessment of good manufacturing practices involving acai-based products.

Analysis of human leukocyte antigens class II-DR in Brazilian children and adolescents with systemic lupus erythematosus

OBJECTIVE: To analyze the frequency of human leukocyte antigens class II-DR in children and adolescents with systemic lupus erythematosus. PATIENTS AND METHODS: Fifty-fiveBrazilian systemic lupus erythematosus children and adolescents and 308 healthy individuals were studied. Gender, race, and age of onset of systemic lupus erythematosus were recorded. The human leukocyte antigens typing of class II-DR was carried out by polymerase chain reaction amplification with sequence-specific primers (PCR-SSP). Data were analyzed statistically using the chi square test with Yates' correction, Fisher's exact test, and Bonferroni's correction. RESULTS: Human leukocyte antigen-DR 15 was the most frequently detected antigen in this group of children and adolescents, and it also occurred more frequently in the female group, in children with onset of systemic lupus erythematosus between 0 and 9 years and between 10 to 14 years, and in the Black race group, but these associations were not statistically significants. CONCLUSION: In this group of children and adolescents with a high degree of racial admixture, we could not verify a significant association between human leukocyte antigens class II-DR and systemic lupus erythematosus.

RAPD em Pau-rosa (Aniba rosaeodora Ducke): adaptação do método para coleta de amostras in situ, ajuste das condições de PCR e apresentação de um processo para selecionar bandas reprodutíveis

O Pau-rosa (Aniba rosaeodora Ducke) é uma espécie importante economicamente para a Região Amazônica, porque sua madeira é fonte de linalol, insumo utilizado pelas perfumarias. Esta espécie foi explorada durante décadas e, ainda assim, o conhecimento acerca da diversidade genética intra-específica é muito restrito. Foram objetivos deste trabalho: 1) validar um protocolo para coleta de folhas de pau-rosa que permitisse preservar a integridade do DNA até a estocagem em "freezer"; 2) selecionar um protocolo para extração de DNA em quantidade e qualidade adequadas para geração de bandas RAPD e 3) desenvolver um critério para avaliar o grau de reprodutibilidade que pudesse auxiliar a seleção de bandas RAPD úteis para análises de diversidade genética. Imediatamente após a coleta, as folhas foram acondicionadas em tubos de polietileno com sílica gel e aí permaneceram por até 10 dias. Foram testados três protocolos para a extração de ácidos nucléicos destas folhas, condições ideais para as PCR e a reprodutibilidade dos padrões RAPD. Critérios para a eliminação das bandas que mais contribuíram para o afastamento dos resultados do ideal da reprodutibilidade total foram desenvolvidos e a significância estatística das diferenças geradas pela aplicação dos critérios ao conjunto de dados foi testada. DNA com qualidade e em quantidade suficiente para a geração de padrões RAPD, nas condições ideais definidas para as PCRs, foi obtido. A eliminação de bandas com reprodutibilidade menor que 70% não diferiu do controle. A eliminação de bandas com reprodutibilidade menor que 90% diferiu dos demais tratamentos em todos os arranjos testados (P < 5%).

Association study of the Ile349val polymorphism of the gene ADH1C and alcohol dependence

OBJECTIVE: The aim of this study was to investigate the polymorphism Ile349Val of the enzyme alcohol dehydrogenase ADH1C gene among individuals with alcohol dependence syndrome (ADS) attending Alcoholics Anonymous (AA) meetings. METHODS: A total of 120 subjects residing in Rio de Janeiro city participated in this study. Subjects were divided into two groups: a group consisting of 54 individuals from the ADS group and 66 individuals that declared not having any alcohol dependence (control group). DNA was extracted from mouth epithelial cells by phenol-chloroform method and further submitted to amplification by polymerase chain reaction (PCR). RESULTS: Our results did not show differences between the genotypes of control individuals and ADS subjects. Nevertheless, we found increased rates of alcoholism in families of ADS subjects as compared to controls. CONCLUSIONS: Our results did not show any genotype difference on the ADH1C gene when control and AA genotypes are compared.

Analysis of the Association Between Apolipoprotein E Polymorphism and Cardiovascular Risk Factors in an Elderly Population with Longevity

OBJECTIVE: To establish the allelic and genotypic frequencies related to apolipoprotein E (ApoE) polymorphism and association of the genotypes with risk factors and cardiovascular morbidity in an elderly population with longevity. METHODS: We analyzed 70 elderly patients aged 80 years or more who were part of the Projeto Veranópolis. We used the gene amplification technique through the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and cleavage with the restriction enzyme Hha I to identify the ApoE genotypes. The most frequent genotypes were compared considering biological variables and cardiovascular risks and morbidity. RESULTS: The frequencies of the E2, E3, and E4 alleles were 0.05, 0.84, and 0.11, respectively, and of the genotypes were as follows: E3E3 (0.70), E3E4 (0.22), E2E3 (0.06), and E2E2 (0.02). Individuals with the E3E4 had a mean age greater than those with the E3E3. No association was observed between the genotypes and the variables analyzed, except for obesity, which was associated with the E3E3 genotype. Individuals with the E3E4 genotype had high levels of LDL-cholesterol and fibrinogen as compared with those with the E3E3 genotype. CONCLUSION: The results suggest that the E4E4 genotype may be associated with early mortality. A balance between the protective or neutral factors and the cardiovascular risk factors may occur among the individuals with different genotypes, attenuating the negative effects of the E4 allele.

Níveis séricos de interleucina-6 (IL-6), interleucina-18 (IL-18) e proteína C reativa (PCR) na síndrome coronariana aguda sem supradesnivelamento do ST em pacientes com diabete tipo 2

FUNDAMENTO: A aterosclerose é uma doença inflamatória e níveis séricos de marcadores inflamatórios, como a interleucina 6 (IL-6), interleucina-18 (IL-18) e proteína C reativa (PCR), são utilizados para avaliação de pacientes em quadros de coronariopatia. No paciente com diabete do tipo 2, a aterosclerose está relacionada a um maior número de eventos como infarto e morte, quando comparado aos pacientes sem diabete. OBJETIVO: Avaliar a resposta inflamatória nos pacientes com diabete e eventos agudos de instabilidade coronariana. MÉTODOS: Selecionamos primariamente dois grupos de pacientes. O primeiro grupo foi composto por pacientes ambulatoriais diabéticos com angina estável (D-SCC) e presença de coronariopatia ao estudo coronariográfico (n = 36). O segundo grupo foi composto por pacientes diabéticos atendidos no pronto-socorro com quadro de síndrome coronariana aguda (D-SCA) sem supradesnivelamento do ST (n = 38). Como controle, foram utilizados pacientes sem diabete com SCA (n = 22) e SCC (n = 16). As concentrações séricas de PCR, IL-6 e IL-18 foram determinadas pelas técnicas de nefelometria (PCR) e ELISA (IL-6 e IL-18). RESULTADOS: Níveis mais elevados de IL-6 foram observados em pacientes com ou sem diabete e SCA em relação ao grupo com SCC. Por sua vez, pacientes com diabete e SCA apresentaram concentrações maiores de PCR em comparação aos outros grupos. Os níveis séricos de IL-18 não diferiram significativamente entre os pacientes estudados. CONCLUSÃO: Os resultados obtidos sugerem uma maior atividade inflamatória no paciente com quadro de instabilidade coronariana. Essa atividade inflamatória, medida pela PCR, parece ser ainda mais intensa no paciente com diabete.

Níveis de PCR são maiores em pacientes com síndrome coronariana aguda e supradesnivelamento do segmento ST do que em pacientes sem supradesnivelamento do segmento ST

FUNDAMENTO: Há grande interesse no uso de proteína C-reativa de alta sensibilidade (PCR-as) para avaliação de risco. Altos níveis de PCR-as no início da síndrome coronária aguda (SCA), antes da necrose tecidual, pode ser um marcador substituto para comorbidades cardiovasculares. OBJETIVO: Dessa forma, nosso objetivo foi estudar diferentes medidas de seguimento de níveis de PCR-as em pacientes com SCA e comparar as diferenças entre infarto do miocárdio sem elevação do segmento ST (NSTEMI) com pacientes apresentando elevação do segmento ST (STEMI). MÉTODOS: Este é um estudo observacional. Dos 89 pacientes recrutados, 60 apresentavam infarto agudo do miocárdio (IAM). Três níveis seriados de PCR-us, a nível basal na hospitalização antes de 12 horas após inicio dos sintomas, níveis de pico 36-48 horas após hospitalização e níveis de acompanhamento após 4 a 6 semanas foram analisados e comparados entre pacientes com (IAMCSST) e sem supradesnivelamento do segmento ST (IAMSSST). RESULTADOS: Pacientes com IAMCSST tinham IMC significantemente mais alta quando comparados com pacientes IAMSSST. Os níveis de creatino quinase fração MB (CK-MB) e aspartato aminotransferase (AST) eram significantemente mais altos em pacientes com IAMCSST quando comparados com pacientes com IAMSSST (p<0,05). Os níveis de PCR a nível basal e no acompanhamento não diferiram de forma significante entre os dois grupos (p=0,2152 e p=0,4686 respectivamente). Houve uma diferença significante nos níveis de pico de PCR entre os dois grupos. No grupo de pacientes com IAMCSST os níveis foram significantemente mais altos quando comparados aos pacientes com IAMSSST (p=0,0464). CONCLUSÃO: Pacientes com IAMCSST apresentam picos significantemente mais elevados de PCR quando comparados a pacientes IAMSSST. Esses dados sugerem que o processo inflamatório tem um papel independente na patogênese do infarto do miocárdio. Dessa forma, os níveis de PCR podem ajudar na estratificação de risco após o infarto do miocárdio.