Nuclei ultrastructural changes of C6/36 cells infected with virus dengue type 2 / Cambios ultraestructurales en núcleos de células C6/36 infectadas con virus dengue de tipo 2

ABSTRACT Introduction: Dengue virus replication has been considered mainly cytoplasmic, however, studies indicate that some flaviviruses may use the intranuclear pathway as part of the machinery that the virus uses to increase infection capacity in the host cell. This paper describes alterations at nuclear level in the cell infected with dengue, which are likely involved in the virus replication processes. Objective: This paper addresses the ultrastructural observations of C6/36 cells of the Aedes albopictus mosquito infected with dengue virus type 2. Materials and methods: C6/36 cells were infected in culture medium with the serum of a patient positively diagnosed for dengue 2. Subsequently, the cells were incubated for 10 days and the cytopathic effect was assessed. The cells were processed for immunofluorescence assays and transmission electron microscopy. Results: The immunofluorescence assays confirmed the presence of viral protein E associated with cellular syncytia in the culture. In the ultrastructural study, the infected cells showed vesicular-tubular structures and dilated cisterns of the endoplasmic reticulum at the cytoplasmic level. Viral particles were found exclusively in cytoplasm localized within the vacuoles. Nuclei of cellular syncytia showed membrane structures arranged in a circular shape and, in some cases, these syncytia displayed lysis; in no case viral particles were observed at the nuclear level. Conclusions: The ultrastructural alterations of nuclei in cells infected with the dengue virus using electron microscopy techniques had not been reported before, as far as we know. It is likely that such modifications are associated with replicative processes at an intranuclear level as an alternate replication mechanism.

RESUMEN Introducción. La replicación del virus del dengue se ha considerado principalmente citoplásmica; sin embargo, en diversos estudios se ha informado que algunos flavivirus pueden utilizar factores intranucleares como parte de la maquinaria que utilizan para aumentar la capacidad de infección en la célula huésped. En este trabajo se describen las alteraciones a nivel nuclear en células infectadas con dengue, probablemente involucradas en procesos de replicación viral. Objetivo. Presentar las observaciones ultraestructurales de células C6/36 de Aedes albopictus infectadas con el virus del dengue de tipo 2. Materiales y métodos. Se infectaron células C6/36 con suero de un paciente con diagnóstico de dengue 2; posteriormente, se mantuvieron en medio de cultivo durante 10 días y se evaluó el efecto citopático. Las células se procesaron para los ensayos de inmunofluorescencia y microscopía electrónica de transmisión, con el fin de hacer el estudio ultraestructural. Resultados. Los ensayos de inmunofluorescencia confirmaron la presencia de la proteína E viral asociada con sincitios celulares en el cultivo. En el estudio ultraestructural, las células infectadas tenían estructuras vesiculares y tubulares, y cisternas dilatadas del retículo endoplásmico en el citoplasma. Las partículas virales se encontraron exclusivamente en vacuolas localizadas en el citoplasma. Los núcleos de los sincitios celulares contenían estructuras de membrana dispuestas en forma circular y, en algunos casos, dichos sincitios presentaban lisis. En ningún caso se observaron partículas virales en el núcleo. Conclusiones. No se habían reportado alteraciones ultraestructurales en los núcleos de células infectadas con el virus del dengue detectadas mediante técnicas de microscopia electrónica. Es probable que tales modificaciones estén asociadas con procesos intranucleares de replicación como un mecanismo alternativo.

Viremia en plasma como factor asociado a gravedad en la infección por el virus del dengue: revisión sistemática de la literatura / Plasma viraemia as a factor associated to severity in dengue virus infection: systematic review of literature

Resumen Introducción: La viremia plasmática es un factor cuyo rol en la gravedad de la infección por el virus del dengue se ha discutido ampliamente en los últimos años, siendo hallados resultados divergentes en los múltiples contextos evaluados. Métodos: Se realizó una revisión sistemática de la literatura utilizando las bases de datos Scopus, EBSCOhost, SpringerLink, Lilacs y Scielo. Se consideraron para su inclusión en la revisión estudios que evaluaran la relación entre la carga viral en plasma y las manifestaciones clínicas o la gravedad de la enfermedad. Resultados: Se obtuvieron 80 referencias en la búsqueda inicial, posterior a la aplicación de los criterios de inclusión/exclusión fueron seleccionados 20 artículos. En el estudio de este fenómeno son múltiples los parámetros desde los cuales debe ser evaluada la viremia, no sólo desde su magnitud, sino también desde su duración post-defervescencia y el día en el que se reporta el valor máximo, entre otros. Discusión: Los resultados de los estudios indican que las características de la viremia pueden jugar un rol de importancia que, junto con otros factores del contexto viral (serotipo del virus, tipo de infección) y del paciente (edad, genotipo, comorbilidades, etc), pueden determinar el desenlace clínico de la infección.

Background: Plasma viremia is a factor whose role in the severity of dengue virus infection has been widely discussed in recent years, with divergent results found in the multiple contexts evaluated. Methods: A systematic review of the literature using the Scopus, EBSCOhost, SpringerLink, Lilacs and Scielo databases was conducted. For inclusion in the review there were considered studies that evaluated the relationship between plasma viral load and clinical manifestations or severity of the disease. Results: We obtained 80 references in the initial search, after the application of the inclusion / exclusion criteria 20 articles were selected. In the study of this phenomenon there are multiple parameters from which viremia should be evaluated, not only from its magnitude, but also from the post-defervescence duration and the day in which the maximum value is reported, among others. Discussion: The results of the studies indicate that the characteristics of viremia may play an important role that, along with other factors of the viral (virus serotype, type of infection) and patient context (age, genotype, comorbidities, etc.), determine the clinical outcome of the infection.

Evaluation of the predisposition and clinical impact of BK virus replication in kidney transplant patients

ABSTRACT The BK virus (BKV) produces a subclinical kidney infection in immunocompetent individuals. However, viremia may occur in kidney transplant patients with ongoing immunosuppression. BKV-associated nephropathy (BKVN) has no specific treatment and is a leading cause of organ transplant loss. In this study, we evaluated the predisposition and the clinical impact of BKV replication in kidney transplant patients during post-transplant monitoring in a reference institution in Brazil. Demographic, clinical and laboratory data generated during routine outpatient follow-up were retrospectively collected. BK viremia was investigated using real-time polymerase chain reaction. Of the 553 participants, 7.4% (n = 41) presented BKV replication. Of these, 16 (39%) lost their kidney graft and interstitial nephritis was identified on kidney biopsy in 50% of the cases. Among the evaluated variables, only the use of the immunosuppressant mycophenolate sodium was identified as a risk factor for viremia (OR 7.96; 95% CI 2.35 to 26.98). The graft survival estimate in BKV-positive patients was significantly reduced (24.8% vs. 85.6%) after 10 years of transplantation. We concluded that defining predisposing factors remains an important challenge for the prevention and control of BKV activity following kidney transplantation, especially considering the development of BKVN and its strong effect on graft maintenance.

Prediction of brain atrophy using three drug scores in neuroasymptomatic HIV-infected patients with controlled viremia

ABSTRACTBACKGROUND: Despite potent antiretroviral therapy, HIV still causes brain damage. Better penetration into the CNS and efficient elimination of monocyte/macrophages reservoirs are two main characteristics of an antiretroviral drug that could prevent brain damage. The aim of our study was to assess efficacy of three antiretroviral drug scores to predict brain atrophy in HIV-infected patients.METHODS:A cross sectional study consisting of 56 HIV-infected patients with controlled viremia, who had no clinically evident neurocognitive impairment. All patients had MRI of the head. A typical T2 transversal slice was analyzed and ventricles-brain ratio (VBr) as an overall brain atrophy index was calculated. Three antiretroviral drug scores were used and correlated with VBr: 2008 and 2010 CNS penetration effectiveness scores (SCPE2008 and SCPE2010) and the recently established monocyte efficacy (SME) score. A p-value <0.05 was considered significant.RESULTS:SCPE2010 was significantly associated with VBr in both univariate (r = -0.285, p = 0.033) and multivariate (ß = -0.299, p = 0.016) regression models, while SCPE2008 was not (r = -0.141, p = 0.300 and ß = -0.156,p = 0.214). SME was associated with VBr in multivariate model only (r = -0.297, p = 0.111 andß = -0.406, p = 0.029). Age and reported duration of HIV infection were also significant predictors of overall brain atrophy in multivariate regression models.CONCLUSIONS:Although based on similar type of research, SCPE2010 is a superior drug score compared to SCPE2008. SME is an efficient drug score in determining brain damage. Both SCPE2010 and SME scores should be taken into account in preventive strategies of brain atrophy and neurocognitive impairment in HIV-infected patients.

Serological and virological evaluation of human T-lymphotropic virus type 1 infection in family groups from Tumaco, Colombia / Evaluación serológica y virológica de la infección por el virus linfotrópico de células T humanas de tipo 1 en grupos familiares de Tumaco, Colombia

Introduction: To date there has been no statistical evaluation of the profiles of immunoglobulin classes and viral replication as variables in the study of HTLV-1 infection and circulation among families in virus-endemic areas of Colombia. Objective: To evaluate the correlation of several immunological and molecular characteristics with the transmission and circulation of HTLV-1 among families in the town of Tumaco. Materials and methods: Plasma levels of HTLV-1 specific immunoglobulin classes IgG, IgM and IgA1, as well as IgG and sIgA in oral fluids, were calculated for 32 members of 10 family groups from Tumaco in which the mother and at least one child were infected with the virus. Levels of the different immunoglobulin classes were correlated with viral RNA circulating in plasma or oral fluids and the proviral burden as detected by RT-PCR. Results: Significant differences were determined between mothers and carrier children for immunoglobulin levels (p=0.037) and proviral burden (p=0.002). The overall estimate of IgG in plasma and sIgA in oral fluids could be correlated with the circulation of free viral RNA in both fluids and high proviral burden, and associated with HAM/TSP mothers. The detection of anti- tax IgG in plasma revealed differences between HAM/TSP mothers and their offspring. Conclusion: The study of immunological and molecular variables permitted the analysis of HTLV-1 circulation among families of Tumaco. The strong correlation between levels of IgM specific for the virus and viral RNA circulating in fluids indirectly confirmed the transmission of HTLV-1 among families.

Introducción. Todavía no hay una evaluación estadística de los perfiles de las clases de inmuno- globulina s y la replicación viral, como variables para estudiar la infección y la circulació n del HTLV-1 en familias de zonas endémicas en Colombia. Objetivo. Evaluar la correlación de varias características inmunológicas y moleculares, con la transmisión y circulación del virus en familias del municipio de Tumaco. Materiales y métodos. Se calcularon los niveles de IgG, IgM e IgA1 en plasma, e IgG y IgA secretoria en fluido oral, de 32 miembros de 10 grupos familiares de Tumaco, en los que la madre y, al menos, un hijo estaban infectados con el virus. La concentración de las diferentes clases de inmunoglobulinas se pudo correlacionar con la circulación de ARN viral libre en plasma y fluido oral, y la carga proviral, según su detección mediante reacción en cadena de la polimerasa de transcripción inversa. Resultados. Se encontraron diferencias significativas en los niveles de inmunoglobulinas (p=0,037) y en la carga proviral (p=0,002) entre madres e hijos portadores. La estimación total de IgG en plasma e IgA secretoria en fluido oral, se pudo correlacionar con la circulación de ARN viral libre en ambos fluidos y una alta carga proviral, y se asoció con las madres paraparesia espástica tropical o mielopatía asociada con el HTLV-1. La detección en plasma de IgG anti-Tax reveló diferencias entre ellas y sus hijos. Conclusión. El estudio de las variables inmunológicas y moleculares permitió analizar la circulación del HTLV-1 en familias de Tumaco. La fuerte asociación entre los niveles de IgM específica para el virus y el ARN viral circulante en los fluidos y la carga proviral, confirmó indirectamente la transmisión intrafamiliar del virus.

Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus)

Rocio virus (ROCV) is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus). The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR). The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC). ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection.

Evaluation of GBV-C / HVG viremia in HIV-infected women / Avaliação da viremia por GBV-C/HGV em mulheres infectadas pelo HIV

The present study aimed at standardizing a real-time quantitative polymerase chain reaction assay to evaluate the presence of GBV-C/HGV RNA. A "TaqMan" assay using primers and probe derived from the 5¢ NCR region was developed and validated. Two hundred and fifty-three plasma samples from HIV-infected women were tested for GBV-C viremia and antibody against the envelope protein 2. GBV-C RNA was detected in 22.5 percent of the patients whereas the antibody was identified in 25.3 percent of the cohort. Detection of viral RNA and of antibodies was mutually exclusive. Viral loads showed a mean of 1,777 arbitrary units / mL, being 1.1 and 13,625 arbitrary units / mL respectively the lowest and highest values measured. We conclude that the real-time quantitative polymerase chain reaction method developed is appropriate for the investigation of GBV-C RNA since it was shown to be highly specific and sensitive, as well as requiring few steps, preventing contamination and providing additional information as to the relative viremia of carriers, a parameter that must be included in studies evaluating the co-factors influencing the clinical outcome of HIV/AIDS.

Este estudo teve como objetivo o desenvolvimento de método de PCR em Tempo Real para a determinação da viremia do vírus GBV-C. Ensaio baseado em primers e sonda "TaqMan" derivados da região 5' não-codificante deste vírus foi padronizado, validado e aplicado em uma série de 253 amostras de plasma de pacientes HIV+. Além do PCR em tempo real, as amostras foram submetidas a um ensaio imunoenzimático anti-E2 e a um nested-PCR. Das 253 amostras testadas, 64 foram positivas para o anticorpo anti-E2 (25,3 por cento), enquanto 57 amostras foram concordantemente RNA positivas pelo nested-PCR e PCR em tempo real (22,5 por cento), perfazendo um índice total de exposição de 48 por cento (25.3 + 22.5). A carga viral teve média de 1.777 UA/mL (13.625 - 1.1UA/mL). Foi obtida metodologia simples, rápida e de boa sensibilidade e especificidade, permitindo a quantificação do RNA do vírus GBV-C com reprodutibilidade. A metodologia permite a análise simultânea de grande número de amostras, sendo apropriada para estudos clínicos. A prevalência de exposição a este agente na população feminina HIV+ estudada é alta, provavelmente decorrente da via sexual comum de transmissão dos agentes.

Detección de Virus Papiloma Humano en el fluido gingival de pacientes con inmunodeficiencia humana y enfermedad periodontal / Detection of Human Papillomavirus in gingival fluid of Venezuelan HIV patients with periodontal disease

Evidencias sugieren que los virus pueden participar en la activación de la enfermedad periodontal, permitiendo el sobrecrecimiento de bacterias periodontopatógenas. El objetivo del estudio fué la detección molecular de VPH en fluido gingival (FG) de pacientes VIH+ con enfermedad periodontal. Se evaluaron muestras de FG de 20 pacientes VIH+ con enfermedad periodontal que asistieron al Centro de Atención de Pacientes con Enfermedades Infecciosas (CAPEI) de la Facultad de Odontología de la Universidad Central de Venezuela, 13 bajo terapia antirretroviral (HAART) y 7 VIH+ sin HAART. Se incluyeron 7 pacientes seronegativos con periodontitis crónica y como grupo control 7 pacientes seronegativos periodontalmente sanos. Se extrajo el ADN, se amplificó la región L1 de VPH con primers MY09 y MY11. Las muestras VPH+ fueron genotipificadas para los tipos 6, 11, 16, 18, 31 y 45. VPH fue detectado en 46% de los pacientes VIH+ bajo terapia. El contaje CD4+ en la población VPH+ no presentó diferencias con el grupo VPH-, y la carga viral mostró valores promedio significativamente mayores (200.470± 324.244 copias/mL) con respecto a los pacientes VPH- (10.246±23.805 copias/mL). Las muestras VPH+ presentaron los genotipos 6 y 11, de los cuales 66,6% estaban coinfectados con ambos tipos. Las condiciones periodontales no presentaron diferencias entre los individuos con doble infección viral por VPH y VIH, y los que solo portaban VIH. VPH fue detectado solamente en fluido gingival de pacientes VIH+ con HAART, indicando que esta terapia puede influir en el estado inmunológico independientemente de las condiciones periodontales.

Evidence suggests that viruses may be involved in the activation of periodontal disease, allowing the overgrowth of periodontal pathogens. The purpose of the present study was to detect the presence of Human Papillomavirus (HPV) in gingival crevicular fluid (GCF) in HIV+ Venezuelan patients with periodontal disease. We evaluated GCF samples from 20 HIV+ patients with periodontal disease from the Infectious Disease Center, Faculty of Dentistry, Central University of Venezuela, and were clinically examined to establish their periodontal conditions, 13 under HAART (antiretroviral therapy) and 7 without HAART. Seven seronegative patients with chronic periodontitis and 7 seronegative patients, without periodontal disease were included. DNA extraction was performed, the consensus primers MY09 and MY11 for the HPV L1 region were used for PCR amplification. Genotipification was made for the 6, 11, 16, 18, 31 and 45 genotypes. HPV were detected in 46% of HIV+ patients under therapy. The CD4 cell counts in the HPV+ patients were not significantly different from the HPV-group. The viral load in the HPV+ group was significantly higher (200,470 ± 324,244 copy/mL) than in the HPV- patients (10,246 ± 23,805 copy/mL). Genotypes 6 and 11 were observed in the HPV positive samples, of which 4/6 (66.6%) presented coinfection with both types. No significant differences in the periodontal conditions were observed between patients with HPV-HIV infection related to patients with only HIV. HPV was detected only in the gingival crevicular fluid of HIV+ patients under HAART independently of the periodontal conditions.

HIV-1 drug resistance genotypic profiles in children with undetectable plasma viremia during antiretroviral therapy

Treatment of HIV-1 infection with highly active antiretroviral therapy has led to sustained viral suppression in the plasma in a large number of children. However, studies have suggested that the integrated provirus in resting CD4+ T lymphocytes could be a source of reactivatable virus and maintain drug-resistant virus. We evaluated the resistance-related mutations in children receiving antiretroviral therapy with prolonged viral suppression. Thirty-two peripheral blood mononuclear cell samples from 16 children with viral loads that had been below detection limits for at least 12 months were obtained at two different time points and the DNAs sequenced. The median CD4 cell count was 1,016 cells/mm³ (347-2,588) and 938 cells/mm³ (440-3,038) at the first and second time points, respectively. The median follow-up time was 15 months (9-27). Six (37.5 percent) and seven (43.75 percent) of the 16 patients showed at least one NRTI-associated mutation in the first and second samples, respectively. Two out of 16 (12.5 percent) had an NNRTI-associated mutation at the first time point and three out of 16 (18.75 percent) at the second. In addition, 14 out of 16 (87.5 percent) had at least one PI-associated mutation at both time points. Despite plasma HIV-1 RNA suppression for at least 12 months, resistance-related mutations from previous antiretroviral failures could still be detected in archival virus. Furthermore, viral evolution occurred at the reverse transcriptase region in spite of viral suppression to levels below 400 copies/mL. Persistence of archival resistant virus may be relevant when considering future treatment options.

Perfil sorológico, viremia e eliminação do circovírus suíno tipo 2 em animais naturalmente infectados pertencentes a granjas com ou sem a síndrome da refugagem multissistêmica / Serological profile, viremia and PCV2 shedding in naturally infected pigs of herds with or without postweaning multisystemic wasting syndrome

Um estudo virológico e sorológico seccional (E1) e outro longitudinal (E2) foram realizados em granjas com (G2 e G3) e sem (G1) a síndrome de refugagem multissitêmica (SRM) no Brasil. Foram coletadas amostras de sangue, soro, swabs nasal e retal de animais de cada categoria do ciclo produtivo: porcas, leitões maternidade, creche, recria e terminação. Em E1, nas granjas G1a e G2, foram amostrados 40 animais de cada categoria. Em E2, nas granjas G1b e G3, 35 leitões na maternidade foram identificados e amostrados ao longo do ciclo produtivo. O soro foi avaliado para presença de anticorpos contra circovírus suíno tipo 2 (CVS2) e sangue e swabs para presença do ácido nucléico viral. Em E1, a categoria porcas possuía altas taxas de animais virêmicos e soropositivos, com porcentagem de porcas com títulos altos superior a G2. Em G1a a queda de imunidade passiva ocorreu entre o final da fase de creche e início da recria com aumento da eliminação viral em swabs e subsequente soroconversão. Em G2 a queda ocorreu entre a fase final da maternidade e início da creche, com diminuição da eliminação viral. Em E2, a queda da imunidade materna ocorreu entre a 1ª e 2ª coleta em G1b; e em G3, entre a 2ª e 3ª coleta. Em ambas as granjas, a queda de imunidade passiva coincidiu com o aumento da viremia e eliminação viral e a soroconversão ocorreu entre a 3ª e 4a coleta em ambas as granjas com aumento da média de título de anticorpos e declínio da viremia. Viremia e eliminação viral foram detectadas em todas as coletas realizadas; 42% dos animais amostrados em E2 foram virêmicos em todas as coletas e todas as amostras de tecido coletadas no abate foram positivas para o CVS2. Este estudo confirma a persistência da viremia mesmo em presença de altos títulos de anticorpos e que o perfil sorológico em um rebanho com e sem a presença da síndrome pode ser diferente, principalmente em relação à duração da imunidade passiva.

A virological and serological cross-sectional study (E1) and a longitudinal study (E2) were performed on herds with (G2 and G3) and without (G1) post weaning multisystemic syndrome (PMWS) in Brazil. Blood, serum, nasal and rectal swabs samples were collected of sows, farrowing piglets, nursery, growing and finishing pigs. In E1, were sampled 40 animals in each category (G1a and G2). In E2, (G1b and G3), 35 farrowing piglets were identified and sampled along the production cycle. Porcine circovirus type 2 (PCV2) antibodies were assayed. A PCR was used to detected PCV2 genome in blood and swabs. In E1, sows had high rates of viremic and seropositives animals, with percentage of sows with high antibodies titers greater than G2. Passive antibodies decline occurred between nursery and growing area with increased viral shedding in swabs and subsequent seroconversion in G1. In G2, the passive antibodies decay occurred in nursery, with a reduction in viral shedding. In E2, the decline of maternal immunity occurred between the 1st and 2nd collection in G1b, and between 2nd and 3rd collections in G3. In both herds, the decay of passive immunity coincided with increased viremia and viral shedding; and seroconversion occurred between the 3rd and 4th collection in both herds with decline of viremia. Viremia and viral shedding was detected in all samples days, 42% of animals sampled in E2 were viremic and all tissue samples collected at slaughterwere positive for PCV2. This study confirms the persistence of viremia even in the presence of high titers of antibodies and the serological profile in a herd with or without PMWS may be different, especially with regard to the passive immunity duration.

Evaluation of the total hepatitis C virus core antigen assay in determining viraemia in Egyptian patients with chronic hepatitis C

Hepatitis C virus [HCV] is an important etiologic agent for chronic liver diseases in Egypt. Positivity of anti-HCV antibodies indicates past or current infection and in contrast to molecular detection of HCV-RNA does not give any indication about viral replication. Several problems still persist when RT-PCR is used for screening of patients. Recently, the availability of an anti-core antigen monoclonal antibody allowed development of an enzyme-linked immunosorbent assay [ELISA] detecting and quantifying total HCV core Ag [C-Ag] in peripheral blood of HCV-infected patients. The aim of this study is to evaluate the usefulness of the total HCV C-Ag in determining viremia compared to HCV-RNA detected by RT-PCR among anti-HCV positive Egyptian patients. In this study, 143 anti-HCV antibody positive patients were tested for HCV-RNA by PCR and HCV-Core antigen to evaluate the sensitivity and specificity of the latter. Among those 116 cases [81.11%] were HCV-RNA+ve and 111 [77.68] cases were HCV-Core Ag +ve, while 27 cases [18.84%] were HCV- RNA negative and 32[22.32%] were HCV-Core Ag -ve. Out of 116 patients who were HCV- RNA +ve, 111 [95.65%] were HCV-Core Ag +ve also, while 5 [4.35%] cases all with low viral load were negative. The sensitivity, specificity, PPV and NPV of HCV-Core Ag when referred to HCV-RNA were 95.7%, 100%, 100% and 84.4% respectively. Thus positive total HCV Core Ag could be a useful test in clinical practice to determine HCV viremia with a sensitivity and specificity close to that of PCR assays. However, a negative test is not reliable to exclude low viral load

Serum zinc concentration in Egyptian patients with chronic hepatitis C

The prevalence of HCV infection varies throughout the world, with the highest number of infections reported in Egypt. Zinc has been closely related to the pathogenesis of chronic hepatitis C. Zinc deficiency causes reduction in glutathione [GSH] which has been implicated in various cellular events. Zinc may play an important role as a negative regulator in HCV replication; zinc supplementation increases the therapeutic response of Interferon [IFN]. To detect serum zinc concentration in adult patients with chronic [compensated] hepatitis C prior to treatment with IFN, and correlate the levels with the degree of inflammation and stage of fibrosis. This study was conducted on 40 adult patients chronically infected with HCV as well as 10 healthy subjects serving as a control group. Participants were subjected to complete clinical and laboratory assessment, quantitative analysis of HCV RNA by PCR technique and abdominal ultrasonography, in addition to estimation of serum Zinc by Graphite Furnace Atomic Absorption Spectrometer. A significant negative correlation was found between the level of zinc and liver fibrosis. The mean value of zinc in patients was 39.5ug/dl, while in the control it was 75ug/dl [normal range: 70-80ug/dl]. There was a non significant negative correlation between serum zinc and serum AST, ALT, disease activity and level of viraemia. Our study suggested the possible important protective role of zinc in patients with chronic hepatitis C, as serum zinc levels showed negative significant correlation with the stage of liver fibrosis

Wild dengue virus types 1, 2 and 3 viremia in rhesus monkeys

Among the flaviviruses, dengue, with its four serotypes, has spread throughout the tropics. The most advanced vaccines developed so far include live attenuated viruses, which have been tested in humans but none has been licensed. Preclinical testing of dengue vaccine candidates is performed initially in mice and in nonhuman primates. In the latter the main criteria used to assay protection are neutralizing antibodies elicited by the vaccine candidate and the magnitude and duration of peripheral viremia upon challenge of previously immunized animals. Towards the identification of wild-type viruses that could be used in challenge experiments a total of 31 rhesus monkeys were inoculated subcutaneously of wild dengue types 1, 2, and 3 viruses. The viremia caused by the different viruses was variable but it was possible to identify dengue viruses useful as challenge strains.

Duración de la viremia y la excreción fecal del virus en niños con Hepatitis A / Duration of viremia and fecal shedding of the virus in Hepatitis A infected children

La infección por el virus de hepatitis A (HAV) es endémica en Argentina. El uso de técnicas moleculares permitió extender la detección del RNA del HAV en sueroy heces en pacientes con diferentes presentaciones clínicas. Comparamos la sensibilidad del protocolo de RT-PCR que usamos con cebadores dirigidos a distintas regiones del genoma, resultando la detección de la región VP3 C terminal la más sensible. Se obtuvieron prospectivamente muestras de suero y materia fecal de 20 niños con hepatitis aguda autolimitada por HAV. El RNA del HAV fue detectado en 18/20 niños en muestras basales y en 19/20 sumando una muestra posterior. El RNA del HAV fue detectable en 9/20 acientes hasta 30 días en suero; en materia fecal en 2/20 hasta 60 días y en 1/20 hasta 90 días. La secuencia genómica para la región VP1/2A en 8 muestras demostró que todas pertenecían al subgenotipo IA, aunque eran diferentes entre sí. Solo en 1/11 niños con falla hepatica fulminante fue posible la detección del RNA del HAV utilizando la región VP3 C terminal y el genotipo fue I. La reciente introducción de la vacunación universal en niños de 1 año de edad en Argentina podría disminuir drásticamente la circulación del virus, emergiendo nuevas fuentes de infección y permitiendo la introducción de nuevos genotipos. Las técnicas moleculares aplicadas al estudio de la historia natural de la infección y a la vigilancia epidemiológica contribuyenal control y la toma de decisiones eficientes en políticas de Salud Pública.

Hepatitis A virus (HAV) infection is endemic in Argentina. Molecular tools have allowed HAV RNA detection to be extent to sera and feces from patients with different clinical backgrounds. We compare the sensitivity of the RT-PCR protocol we follow using primers targeting different genomic regions and VP3 C terminal was the most sensitive. Sequential sera and fecal samples were obtained from 20 children with acute self limited Hepatitis A. HAV RNA was detectable in 18/20 children if sera and stool specimens were collected at the onset of symptoms and in 19/20 if a later sample was considered. HAV RNA was detectable in serum from 9/20 patients until day 30 and in feces from 2 patients until day 60 and until day 90 in one. Genomic sequences from VP1/2A region in 8 samples showed they all belong to subgenotype IA although they were different between them. HAV RNA was detectable only in 1/11 sera from children with acute liverfailure when VP3 C terminal fragment was searched and it belonged to genotype I. Universal vaccination in one year old children was recently implemented in Argentinaand it will dramatically enable the decrease of the viral circulation, making new sources of infection emerge and allowing the introduction of new genotypes. The application of molecular tools to the study of the natural history of infection and to the epidemiologicsurveillance may contribute to efficient control and lead to rational decisions in public health policies.

Aplicação de PCR em tempo real para avaliar viremia em humanos e primatas não humanos, imunizados com a vacina de febre amarela cepa 17DD e o vírus recombinante 17D-DENV / Application of PCR in real time to evaluate viremia in human beings and not human primates, immunized with the vaccine of yellow fever cepa 17DD and recombinant virus 17D-DENV

Um dos parâmetros principais para o estudo de atenuação de flavivírus é a determinação de sua capacidade de replicação. Neste contexto, empregamos o método de RT-PCR em tempo real que permite a detecção de ácidos nucléicos diretamente de amostras biológicas. Neste trabalho, utilizamos, então, esta tecnologia, para determinar a replicação de vírus de febre amarela 17DD e 17D-recombinantes, que expressam a proteína do envelope dos vírus dengue para validação destes como candidatos a vacinas. Esta metodologia foi empregada de forma complementar ao teste clássico de titulação viral por plaqueamento em culturas de células de vertebrados. Os materiais clínicos incluem soros de macacos rhesus previamente inoculados com estes vírus por via intracerebral ou subcutânea e soros de 32 indivíduos vacinados com vírus 17DD. O método demonstrou boa reprodutibilidade para amostras com título viral de até 2 Log10 PFU/mL com limite de detecção de até 10 PFU/mL ou 143 cópias /mL, sendo esta sensibilidade comparável ao que foi descrito para outros vírus analisados pela mesma tecnologia. Nesta faixa de concentração, os resultados obtidos pela metodologia de RT-PCR em tempo real mostraram-se compatíveis com os obtidos pelas técnicas de RT-PCR “semi nested” e pelo plaqueamento em soros de macacos inoculados com vírus 17DD. A análise comparativa da quantificação viral em espécimes clínicos de macacos sugere a limitada capacidade de replicação do vírus 17DD independente da via de inoculação. Os vírus 17D-recombinantes, avaliados neste estudo, demonstraram sua atenuação em relação ao vírus 17DD, tanto pela metodologia de RT-PCR em tempo real como por plaqueamento. Assim sendo, consideramos que a tecnologia empregada para o estudo da capacidade replicativa dos vírus 17DD e 17D-recombinantes em diferentes hospedeiros, demonstrou o perfil de atenuação dos vírus testados. Com relação aos vírus recombinantes 17D-dengue, estes resultados embasam a continuação do seu desenvolvimento como cand...

Epidemiologia molecular do vírus da hepatite C (VHC) na cidade de Salvador-BA / Molecular epidemiology of hepatitis C virus (HCV) in the city of Salvador-BA

O VHC é um vírus RNA (VHC-RNA) hepatotrópico associado com um alto risco à cronificação, cirrose e carcinoma hepatocelular. Nos últimos anos, técnicas moleculares para a detecção do VHC-RNA e a genotipagem têm se tomado ferramentas indispensáveis para a avaliação de pacientes com hepatite crônica C. O objetivo principal foi realizar um estudo de epidemiologia molecular para determinar a prevalência de infecção e a distribuição dos genótipos do VHC em uma amostra de base populacional e em populações sob risco. Para o estudo de base populacional foram coletados 1308 amostras de soros de indivíduos selecionados através de uma aleatorização estratificada da população de Salvador (amostra do Programa Bahia Azul). Para o estudo em grupos sob risco, foram coletadas amostras de soro de 127 pacientes com anticorpo anti- VHC positivo provenientes do HUPESIUFBA, de 66 hemofilicos da HEMOBA e de 97 hemodialisados de sete clínicas de Salvador, voluntários na pesquisa. A prevalência de infecção pelo VHC foi estimada a partir da avaliação dos resultados de soroprevalência do anticorpo anti-VHC obtidos através do ELISA de 2ana. geração e confirmação pelo RIBA e/ou RT-PCR, método in-house. Quando possível, foi colhida uma segunda amostra de soro para confirmação dos resultados de RIBA ou RT-PCR negativos. Nas amostras VHC-RNA positivas foi realizada a genotipagem através da R T - PCR, utilizando-se primers genótipo-específicos dirigidos contra a região do core ou através da análise do RFLP da região 5'UTR do VHC-RNA. A prevalência de infecção pelo VHC na população em geral de Salvador, em hemofilicos e hemodialisados foi de 1,5%, 32,6% e 7,1 %, respectivamente. A infecção na população em geral foi associada com idade superior a 20 anos (p<0,05), 12 ou mais anos de escolaridade p<0,01), nível de renda mediano (p<0,05) e residência no bairro da Barra (p<0,05), uma vizinhança de classe média-alta da cidade de Salvador...