Identification of enteric viruses circulating in a dog population with low vaccine coverage

ABSTRACT Although the use of vaccines has controlled enteric diseases in dogs in many developed countries, vaccine coverage is still under optimal situation in Brazil. There is a large population of nonimmunized dogs and few studies about the identification of the viruses associated with diarrhea. To address this situation, stool samples from 325 dogs were analyzed by polymerase chain reaction for the detection of common enteric viruses such as Canine adenovirus (CAdV), Canine coronavirus (CCoV), Canine distemper virus (CDV), Canine rotavirus (CRV) and Carnivorous protoparvovirus 1 (canine parvovirus 2; CPV-2). At least one of these species was detected in 56.6% (184/325) of the samples. The viruses detected most frequently in either diarrheic or nondiarrheic dog feces were CPV-2 (54.3% of the positive samples), CDV (45.1%) and CCoV (30.4%), followed by CRV (8.2%) and CAdV (4.9%). Only one agent was detected in the majority of the positive samples (63%), but co-infections were present in 37% of the positive samples and mainly included CDV and CPV-2. The data presented herein can improve the clinical knowledge in regions with low vaccine coverage and highlight the need to improve the methods used to control these infectious diseases in domestic dogs.

Increasing prevalence and dissemination of invasive nontyphoidal Salmonella serotype Typhimurium with multidrug resistance in hospitalized patients from southern Brazil

ABSTRACT Introduction: Nontyphoidal Salmonella serotypes are the main cause of human food-borne infection, including several hospitalization cases in the developing countries. Aim: To detect the main serotypes and to characterize the antibiotic resistance of human non-enteric and enteric nontyphoidal Salmonella from clinical isolates in Brazil. Methods: Salmonella serotypes were identified by microbiological and molecular methods. Susceptibility testing to antibiotics was performed by agar disk diffusion. Real-time PCRs were carried out for the detection of the genus Salmonella as well as serotypes Typhimurium and Enteritidis. Results: A total of 307 nontyphoidal Salmonella were isolated from 289 different patients in a reference laboratory (LACEN-RS) from Southern Brazil in a six-year period (2010-2015). There were 45 isolates from emerging cases and 244 from sporadic cases in hospitalized patients. Non-enteric isolates were detected in 42.6% of the patients from sources such as urine, blood and other clinical fluids. Serological and PCR-specific tests demonstrated that Typhimurium (48.4%) and Enteritidis (18.3%) were the most frequent serotypes. Typhimurium isolates were generally resistant to three or more antibiotic classes, while Enteritidis isolates to one or two classes. Typhimurium was the most frequent serotype in all samples (48.4%), mainly among the hospitalized patients (55.6%), and presented the highest rates of multidrug resistance (59.3% of the isolates of this serotype). Further, the prevalence of this serotype increased along the years of the study in comparison to other nontyphoidal Salmonella serotypes. Conclusion: Greater public health attention should be given to prevent salmonellosis in the community and in hospital settings to reduce the rates of Typhimurium strains with multidrug resistance.

Human metapneumovirus in Southern Brazil

INTRODUCTION Infections caused by respiratory viruses are important problems worldwide, especially in children. Human metapneumovirus (hMPV) is a respiratory pathogen and causes severe infections with nonspecific symptoms. This study reports the hMPV occurrence and dissemination in southern Brazil and compares the frequency of occurrence of this virus and the human respiratory syncytial virus (hRSV) in the epidemiological weeks in a three-year period (2009-2011). METHODS: In total, 545 nasopharyngeal (NP) specimens from individuals with Severe Acute Respiratory Syndrome (SARS) who were negative for other seven respiratory viruses were analyzed for the presence of hMPV. Human metapneumovirus was detected by direct immunofluorescence and real-time reverse transcription polymerase chain reaction. RESULTS: hMPV was detected in 109 patients from the main geographic regions of the southernmost state of Brazil, presenting similar overall prevalence in males (46.8%) and females (53.2%). Among children who were less than six years old, hMPV was detected in 99 samples of all age groups, with a higher frequency in infants who were less than one year old (45.7%) compared to all other age groups until six years. hMPV and hRSV infection occurred in almost the same epidemiological weeks (EWs) of each year, with peaks of incidence between EW 31/37 and EW 26/38 for the years 2009 and 2011, respectively. hMPV was further detected in several cases of SARS and it was the only virus detected in three deaths. CONCLUSIONS These findings indicate that hMPV is in circulation in southern Brazil and highlight the importance of diagnosing hMPV for influenza-like illness in the population. (AU)

Valor prognóstico dos níveis de DNA circulante em pacientes graves e em pacientes com trauma / Prognostic value of circulating DNA levels in critically ill and trauma patients

Nos últimos anos, o número de estudos que investigam os ácidos nucleicos circulantes como potenciais biomarcadores tem aumentado. A detecção desses biomarcadores é uma alternativa minimamente invasiva para o diagnóstico e o prognóstico de diversas condições clínicas. O valor dos níveis de DNA circulante como biomarcador preditivo foi demonstrado em pacientes com numerosas patologias agudas que apresentam riscos elevados de necessitar cuidados intensivos e de sofrer mortalidade hospitalar. Os mecanismos pelos quais os níveis de DNA circulante aumentam em pacientes com essas condições ainda são obscuros. Nesta revisão, focalizamos o potencial uso deste biomarcador para predição prognóstica em pacientes graves e pacientes com trauma. A revisão da literatura foi feita por meio de busca no MedLine utilizando o PubMed em inglês.

The number of studies investigating circulating nucleic acids as potential biomarkers has increased in recent years. The detection of such biomarkers is a minimally invasive alternative for the diagnosis and prognosis of various clinical conditions. The value of circulating DNA levels as a predictive biomarker has been demonstrated in patients suffering from numerous acute pathologies that have a high risk of intensive care needs and in-hospital deaths. The mechanism by which circulating DNA levels increase in patients with these conditions remains unclear. In this review, we focused on the potential use of this biomarker for prognosis prediction in critically ill and trauma patients. The literature review was performed by searching MedLine using PubMed in the English language.

A complete molecular biology assay for hepatitis C virus detection, quantification and genotyping

Introduction Molecular biology procedures to detect, genotype and quantify hepatitis C virus (HCV) RNA in clinical samples have been extensively described. Routine commercial methods for each specific purpose (detection, quantification and genotyping) are also available, all of which are typically based on polymerase chain reaction (PCR) targeting the HCV 5′ untranslated region (5′UTR). This study was performed to develop and validate a complete serial laboratory assay that combines real-time nested reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) techniques for the complete molecular analysis of HCV (detection, genotyping and viral load) in clinical samples. Methods Published HCV sequences were compared to select specific primers, probe and restriction enzyme sites. An original real-time nested RT-PCR-RFLP assay was then developed and validated to detect, genotype and quantify HCV in plasma samples. Results The real-time nested RT-PCR data were linear and reproducible for HCV analysis in clinical samples. High correlations (> 0.97) were observed between samples with different viral loads and the corresponding read cycle (Ct - Cycle threshold), and this part of the assay had a wide dynamic range of analysis. Additionally, HCV genotypes 1, 2 and 3 were successfully distinguished using the RFLP method. Conclusions A complete serial molecular assay was developed and validated for HCV detection, quantification and genotyping. .

Oseltamivir-resistant influenza A(H1N1)pdm09 virus in southern Brazil

The neuraminidase (NA) genes of A(H1N1)pdm09 influenza virus isolates from 306 infected patients were analysed. The circulation of oseltamivir-resistant viruses in Brazil has not been reported previously. Clinical samples were collected in the state of Rio Grande do Sul (RS) from 2009-2011 and two NA inhibitor-resistant mutants were identified, one in 2009 (H275Y) and the other in 2011 (S247N). This study revealed a low prevalence of resistant viruses (0.8%) with no spread of the resistant mutants throughout RS.

A Multiplex real-time PCR for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from Brazilian commercial poultry flocks

Mycoplasma gallisepticum (MS) and Mycoplasma synoviae (MS) are important avian pathogens and cause economic losses to the poultry industry. Molecular biology techniques are currently used for a rapid detection of these pathogens and the adoption of control measures of the diseases. The aim of this study was to develop and validate a technique for simultaneous detection of MG and MS by multiplex real time polymerase chain reaction (PCR). The complete assay (Multiplex MGMS) was designed with primers and probes specific for each pathogen and developed to be carried out in a single tube reaction. Vaccines, MG and MS isolates and DNA from other Mycoplasma species were used for the development and validation of the method. Further, 78 pooled clinical samples from different poultry flocks in Brazil were obtained and used to determine the sensitivity and specificity of the technique in comparison to 2 real time PCR assays specific for MG (MG PCR) and MS (MS PCR). The results demonstrated an agreement of 100% (23 positive and 44 negative samples) between Multiplex MGMS and MG PCR in the analysis of 67 samples from MG positive and negative poultry flocks, and an agreement of 96.9% between Multiplex MGMS and MS PCR in the analysis of 64 samples from MS positive and negative poultry flocks. Considering the single amplification tests as the gold standard, the Multiplex MGMS showed 100% of specificity and sensitivity in the MG analysis and 94.7% sensitivity and 100% specificity in the MS analysis. This new assay could be used for rapid analysis of MG and MS in the poultry industry laboratories.

Human papillomavirus detection and typing using a nested-PCR-RFLP assay

BACKGROUND: It is clinically important to detect and type human papillomavirus (HPV) in a sensitive and specific manner. OBJECTIVES: Development of a nested-polymerase chain reaction-restriction fragment length polymorphism (nested-PCR-RFLP) assay to detect and type HPV based on the analysis of L1 gene. METHODS: Analysis of published DNA sequence of mucosal HPV types to select sequences of new primers. Design of an original nested-PCR assay using the new primers pair selected and classical MY09/11 primers. HPV detection and typing in cervical samples using the nested-PCR-RFLP assay. RESULTS: The nested-PCR-RFLP assay detected and typed HPV in cervical samples. Of the total of 128 clinical samples submitted to simple PCR and nested-PCR for detection of HPV, 37 (28.9 percent) were positive for the virus by both methods and 25 samples were positive only by nested-PCR (67.5 percent increase in detection rate compared with single PCR). All HPV positive samples were effectively typed by RFLP assay. CONCLUSION: The method of nested-PCR proved to be an effective diagnostic tool for HPV detection and typing.

Hepatitis C in hemodialysis: the contribution of injection drug use

BACKGROUND: Hepatitis C virus (HCV) infection is the most common cause of acute or chronic hepatitis in patients on hemodialysis (HD). The purpose of this study was to describe the prevalence of positive HCV RNA and investigate injection drug use as an emerging risk factor in patients with chronic renal disease on HD. METHODS: This was a multicenter cross-sectional study with 325 patients with chronic renal disease on HD in the period between August 1, 2005 to August 30, 2006, receiving care at four institutions in the city of Porto Alegre, Southern Brazil. Epidemiological data were collected by means of a structured questionnaire. The following laboratory tests were performed: alanine aminotransferase (ALT), anti-hepatitis C virus antibodies (anti-HCV), and qualitative polymerase chain reaction (PCR). RESULTS: Of 325 patients, 68 had positive HCV RNA results. The comparison between patients with positive and negative PCR results revealed significant differences in duration of HD (mean = 71 versus 52.4 months; p = 0.02); previous blood transfusion (92 percent versus 72 percent; p < 0.01); injection drug use (13 percent versus 0.7 percent; p < 0.01); anti-HCV positivity at start of HD therapy (60 percent versus 4 percent; p < 0.01); and mean ALT value (39 versus 26.5; p < 0.01). Logistic regression analysis showed a positive HCV RNA independently associated to being on HD for more than five years [OR: 2.1 (95 percent CI 1.2 -3.8)]; previous blood transfusion [OR: 3.7 (95 percent CI 1.4 - 9.5)]; and injection drug use [OR: 22.6 (95 percent CI 4.2 - 119.6)]. CONCLUSION: Injection drug use was an independent risk factor for HCV infection among chronic renal disease patients on HD.

Biomarcadores prognósticos no traumatismo crânio-encefálico grave: [revisão] / Outcome biomarkers following severe traumatic brain injury: [review]

O trauma é a principal causa de morte em pessoas entre 1 e 44 anos de idade. O traumatismo crânio-encefálico é o principal fator determinante da mortalidade e da morbidade decorrentes do trauma. A predição do prognóstico é um dos principais problemas associados ao traumatismo crânio-encefálico grave, já que o valor preditivo variável da avaliação clínica complica a identificação de pacientes com maior risco para desenvolvimento de lesões secundárias e desfecho fatal. Devido a estas questões, há considerável interesse no desenvolvimento de biomarcadores que reflitam a gravidade do dano cerebral e que se correlacionem com mortalidade e prognóstico funcional em longo prazo. As proteínas S100B e enolase neuronal específica estão entre os marcadores mais estudados para este fim, mas há também estudos com a proteína glial fibrilar ácida, a creatinino quinase cerebral, a proteína mielina básica, o ácido desoxirribonucléico plasmático, a proteína de choque quente 70, o fator von Willebrand, as metaloproteinases, o fator neurotrófico derivado do cérebro, dentre outros. Evidências sugerem que a inflamação, o estresse oxidativo, a excitotoxicidade, as respostas neuroendócrinas e a apoptose têm um importante papel no desenvolvimento de lesões secundárias. Marcadores envolvidos nestes processos também estão sendo estudados no traumatismo crânio-encefálico. Revisamos estes marcadores, muitos dos quais apresentam resultados promissores para uma futura aplicação clínica.

Trauma is the leading cause of death of people from 1 to 44 years of age. Traumatic brain injury is the main determinant for mortality and morbidity caused by trauma. Outcome prediction is one of the major problems related to severe traumatic brain injury because clinical evaluation has an unreliable predictive value and complicates identification of patients with higher risk of developing secondary lesions and fatal outcome. That is why, there is considerable interest in development of biomarkers that reflect the severity of brain injury and correlate with mortality and functional outcome. Proteins S100B and neuron specific enolases are among the markers most studied for this purpose, however some studies are investigating glial fibrillary acidic protein, creatinine phospokinase, isoenzime B, myelin basic protein, plasma desoxiribonucleic acid, heat shock protein 70, von Willebrand factor, metalloproteinases and brain-derived neurotrophic factor, among others. Evidence suggests that inflammation, oxidative stress, excitotoxicity, neuroendocrine responses and apoptosis play an important role in the development of secondary lesions. Markers involved in these processes are being studied in traumatic brain injury. We reviewed these biomarkers, some of which present promising results for future clinical application.

Prevalence of hepatitis C virus in alcoholic patients: role of parenteral risk factors / Prevalência do vírus da hepatite C em alcoolistas: a atuação dos fatores de risco parenterais

BACKGROUND: The prevalence of hepatitis C virus (HCV) infection is elevated in alcoholic patients, but the risk factors are unclear. The role of parenteral risk factors are indeterminated in this population. AIMS: To determine the prevalence of hepatitis C virus infection in alcoholic patients admitted to a detoxification unit and to evaluate the presence of underlying parenteral risk factors. METHODS: A total of 114 consecutive unselected alcoholic patients admitted to a single chemical dependency unit during 14 month were included. Epidemiological data and history of parenteral risk factors for hepatitis C virus infection were obtained with a standardized questionnaire. Blood was collected for determination of aminotransferases and anti-hepatitis C virus antibodies (ELISA-3). Positive samples were confirmed by polymerase chain reaction and tested for genotype. RESULTS: Among the 114 alcoholics, 17 (15 percent) were anti-hepatitis C virus positive. Of these, 12 (71 percent) had detectable serum HCV-RNA by PCR. Genotype 1 was found in six cases and genotype 3 in five (one patient was undetermined). Forty-nine (43 percent) patients had elevated serum ALT and/or AST at baseline. The comparison between the 17 positive and the 97 negative patients showed significant differences in mean serum ALT levels (42 ± 41 IU/L vs. 22 ± 20 IU/L), rate of elevated ALT (65 percent vs. 34 percent), and presence of parenteral risk factors (94 percent vs. 10 percent). Comparison between alcoholic patients with and without elevated aminotransferases showed significant difference only in the rate of positive anti-hepatitis C virus antibodies (24 percent vs. 7 percent). Furthermore, among the 17 anti-hepatitis C virus positive patients, the rate of detectable HCV-RNA was significantly higher in the 12 with elevated aminotransferases versus the 5 with normal aminotransferases (92 percent vs. 20 percent). CONCLUSIONS: There was a high prevalence of anti-hepatitis C virus antibodies in alcoholics and the majority was confirmed by the presence of detectable HCV-RNA. Intravenous drug use was the main risk factor for hepatitis C virus infection in this population.

RACIONAL: A prevalência da infecção pelo vírus da hepatite C (VHC) é elevada em pacientes alcoolistas, porém os fatores de risco não estão bem estabelecidos. O papel dos fatores de risco parenterais permanece ainda indefinido nessa população. OBJETIVOS: Determinar a prevalência da infecção pelo VHC em alcoolistas internados em uma unidade de desintoxicação, e avaliar a presença de fatores de risco parenteral subjacentes. PACIENTES E MÉTODOS: Foram estudados 114 alcoolistas, não selecionados, consecutivamente admitidos em uma unidade de dependência química durante 14 meses. Através de questionário estruturado, obtiveram-se os dados epidemiológicos e história de fatores de risco parenteral para infecção pelo VHC. Foi coletado sangue para determinação de aminotransferases e anticorpos anti-VHC (ELISA-3). As amostras positivas foram confirmadas pela PCR e determinado o genótipo. RESULTADOS: Entre os 114 alcoolistas, 17 (15 por cento) eram anti-VHC positivos. Doze (71 por cento) tinham RNA do VHC detectável por PCR no soro. O genótipo 1 foi encontrado em seis casos e o genótipo 3 em cinco (em um paciente foi indeterminado). Quarenta e quatro (43 por cento) pacientes tinham ALT e/ou AST elevadas. A comparação entre os 17 pacientes positivos e os 97 negativos mostrou diferenças significativas na média do nível da ALT (42 ± 41 UI/L vs. 22 ± 20 UI/L), na taxa de ALT elevada (65 por cento vs. 34 por cento), e na presença de fatores de risco parenteral (94 por cento vs. 10 por cento). A comparação entre alcoolistas com e sem aminotransferases elevadas mostrou diferença significativa apenas na taxa de anti-VHC positivo (24 por cento vs. 7 por cento). Entretanto, entre os 17 pacientes anti-VHC positivos, a taxa de RNA do VHC detectável no soro foi significativamente maior entre os 12 com aminotransferases elevadas do que entre os 5 com aminotransferases normais (92 por cento vs. 20 por cento). CONCLUSÃO: A prevalência de anti-VHC foi elevada em alcoolistas, sendo a maioria confirmada pela presença do RNA do VHC no soro. O uso de drogas injetáveis foi o principal fator de risco para infecção pelo VHC nesta população.

Isolamento de Arcobacter butzleri de músculos de carcaças de suínos de terminaçäo e de matrizes descartadas abatidos em um matadouro no Estado do Rio Grande do Sul, Brasil / Isolation of Arcobacter butzleri from muscles of the carcasses of fettening pigs and culled sows slaughtered in abattoir in the State of Rio Grande do Sul, Brazil

Foram colhidos fragmentos de músculos da carcaça de 74 suínos de abate e de 150 porcas descartadas de granjas de ciclo completo, abatidos em frigorífico no Rio Grande do Sul. Os materiais foram inoculados em meio EMJH (Ellinghausen, MacCullough, Johnson & Harris), incubados a 30ºC e o cultivo foi passado a placas de agar sangue através de membrana de 0,45µm. Foram obtidas 69 amostras de Arcobacter sp. classificadas por PCR como A. butzleri, respectivamente de suínos de abate (21 isolamentos, 29,3 por cento) e matrizes descartadas (48 isolamentos, 32 por cento).

Caracterizaçäo antigênica e fenotípica de cepas de Pasteurella multocida isoladas de pulmöes de suínos com pneumonia e/ou pleurite / Antigenic and phenotypic characterization of Pasteurella multocida strains isolated from the lungs of pigs with pneumonia and/or pleuritic lesions

Foi analisada a variabilidade antigênica e fenotípica de 22 cepas de Pasteurella multocida isoladas de pulmöes de suínos com pneumonia e/ou pleurite. Os testes fenotípicos foram realizados pela determinaçäo de características bioquímicas e sensibilidade a agentes antimicrobianos. Todos os isolados fermentaram manitol e sorbitol, mas nenhum arabinose; 14 foram capazes de metabolizar xilose, quatro trealose, dois dulcitol e um maltose. A análise destas características permitiu agrupar os isolados em 5 padröes bioquímicos distintos. Quanto à sensibilidade a nove agentes antimicrobianos, verificou-se grande variaçäo, com apenas 50 por cento dos isolados sensíveis a pelo menos sete dos nove antibióticos testados. Nenhum princípio ativo foi capaz de inibir todos os isolados. A melhor eficiência foi observada com a amoxicilina (30 mg); 72,7 por cento dos isolados se mostraram sensíveis. A menor eficiência foi demonstrada pela espectinomicina (100 mg) com 45,5 por cento. A caracterizaçäo antigênica consistiu na sorotipagem capsular e determinaçäo de variabilidade do gene de proteína de membrana externa (ompH) pela reaçäo em cadeia da polimerase (PCR) e digestäo com cinco enzimas de restriçäo. Das 22 cepas, 21 foram compatíveis com sorotipo capsular A e uma com D. A caracterizaçäo do gene ompH agrupou os isolados em sete padröes distintos que apresentaram boa correlaçäo com os testes bioquímicos

Isolation of Arcobacter spp from poultry carcasses, in Brazil

Foram isoladas 48 amostras de Arcobacter spp de 37 carcaças de frangos colhidas em frigorífico, prontas para consumo, entre 80 carcaças examinadas. Foram feitas tentativas de cultivo a partir de pele e de músculo, sendo obtidas 25 cultivos positivos de músculo e 23 de pele. As bactérias foram classificadas pelas características fenotípicas e pelo teste de PCR e PCR múltiplo, obtendo-se 41 amostras classificadas como Arcobacter butzleri e 07 com classificaçäo a nível de gênero Arcobacter sp. Estes säo os primeiros relatos sobre a ocorrência das bactérias em carcaças de animais no Brasil.

Resposta sustentada após 1 ano de interferon isolado ou associado a ribavirina em pacientes com cirrose pelo vírus C / Sustained response after one-year treatment with interferon, exclusively or in association with ribavirin in positive hepatitis c virus cirrhotic patients

O trabalho teve por objetivo avaliar a taxa de resposta completa ao final do tratamento e de resposta sustentada em pacientes com cirrose pelo vírus da hepatite C tratados com interferon isolado ou combinado com ribavirina. Um total de 33 pacientes cirróticos ambulatoriais, VHC positivos, sem outras causas identificáveis de doença hepática, compensados e classificados como Child-Pugh A, foram divididos em dois grupos, de forma não randomizada...

Prevalência de diabetes mellitus em pacientes com hepatite C / Prevalence of diabetes mellitus in patients with chronic hepatitis C

O vírus da hepatite C pode cursar com manifestações extra-hepáticas relacionadas a distúrbio imunológico. Este trabalho objetivou verificar a prevalência de Diabetes mellitus em pacientes com hepatite crônica C. Entre novembro de 1992 e dezembro de 1997 foram avaliados prospectivamente 140 pacientes ambulatoriais e consecutivos, com anti-VHC (ELISA-2) e PCR positivos...

Detecção e tipagem molecular de Papilomavírus Humano (HPV) em 460 amostras e colo uterino: estudo comparativo com exaes citopatológico e colposcópico / Human Papillomavirus (HPV) detection and molecular typing from 460 cervical samples: comparative study between colposcopy and citology (Papanicolaou smears)

A Reação em Cadeia da Polimerase (PCR – Polymerase Chain Reaction) é uma técnica de amplificação enzimática de sequências específicas de ácidos nucléicos. Essa técnica tem sido amplamente descrita para detecção e tipagem do Papilomavírus Humano (HPV – Human Papillomavirus). Neste trabalho, 460 amostras de colo uterino foram avaliadas para a presença do DNA do HPV pela técnica de PCR. Amostras positivas foram subsequentemente tipadas por RFLP (Restriction Fragment Polymorphism). Os resultados de PCR-RFLP foram comparados com os exames colposcópico e citopatológico (Papanicolaou). O PCR-RFLP demonstrou ser uma técnica eficaz na detecção e tipagem virais, apresentando maior sensibilidade do que os exames citopatológico e colposcópico.