Hepatitis C in Hemodialysis Units: diagnosis and therapeutic approach / Hepatite C nas Unidades de Hemodiálise: diagnóstico e abordagem terapêutica

Abstract According to data from the last census of the Brazilian Society of Nephrology (SBN), the prevalence of hepatitis C virus (HCV) in Brazilian hemodialysis units (HU) is 3.3%, about three times higher than what is reported for the Brazilian general population. Often, professionals working in HU are faced with clinical situations that require rapid HCV diagnosis in order to avoid horizontal transmission within the units. On the other hand, thanks to the development of new antiviral drugs, the cure of patients with HCV, both in the general population and in patients with chronic kidney disease and the disease eradication, appear to be very feasible objectives to be achieved in the near future . In this scenario, SBN and the Brazilian Society of Hepatology present in this review article a proposal to approach HCV within HUs.

Resumo De acordo com os dados do último censo da Sociedade Brasileira de Nefrologia (SBN), a prevalência de portadores do vírus da hepatite C (HCV) nas unidades de hemodiálise (UH) no Brasil é de 3,3%, cerca de três vezes maior do que é observado na população geral brasileira. Muitas vezes, os profissionais que trabalham nas UH deparam-se com situações clínicas que demandam rápido diagnóstico do HCV, a fim de evitar uma transmissão horizontal dentro das unidades. Por outro lado, a cura dos pacientes portadores do HCV, tanto na população geral como na portadora de doença renal crônica e a erradicação da doença, em virtude do desenvolvimento de novas drogas antivirais, parecem ser objetivos bastante factíveis, a ser alcançados em futuro próximo. Nesse cenário, a SBN e a Sociedade Brasileira de Hepatologia apresentam neste artigo de revisão uma proposta de abordagem do HCV dentro das UH.

First report of treatment failure in a patient with cutaneous leishmaniasis infected by Leishmania (Viannia) naiffi carrying Leishmania RNA virus: a fortuitous combination?

Abstract We report the case of a 32-year-old man from Rio de Janeiro, who was infected in the Amazon region of Brazil by Leishmania (Viannia) naiffi. Generally, patients with L. naiffi cutaneous leishmaniasis exhibit a good therapeutic response to either pentavalent antimonials or pentamidine. However, after pentamidine treatment, this patient's infection evolved to therapeutic failure. To understand this clinical outcome, we investigated the presence of the Leishmania RNA virus (LRV) in parasites isolated from the cutaneous lesion; herein, we discuss the possible association between a poor response to pentamidine therapy and the presence of the LRV.

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

Correlation between presence of Leishmania RNA virus 1 and clinical characteristics of nasal mucosal leishmaniosis / Correlação entre a presença de Leishmania RNA Vírus 1 e as características clínicas da leishmaniose de mucosa nasal

ABSTRACT INTRODUCTION: Mucosal leishmaniosis (ML) is a severe clinical form of leishmaniosis. Complex factors related to the parasite and the host are attributed to the development of mucosal lesions. Leishmania RNA virus 1 (LRV1) can disrupt immune response, and may be the main determinant of severity of the disease; it should be investigated. OBJECTIVE: To study the existence of clinical differences between patients with ML with endosymbiosis by LRV1 and. those without it. METHODS: A cross-sectional cohort study with clinical evaluation, polymerase chain reaction (PCR) detection of Leishmania, species classification, and search of LRV1 was performed. Only patients with confirmed diagnosis of ML by positive PCR and with nasal mucosa injuries were included in this analysis. RESULTS: Out of 37 patients, 30 (81.1%) were diagnosed with Leishmania braziliensis, five (13.5%) with Leishmania guyanensis, and two (5.4%) with mixed infection of L. braziliensis and L. guyanensis. LVR1 virus was present in 26 (70.3%) of the cases. CONCLUSION: Correlation between clinical phenotype and presence of LRV1 was not observed, although the frequency of the virus is two-fold higher in mucosal lesions than that found in the literature on skin lesions in the same geographical area.

RESUMO Introdução: A leishmaniose de mucosa (LM) é uma forma clínica grave da leishmaniose. Fatores complexos ligados ao parasita e ao hospedeiro são atribuídos ao desenvolvimento das lesões de mucosa. Leishmania RNA Vírus 1 (LRV1) pode subverter a resposta imune, podendo ser o principal determinante da gravidade da doença e deve ser pesquisado. Objetivo: Estudar a existência de diferenças clínicas entre pacientes portadores de LM com endosimbiose por LRV1 e as que não possuem. Métodos: Foi realizado um estudo de coorte histórica com corte transversal com avaliação clínica, detecção da Leishmania por técnica de PCR, classificação da espécie e pesquisa de LRV1. Foram incluídos na análise da pesquisa somente os pacientes com diagnóstico confirmado de LM com PCR positivo, com lesão de mucosa nasal. Resultados: Dos 37 pacientes, 30 (81,1%) foram diagnosticados com L. braziliensis, 5 (13,5%) com L. guyanensis e 2 (5,4%) com infecção mista de L. braziliensis e L. guyanensis. O vírus LVR1 estava presente em 26 casos (70,3%). Conclusão: A correlação entre o fenótipo clínico e a presença do LRV1 não foi constatada, porém a frequência do vírus é duas vezes maior em lesão de mucosa do que encontrado em trabalho, da mesma região, sobre lesão cutânea.

Severity of tegumentary leishmaniasis is not exclusively associated with Leishmania RNA virus 1 infection in Brazil

Leishmania RNA virus (LRV) has been shown to be a symbiotic component of Leishmania parasites in South America. Nested retro-transcription polymerase chain reaction was employed to investigate LRV1 presence in leishmaniasis lesions from Brazil. In endemic areas of Rio de Janeiro (RJ), no LRV1 infection was observed even with mucosal involvement. LRV1 was only detected in Leishmania (V.) guyanensis cutaneous lesions from the northern region, which were obtained from patients presenting with disease reactivation after clinical cure of their primary lesions. Our results indicated that the severity of leishmaniasis in some areas of RJ, where Leishmania (V.) brazi-liensis is the primary etiological agent, was not associated with Leishmania LRV1 infection.

Construção e caracterização de vírus recombinante de febre amarela expressando o gene repórter da Gaussialuciferase / Construction and characterization of a recombinant vírus of Yellow Fever expressing the repórter gene of Gaussia Luciferase

O vírus da febre amarela (YFV, Yellow Fever Virus), um arbovírus da família Flaviridae,é o agente causador da febre amarela (FA), uma doença aguda, febril, não contagiosa, hemorrágica e potencialmente fatal. O YFV é endêmico em regiões tropicais da América do Sul e África. Apesar de sua significância como um problema de saúde pública, muitos mecanismos moleculares da biologia do YFV, como replicação do genoma e patogênese viral ainda não foram bem compreendidos. Avanços em genética reversa viral tem permitido a elucidação de mecanismos da biologia e comportamento viral, bem como a construção de vetores vacinais e desenvolvimento de drogas antivirais. No presente trabalho, descrevemos a construção e caracterização de um vírus recombinante de FA expressando o gene repórter da Gaussialuciferase (GLuc). Utilizando o sistema de recombinação homóloga em levedura, o gene repórter da Proteína Fluorescente Amarela (YFP, Yellow Fluorescent Protein) do vírus recombinante YFV-YFP-DENV1linker, previamente construído em nosso laboratório, foi substituído pelo gene repórter GLuc. A construção foi confirmada por PCR. Os RNAs virais genômicos foram sintetizados in vitro, e posteriormente transfectados em células BHK-21.As células transfectadas foram avaliadas por imunofluorescência indireta e mensuração do gene repórter GLuc. Dois clones foram recuperados e caracterizados em cultivo celular. Nós acreditamos que este vírus repórter deverá ser útilna triagem e desenvolvimento de drogas antivirais específicas, estudos de replicação virale competência vetorial, além da possível utilização como vetor viral vacinal.

Acute respiratory viral infections in children in Rio de Janeiro and Teresópolis, Brazil / Infecções respiratórias agudas causadas por vírus em crianças do Rio de Janeiro e de Teresópolis, Brasil

The frequency of viral pathogens causing respiratory infections in children in the cities of Rio de Janeiro and Teresópolis was investigated. Nasal swabs from children with acute respiratory illnesses were collected between March 2006 and October 2007. Specimens were tested for viral detection by conventional (RT)-PCR and/or real time PCR. Of the 205 nasal swabs tested, 64 (31.2%) were positive for at least one of the viral pathogens. Single infections were detected in 56 samples, 50 of those were caused by RNA viruses: 33 samples tested positive for rhinovirus, five for influenza A, five for metapneumovirus, four for coronavirus and, three for respiratory syncytial virus. For the DNA viruses, five samples were positive for bocavirus and one for adenovirus. Co-infections with these viruses were detected in eight samples. Our data demonstrate a high frequency of viral respiratory infections, emphasizing the need for a more accurate diagnosis particularly for the emerging respiratory viruses. The fact that the emerging respiratory viruses were present in 9.2% of the tested samples suggests that these viruses could be important respiratory pathogens in the country.

Neste estudo foi investigada a frequência de patógenos virais causando infecção em crianças nas cidades do Rio de Janeiro e Teresópolis. Foram coletados 205 swabs nasais de crianças com infecção aguda do trato respiratório no período de março de 2006 a outubro de 2007. Os espécimes foram testados para detecção de vírus através de (RT)-PCR e/ou PCR em tempo real. Dentre as 205 amostras testadas, 64 (31,2%) foram positivas para pelo menos um vírus. Infecções causadas por um único agente viral foram detectadas em 56 amostras, 50 das quais eram causadas por vírus de RNA: 33 amostras foram positivas para rinovírus, cinco amostras foram positivas para influenza A, cinco amostras foram positivas para metapneumovírus, quatro amostras foram positivas para coronavírus e três amostras foram positivas para vírus respiratório sincicial. Para os vírus de DNA foram detectadas cinco amostras positivas para bocavírus humano e uma amostra positiva para adenovírus. Foram identificados oito casos de co-infecção. Nossos dados demonstram frequência elevada de infecções respiratórias virais, enfatizando a necessidade de um diagnóstico mais acurado destes patógenos, principalmente os vírus considerados emergentes. O fato de alguns vírus respiratórios emergentes terem sido detectados em 9,2% das amostras testadas sugere que estes vírus podem ser patógenos respiratórios importantes no país.

The presence of dsRNA virus in Trichomonas vaginalis isolates from symptomatic and asymptomatic Indian women and its correlation with in vitro metronidazole sensitivity.

Purpose: Trichomonas vaginalis, a protozoan parasite, is the causative agent of human trichomoniasis, the most common non-viral sexually transmitted disease. The infection encompasses from a complete asymptomatic presentation to severe sequelae; yet, the virulence markers have been poorly understood. It is suggested that the presence of Trichomonas vaginalis virus (TVV) in T. vaginalis may have an impact on its virulence, and its relatedness to in vitro metronidazole resistance has been reported. The aim of the study was to assess the presence of TVV in fresh and Long -Term Cultivated ( LTC) maintained T. vaginalis isolates from symptomatic (S) and asymptomatic (AS) Indian women and its relatedness, if any, with symptomatology and in vitro drug sensitivity. Materials and Methods: One thousand women (537 S and 463 AS) were screened for the presence of T. vaginalis by wet smear and culture examination of vaginal swab and urine sample. Fresh and LTC (6 months-2 years) maintained 15 isolates each from 15 S and 15 AS women were subjected to agarose gel electrophoresis following total cellular RNA extraction to evaluate the presence of double stranded (ds) RNA viral infection. The susceptibility of isolates to metronidazole was determined in vitro. Results: On agarose gel electrophoresis, three bands (5.5, 2.5 and 1.5 kb) were observed in all the 30 fresh isolates from 15 S and 15 AS women and only in 7 LTC isolates from 3 S and 4 AS women. All the fresh isolates harbouring TVV were found to be sensitive to metronidazole in vitro irrespective of the symptomatology of subjects, and out of seven LTC isolates harbouring TVV, six were sensitive to metronidazole and one showed borderline resistance. Conclusions: The results suggest that the presence of TVV alone may not be a virulence marker and loss of TVV on LTC appears to be related to drug resistance. The T. vaginalis Indian isolates are sensitive to metronidazole.

Concurrent dengue and malaria in the Amazon region / Co-infecção por dengue e malária na região Amazônica

INTRODUCTION: The Amazon region has extensive forested areas and natural ecosystems, providing favorable conditions for the existence of innumerous arboviruses. Over 200 arboviruses have been isolated in Brazil and about 40 are associated with human disease. Four out of 40 are considered to be of public health importance in Brazil: Dengue viruses (1-4), Oropouche, Mayaro and Yellow Fever. Along with these viruses, about 98 percent of the malaria cases are restricted to the Legal Amazon region. METHODS: This study aimed to investigate the presence of arboviruses in 111 clinical serum samples from patients living in Novo Repartimento (Pará), Plácido de Castro (Acre), Porto Velho (Rondônia) and Oiapoque (Amapá). The viral RNA was extracted and RT-PCR was performed followed by a Multiplex-Nested-PCR, using Flavivirus, Alphavirus and Orthobunyavirus generic and species-specific primers. RESULTS: Dengue virus serotype 2 was detected in two patients living in Novo Repartimento (Pará) that also presented active Plasmodium vivax infection. CONCLUSIONS: Despite scant data, this situation is likely to occur more frequently than detected in the Amazon region. Finally, it is important to remember that both diseases have similar clinical findings, thus the diagnosis could be made concomitantly for dengue and malaria in patients living or returning from areas where both diseases are endemic or during dengue outbreaks.

INTRODUÇÃO: A região Amazônica possui extensas áreas florestadas e ecossistemas naturais, provendo condições favoráveis para a existência de diversos arbovírus. Aproximadamente, 200 arbovírus foram isolados no Brasil, e 40 estão associados com doenças em humanos. Quatro destes 40 são considerados ser de importância para a saúde pública no Brasil: vírus da dengue (1-4), Oropouche, Mayaro e febre amarela. Juntamente com estes vírus, aproximadamente 98 por cento dos casos de malária estão restritos à região da Amazônia Legal. MÉTODOS: O objetivo deste estudo foi investigar a presença de arbovírus em 111 amostras clínicas de sangue de pacientes que residiam em Novo Repartimento (Pará), Plácido de Castro (Acre), Porto Velho (Rondônia) and Amapá (Macapá). O RNA viral foi extraído, RT-PCR foi realizada seguida de uma Multiplex-Nested-PCR, usando primers genéricos e espécie-específicos para Flavivirus, Alphavirus and Orthobunyavirus. RESULTADOS: Detectamos o vírus da dengue, sorotipo 2, em dois pacientes que residiam em Novo Repartimento (Pará), que também tinham infecção por Plasmodium vivax. CONCLUSÕES: Apesar de dados escassos, esta situação, provavelmente, ocorre mais frequência que a detectada na região Amazônica. Definitivamente, é importante lembrar que ambas as doenças possuem achados clínicos similares, assim o diagnóstico deveria ser feito concomitantemente para dengue e malária em pacientes que residem ou estão voltando de áreas onde ambas as doenças são endêmicas.

Double-stranded RNA viral infection in Cuban Trichomonas vaginalis isolates

Trichomonas vaginalis can be infected with double-stranded RNA (dsRNA) viruses designated T. vaginalis virus (TVV), which may have important implications for trichomonal virulence and disease pathogenesis. We tested for TVV in 40 fresh T. vaginalis isolates from Cuban patients by total extraction of nucleic acids (DNA and RNA). TVV was detected in 22 (55 percent) of the 40 T. vaginalis isolates. This gives an estimate of the infection rate of Cuban T. vaginalis isolates by the dsRNA virus. Future research should focus on the association between trichomonosis symptoms and the presence of TVV.

Blackgram mottle virus RNA and its in vitro translation.

In vitro translation of blackgram mottle virus RNA in rabbit reticulocyte lysate resulted in synthesis of five major virus specific polypeptides with mol wt 90,000(p90), 82,000(p82), 42,000(p42), 39,000(p39) and 32,000(p32), respectively. The polypeptide p39 was identified as coat protein based on its electrophoretic mobility and immunoprecipitation with BMoV-antiserum.

Early events in virus-plant interactions

1. Plant viruses can only enter their host through a wounded plant cell. Once in the cytoplasm, the virion must be disassembled, and for certain viruses with a "+" RNA genome, cotranslational disassembly of virus particles has been described. 2. Subsequent to viral protein synthesis which requires the host translational machinery, the "+" RNA genome is replicated in the cytoplasm. Viral genome amplification requires at least one viral-coded non-structural protein in conjunction with one or more host factors. 3. Early events in virus infection can be studied in systems that hinder these events. This is the case of natural hosts that are resitant to viruses: mutant viruses which overcome such resistance have been described. It is also the case of genetically engineered plants that are protected from virus infection. Both types of systems should help in determining the mode of interaction involved, and possibly also the host factor(s) involved in the various steps of virus infection