Vagner Ricardo Lunge

IL28B Polymorphism Associated with Spontaneous Clearance of Hepatitis C Infection in a Southern Brazilian HIV Type 1 Population

About one-third of people infected with human immunodeficiency virus-1 (HIV-1) are coinfected with hepatitis C virus (HCV) because of shared transmission routes. Studies report that HIV-1 complicates hepatitis C infection by increasing HCV viral load and reducing spontaneous clearance. Single nucleotide polymorphisms (SNPs) upstream of the IL28B gene have been associated with spontaneous and treatment-induced clearance of HCV infection. The aim of this study was to evaluate the association between the SNP rs12979860 of the IL28B gene and spontaneous clearance of HCV infection in a Brazilian HIV-1 population. The SNP was analyzed by polymerase chain reaction (PCR) followed by restriction digestion in 138 anti-HCV-positive patients. Spontaneous clearance was observed in 34 subjects (24.6%). Genotype distribution was significantly different between spontaneous clearance and HCV chronic patients. The CT/TT genotypes conferred a nearly 3-fold increased odds to chronic HCV infection relative to the CC genotype (odds ratio, 2.78; 95% confidence interval, 1.16-6.64; p=0.011). In conclusion, the rs12979860 polymorphism is associated with spontaneous clearance of HCV in HIV-1 Brazilian infected patients.

Prevalence of hepatitis C virus in alcoholic patients: role of parenteral risk factors

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.

Genotyping of canine distemper virus strains circulating in Brazil from 2008 to 2012

Canine distemper virus (CDV) is a major pathogen of dogs and represents a serious threat to both unvaccinated and vaccinated animals. This study surveyed dogs with or without clinical signs related to canine distemper from different regions of Brazil from 2008 to 2012. A total of 155 out of 386 animals were found to be CDV positive by RT-PCR; 37 (23.8%) dogs were asymptomatic at the time of sampling, and 90 (58%) displayed clinical signs suggestive of distemper. Nineteen (12.2%) dogs had a record of complete vaccination, 15 (9.6%) had an incomplete vaccination protocol, and 76 (49%) had no vaccination record. Based on the sequence analysis of the complete hemagglutinin gene of 13 samples, 12 of the strains were characterized as Genotype South America-I/Europe. Considering criteria of at least 95% nucleotide identity to define a genotype and 98% to define a subgenotype, South America-I/Europe sequences segregated into eight different phylogenetically well-defined clusters that circulated or co-circulated in distinct geographical areas. Together, these findings highlight the relevance of CDV infection in Brazilian dogs, demonstrate the predominance of one genotype in Brazil and support the need to intensify the current control measures.

Emergence of a New Genotype of Avian Infectious Bronchitis Virus in Brazil

SUMMARY Infectious bronchitis virus (IBV) is the agent of a highly contagious disease that affects domestic fowl (Gallus gallus). Recent reports showed a high prevalence of one main IBV genotype (Brazil or BR-I) with low genetic diversity in commercial poultry flocks from Brazil. This research analyzed IBV positive poultry flocks from different rearing regions to verify the S1 gene variability and geographic distribution of variant IBV strains in recent years (2010 and 2011). Samples of IBV-positive flocks were obtained from 60 different farms. Forty-nine partial S1 gene sequences were determined and aligned for phylogenetic and amino acid similarity analyses. Eleven samples (22.4%) were similar to Massachusetts vaccine strains (Mass genotype) and 34 samples (69.4%) to the previously characterized Brazilian BR-I genotype. Interestingly, the remaining four samples (8.2%) clustered into a new IBV variant genotype (Brazil-II or BR-II), divergent from the BR-I. A unique nucleotide sequence insertion coding for five amino acid residues was observed in all the Brazilian variant viruses (BR-I and BR-II genotypes). These results show a higher genetic diversity in Brazilian IBV variants than previously described. RESUMEN Aparición de un nuevo genotipo del virus de la bronquitis infecciosa aviar en Brasil. El virus de la bronquitis infecciosa (IBV) es el agente de una enfermedad altamente contagiosa que afecta a las gallinas domésticas (Gallus gallus). Los reportes recientes muestran una alta prevalencia de un genotipo principal de este virus (Brasil o BR-I) con baja diversidad genética en las parvadas avícolas comerciales de Brasil. Esta investigación analiza las parvadas avícolas positivas a la presencia de este virus en diferentes regiones, para verificar la variabilidad del gene S1 y la distribución geográfica de las cepas variantes de este virus en los últimos años (2010 y 2011). Se recolectaron muestras del virus de bronquitis de parvadas positivas en 60 granjas diferentes. Se determinaron 49 secuencias parciales del gene S1 y se alinearon para llevar a cabo análisis filogenéticos y de similitud de aminoácidos. Once muestras (22.4%) fueron similares a la cepa vacunal Massachusetts (Mass) (genotipo Mass) y 34 muestras (69.4%) fueron similares a la cepa brasileña previamente caracterizada, genotipo BR-I. De manera interesante, las cuatro muestras restantes (8.2%) se agruparon en un nuevo genotipo variante (Brasil-II o BR-II), que es divergente de la BR-I. Una inserción única en la secuencia de nucleótidos que codifica para cinco residuos de aminoácidos se observó en todos los virus variantes de Brasil (genotipos BR-I y BR-II). Estos resultados muestran una mayor diversidad genética de las cepas variantes brasileñas del virus de la bronquitis infecciosa a diferencia de lo que se había descrito anteriormente.

Infectious bronchitis virus in different avian physiological systems—A field study in Brazilian poultry flocks

Abstract Avian infectious bronchitis is a highly contagious viral disease with economic effects on poultry agribusiness. The disease presents multi-systemic clinical signs (respiratory, renal, enteric, and reproductive) and is caused by one coronavirus (infectious bronchitis virus, IBV). Infectious bronchitis virus is classified into different serotypes and genotypes (vaccine strains and field variants). This study aimed to evaluate the occurrence of IBV in commercial poultry flocks from 3 important producing regions in Brazil and to determine the tropism of the main circulating genotypes to 3 different avian physiological systems (respiratory, digestive, urinary/reproductive). Clinical samples with suggestive signs of IBV infection were collected from 432 different poultry commercial flocks (198 from broilers and 234 from breeders). The total number of biological samples consisted of organ pools from the 3 above physiological systems obtained of farms from 3 important producing regions: midwest, northeast, and south. Infectious bronchitis virus was detected by reverse-transcription, real-time PCR of the 5′ untranslated region. The results showed 179 IBV-positive flocks (41.4% of the flocks), with 107 (24.8%) from broilers and 72 (16.8%) from breeders. There were similar frequencies of IBV-positive flocks in farms from different regions of the country, most often in broilers (average 54%) compared with breeders (average 30.8%). reverse-transcription was more frequently detected in the digestive system of breeders (40%), and in the digestive (43.5%) and respiratory (37.7%) systems of broilers. Infectious bronchitis virus genotyping was performed by a reverse-transcription nested PCR and sequencing of the S1 gene from a selection of 79 IBV-positive flocks (45 from broilers and 34 from breeders). The majority of the flocks were infected with Brazilian variant genotype than with Massachusetts vaccine genotype. These results demonstrate the predominance of the Brazilian variant (mainly in the enteric tract) in commercial poultry flocks from 3 important producing regions in Brazil.

Detection and differentiation of field and vaccine strains of canine distemper virus using reverse transcription followed by nested real time PCR (RT-nqPCR) and RFLP analysis.

Abstract Canine distemper virus (CDV) is the cause of a severe and highly contagious disease in dogs. Practical diagnosis of canine distemper based on clinical signs and laboratory tests are required to confirm CDV infection. The present study aimed to develop a molecular assay to detect and differentiate field and vaccine CDV strains. Reverse transcription followed by nested real time polymerase chain reaction (RT-nqPCR) was developed, which exhibited analytical specificity (all the samples from healthy dogs and other canine infectious agents were not incorrectly detected) and sensitivity (all replicates of a vaccine strain were positive up to the 3125-fold dilution – 100.7 TCID50). RT-nqPCR was validated for CDV detection on different clinical samples (blood, urine, rectal and conjunctival swabs) of 103 animals suspected to have distemper. A total of 53 animals were found to be positive based on RT-nqPCR in at least one clinical sample. Blood resulted in more positive samples (50 out of 53, 94.3%), followed by urine (44/53, 83.0%), rectal (38/53, 71%) and conjunctival (27/53, 50.9%) swabs. A commercial immunochromatography (IC) assay had detected CDV in only 30 conjunctival samples of these positive dogs. Nucleoprotein (NC) gene sequencing of 25 samples demonstrated that 23 of them were closer to other Brazilian field strains and the remaining two to vaccine strains. A single nucleotide sequences difference, which creates an Msp I restriction enzyme digestion, was used to differentiate between field and vaccine CDV strains by restriction fragment length polymorphism (RFLP) analysis. The complete assay was more sensitive than was IC for the detection of CDV. Blood was the more frequently positive specimen and the addition of a restriction enzyme step allowed the differentiation of vaccine and Brazilian field strains.

Prevalence and genotypic diversity of cervical human papillomavirus infection among women from an urban center in Brazil.

Human papillomavirus (HPV) infection is a common viral sexually transmitted infection and the main cause of cervical cancer in women worldwide. Epidemiological data on the prevalence of HPV in a given population is essential for the establishment of effective prevention strategies. The aim of this study was to determine HPV prevalence in women who attended a public health service within an urban center in Brazil. Cervical samples were collected from 337 women recruited from a primary public health care clinic in the city of Cruz Alta located in Rio Grande do Sul, the southernmost State of Brazil. Samples were analyzed for HPV DNA and with Pap smear screening tests. HPV was detected in 114 (34%) women. HPV type analysis revealed that 95 (83.3%) of those represented infections with a single genotype, while 19 (16.7%) were mixed genotype infections. High- and low-risk HPV genotypes were detected in 83 (72.8%) and 48 (42.1%) samples, respectively. Furthermore, a great diversity of HPV genotypes was observed (18 high-risk, 12 low-risk, and 1 indeterminate). The most commonly identified low-risk types were candHPV62 (7.9%) and 61 (5.3%), while the most common high-risk types were 16 and 33 (8.8% each). Abnormal cytology was observed in 10 (3.0%) women, 9 of which were infected with HPV. Of the remaining 327 women with normal cytology results, 107 (32.7%) were positive for HPV DNA. HPV infection was correlated with younger age (less than 40 years), a first Pap smear, and other vaginal infections.

Prevalência de subtipos do HIV-1 em amostra de pacientes de um centro urbano no sul do Brasil

OBJECTIVE To estimate the prevalence of HIV-1 subtypes and analyze factors associated. METHODS A cross-sectional study was performed with a convenience sample of 80 adult HIV-positive patients, users of an AIDS/STD specialized service, in the city of Canoas, Southern Brazil, between July 2008 and January 2009. Determination of HIV subtypes was performed with the amplification of viral genome fragment, using polymerase chain reaction, followed by sequencing of the amplified fragments. Sociodemographic, clinical and behavioral variables were collected in a structured questionnaire. Univariate statistical analysis was performed, using chi-square test and Students t-test. RESULTS A higher prevalence of subtype C was found (43.8%; 95% CI: 32.9;54.6), followed by CRF31_BC (35.0%; 95% CI: 24.6;45.5) and subtypes B (18.8%; 95% CI: 10.2;27.3) and F (2.4%; 95% CI: 0;5.9). Other HIV-1 subtypes were not observed. Patients infected with CRF31_BC were diagnosed more recently than patients infected with subtype B (p<0.05). In addition, there was a higher frequency of co-infection with other viruses (hepatitis B and C and human T-lymphotropic viruses) in individuals with CRF31_BC, compared to other subtypes. With regard to sociodemographic aspects, there were no differences in the distribution of subtypes and recombinant forms, in terms of gender and sexual practices. CONCLUSIONS Results obtained indicate a higher frequency of subtype C and CRF31_BC in this urban center of Southern Brazil, with possible different ways of transmission.OBJETIVO: Estimar la prevalencia de los subtipos de VIH-1 y analizar factores asociados. METODOS: Se realizo un estudio transversal con muestra de conveniencia de 80 pacientes adultos VIH-positivos atendidos en servicio especializado en DST/Sida en Canoas, Sur de Brasil, en el periodo de julio de 2008 a enero de 2009. La determinacion de los subtipos de VIH fue realizada por amplificacion de fragmento del genoma viral por la reaccion en cadena de la polimerasa seguida de la secuenciacion de los fragmentos amplificados. Variables sociodemograficas, clinicas y conductuales fueron colectadas en cuestionario estructurado. Se realizo analisis estadistico univariado utilizando las pruebas de chi-cuadrado y t de Student. RESULTADOS: Se observo una prevalencia mayor del subtipo C (43,8%; IC 95%:32,9;54,6), seguido de la CRF31_BC (35,0%; IC 95%: 24,6;45,5) y subtipos B (18,8%; IC 95%: 10,2;27,3) y F (2,4%; IC 95%: 0;5,9). Otros subtipos de VIH-1 no fueron observados. Pacientes infectados con CRF31_BC presentaron diagnostico mas reciente que los pacientes infectados con el subtipo B (p<0,05). Se observo tambien una mayor frecuencia de co-infeccion con otros virus (hepatitis B y C y T-linfotropicos humanos) en los individuos portadores del CRF31_BC en comparacion con los demas subtipos. Con relacion a los aspectos sociodemograficos, no fueron observadas diferencias en la distribucion de los subtipos y formas recombinantes con relacion al sexo y practicas sexuales. CONCLUSIONES: Los datos obtenidos en el presente estudio indican una mayor frecuencia del subtipo C y de la CRF31_BC en este centro urbano del sur de Brasil y sugieren que dichos subtipos pueden presentar vias de transmision diferentes.

Identification of porcine intestinal spirochetes by PCR-restriction fragment length polymorphism analysis of ribosomal DNA encoding 23S rRNA

The Brachyspira (formerly Serpulina) species rrl gene encoding 23S ribosomal RNA (rRNA) was used as a target for amplification of a 517bp DNA fragment by polymerase chain reaction (PCR). The primers for PCR amplification had sequences that were conserved among Brachyspira 23S rRNA gene and were designed from nucleotide sequences of Brachyspira hyodysenteriae, Serpulina intermedia, Brachyspira innocens and Brachyspira pilosicoli available from the GenBank database. Digestion of PCR-generated products from reference and field isolates of swine intestinal spirochetes with restriction enzymes Taq I and Alu I revealed five restriction fragment length polymorphism (RFLP) patterns. Each RFLP pattern corresponded to previously established genetic groups including B. hyodysenteriae (I), S. intermedia/B. innocens (II), Brachyspira murdochii (III), B. pilosicoli (IV) and B. alvinipulli (V). The 23S rRNA PCR/RFLP provided a relatively simple genotypic method for identification of porcine pathogenic B. hyodysenteriae and B. pilosicoli.

Caracterização antigênica e fenotípica de cepas de Pasteurella multocida isoladas de pulmões de suínos com pneumonia e/ou pleurite

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.