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.

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.

Abstract 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.

ABSTRACT INTRODUCTION: Human metapneumovirus (hMPV) is an emergent human respiratory pathogen. This study aimed to evaluate the performance of direct immunofluorescence (DIF) to detect hMPV in a clinical laboratory setting. METHODS: Nasopharyngeal aspirate samples (448) of children and adults with respiratory illness were used to detect hMPV by using DIF and real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. RESULTS: In all, 36 (8%) samples were positive by DIF and 94 (21%) were positive by qRT-PCR. Direct immunofluorescence specificity was 99% and sensitivity was 38%. CONCLUSIONS: DIF is not very sensitive under clinical laboratory settings.

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.

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.

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.

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.

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%) were positive for the virus by both methods and 25 samples were positive only by nested-PCR (67.5% 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.

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% versus 72%; p < 0.01); injection drug use (13% versus 0.7%; p < 0.01); anti-HCV positivity at start of HD therapy (60% versus 4%; 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% CI 1.2 -3.8)]; previous blood transfusion [OR: 3.7 (95% CI 1.4 - 9.5)]; and injection drug use [OR: 22.6 (95% CI 4.2 - 119.6)]. CONCLUSION: Injection drug use was an independent risk factor for HCV infection among chronic renal disease patients on HD.

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.

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%) eram anti-VHC positivos. Doze (71%) 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%) 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% vs. 34%), e na presença de fatores de risco parenteral (94% vs. 10%). A comparação entre alcoolistas com e sem aminotransferases elevadas mostrou diferença significativa apenas na taxa de anti-VHC positivo (24% vs. 7%). 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% vs. 20%). 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.

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%) were anti-hepatitis C virus positive. Of these, 12 (71%) 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%) 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% vs. 34%), and presence of parenteral risk factors (94% vs. 10%). Comparison between alcoholic patients with and without elevated aminotransferases showed significant difference only in the rate of positive anti-hepatitis C virus antibodies (24% vs. 7%). 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% vs. 20%). 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.

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%) e matrizes descartadas (48 isolamentos, 32%).

Portions of muscle from carcasses of 74 fattening pigs and 150 culled sows were collected from abattoir in the State of Rio Grande do Sul, Brazil. The samples were inoculated into EMJH (Ellinghausen, MacCullough, Johnson & Harris) medium and the culture filtered through 0.45µm membrane on blood agar. Arcobacter sp were isolated, 69 isolates, typed by PCR as Arcobater butzleri, respectively from fattening pigs (21 isolates, 29.3%) and culled sows (48 isolates, 32%).

Foi analisada a variabilidade antigênica e fenotípica de 22 cepas de Pasteurella multocida isoladas de pulmões de suínos com pneumonia e/ou pleurite. Os testes fenotípicos foram realizados pela determinação 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 padrões bioquímicos distintos. Quanto à sensibilidade a nove agentes antimicrobianos, verificou-se grande variação, com apenas 50% 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% dos isolados se mostraram sensíveis. A menor eficiência foi demonstrada pela espectinomicina (100 mg) com 45,5%. A caracterização antigênica consistiu na sorotipagem capsular e determinação de variabilidade do gene de proteína de membrana externa (ompH) pela reação em cadeia da polimerase (PCR) e digestão com cinco enzimas de restrição. Das 22 cepas, 21 foram compatíveis com sorotipo capsular A e uma com D. A caracterização do gene ompH agrupou os isolados em sete padrões distintos que apresentaram boa correlação com os testes bioquímicos.

The antigenic and phenotypic variation among 22 strains of Pasteurella multocida isolated from pig lungs with pneumonia and/or pleuritic lesions was studied. Phenotypic tests consisted of biochemical assays and antimicrobial sensitivity tests. All isolates fermented mannitol and sorbitol, but none fermented arabinose. Fourteen fermented xylose, 4 trehalose, 2 dulcitol and 1 maltose. Analyzing these characteristics, 5 different biochemical groups were revealed. There was a wide variation in the results of antimicrobial sensitivity testing to 9 products, with 50% of isolates showing sensitivity at least to 7. No antimicrobial agent was able to inhibit all strains. The highest efficiency (72% sensitivity) was observed with amoxycillin (30 mg). Spectinomycin (100 mg) presented the lowest efficiency, 45.5%. Antigenic characterization consisted in capsular serotyping and assessment of variability of an outer membrane protein gene (ompH), using polymerase chain reaction (PCR) and digestion with 5 endonuclease enzymes (restriction fragment length polymorphism, RFLP). Out of 22 strains, 21 were classified as capsular type A and one as type D. Characterization of ompH revealed 7 different patterns. They corresponded well to previously established biochemical groups.