Polyomaviruses BK and JC DNA infection in peripheral blood cells from blood donors

ABSTRACT Objectives: To investigate the prevalence of human polyomavirus (BK and JC viruses) infection in peripheral blood mononuclear cells of healthy blood donors. Methods: The study included 250 healthy blood donors. Five-milliliter blood was drawn into sterile EDTA tubes and PBMCs were isolated from whole blood. The isolated PBMCs were counted and stored at −70 °C for future investigation. DNA was extracted and subjected to simple, sensitive and specific semi-nested PCR as well as QPCR using both general and specific primers for different assays. Results: Of 250 blood samples, 66 (26.4%) were positive for BKV DNA (146-34,514 copies/106 cells). JC DNA was found in 45 (18%) blood samples (65-21,250 copies/106 cells). Co-infection with these viruses were found in 11 (4.4%) out of 250 blood samples. Discussion: Our study provides important data on polyomavirus infection in peripheral blood mononuclear leukocytes in immunocompetent individuals. These data indicate significant differences between the prevalence of BKV and JCV infection in healthy blood donors. The prevalence of BK and JC virus infection is higher in the age range 30-39 years compared to other age ranges.

Human polyomavirus infection: Cytological and molecular diagnosis / Infecção pelo poliomavírus humano: diagnóstico citológico e molecular

Summary Few studies directly compare urinary cytology with molecular methods for detecting BK and JC polyomaviruses. Reactivation of BKV infection is the main risk factor for the development of nephropathy in immunocompromised individuals. The limitation of the cytological method can be attributed to the stage where the infected cell does not have specific and sufficient morphological characteristics for a conclusive diagnosis and can be easily interpreted as degenerative alteration. Moreover, morphologically, it is not possible to differentiate the two types of viruses. Polymerase chain reaction (PCR), not only is a sensitive method, but also allows differentiation of viral types without quantification, and therefore is not indicative of nephropathy. According to the American Society of Nephrology, real-time PCR would be the gold standard to indicate nephropathy because it allows quantifying the number of viral copies.

Resumo Poucos estudos comparam diretamente a citologia urinária com métodos moleculares para detecção de poliomavírus BK e JC. A reativação da infecção por BKV é o principal fator de risco para o desenvolvimento de nefropatia em indivíduos imunocomprometidos. A limitação do método citológico pode ser atribuída ao estágio em que a célula infectada não possui características morfológicas específicas e suficientes para um diagnóstico conclusivo, podendo ser facilmente interpretada como alteração degenerativa. Além do mais, morfologicamente, não é possível diferenciar os dois tipos virais. A reação em cadeia pela polimerase (PCR), além de ser um método sensível, permite diferenciar os tipos virais sem quantificá-los, não sendo, portanto, indicativa de nefropatia. Segundo a American Society of Nephrology, a PCR em tempo real seria o padrão-ouro para indicar nefropatia, pois permite quantificar o número de cópias virais.

Distribution and genetic diversity of the human polyomaviruses JC and BK in surface water and sewage treatment plant during 2009 in Porto Alegre, Southern Brazil / Distribuição e diversidade genética dos poliomavírus humanos JC e BK em águas superficiais e de estação de tratamento de esgoto durante 2009 em Porto Alegre, sul do Brasil

Abstract The human polyomaviruses JC and BK (JCPyV and BKPyV) are ubiquitous, species-specific viruses that belong to the family Polyomaviridae. These viruses are known to be excreted in human urine, and they are potential indicators of human wastewater contamination. In order to assess the distribution of both JCPyV and BKPyV in urban water samples collected from a sewage treatment plant (STP) and from a canalized water stream of Porto Alegre, Brazil, two nested-PCR assays were optimized and applied to the samples collected. The amplicons obtained were submitted to sequencing, and the sequences were analyzed with sequences of human polyomaviruses previously deposited in GenBank. Twelve out of 30 water samples (40%) were JCPyV positive, whereas six samples (20%) were BKPyV positive. The sequencing results confirmed the presence of JCPyV subtypes 1 and 3, whereas only BKPyV Ia and Ib were found. This study shows for the first time the presence of human polyomaviruses in surface water and in samples collected in a sewage treatment plant in southern Brazil.

Resumo Os poliomavírus humanos JC e BK (JCPyV e BKPyV) são virus ubíquos, espécie-específicos, pertencentes à família Polyomaviridae. Estes vírus são conhecidos por serem excretados pela urina humana, sendo considerados potenciais indicadores de contaminação por águas residuais urbanas. Buscando acessar a distribuição de JCPyV e BKPyV em amostras de águas coletadas de uma estação de tratamento de esgoto e de um arroio canalizado de Porto Alegre, Brasil, duas técnicas de nested-PCR foram otimizadas e aplicadas às amostras coletadas. Os amplificados obtidos foram submetidos ao sequenciamento e suas sequências analisadas com base em sequências de poliomavírus humanos previamente depositadas no GenBank. Doze de 30 amostras de água (40%) foram positivas para JCPyV, enquanto 6 amostras (20%) foram positivas para BKPyV. Os resultados do sequenciamento confirmaram a presença dos subtipos 1 e 3 de JCPyV, enquanto apenas os BKPyV Ia e Ib foram encontrados. Este estudo demonstra pela primeira vez a presença de poliomavírus humanos em águas superficiais e em amostras coletadas em uma estação de tratamento de esgoto na região sul do Brasil.

JC virus/human immunodeficiency virus 1 co-infection in the Brazilian Amazonian region

Abstract JC virus (JCV) is a member of the Polyomaviridae family and is associated to a severe disease known as progressive multifocal leukoencephalopathy, PML, which is gradually increasing in incidence as an opportunistic infection among AIDS patients. The present study aimed to investigate the occurrence of JCV among HIV-1 carriers including their types and molecular subtypes and the possible association with disease. Urine samples from 66 HIV-1 infected subjects were investigated for the presence of the virus by amplifying VP1 (215 bp) and IG (610 bp) regions using the polymerase chain reaction. JCV was detected in 32% of the samples. The results confirmed the occurrence of type B (subtype Af2); in addition, another polyomavirus, BKV, was also detected in 1.5% of samples of the HIV-1 infected subjects. Apparently, there was no significant difference between mono- (HIV-1 only) and co-infected (HIV-1/JCV) subjects regarding their TCD4+/TCD8+ lymphocyte counts or HIV-1 plasma viral load. Self admitted seizures, hearing and visual loses were not significantly different between the two groups.

Detección de polyomavirus BK y JC en extractos leucocitarios de sangre periférica de pacientes con infección por VIH del área norte de Santiago / BK and JC polyomavirus detection in leukocyte extracts of peripheral blood samples of HIV+ patients from the north area of Santiago

Introducción: Polyomavirus BK (BKPyV) y JC (JCPyV) son patógenos persistentes con capacidad de reactivación en inmunocomprometidos, afectando principalmente el sistema urinario y el sistema nervioso central, respectivamente. No existen estudios chilenos en población infectada por VIH. Objetivo: Detectar la presencia de BKPyV y JCPyV en muestras de sangre de pacientes adultos, chilenos, con infección por VIH y correlacionar los resultados con variables clínicas. Materiales y Métodos: Analizamos 96 muestras de extractos leucocitarios de pacientes del área norte de Santiago. El genoma viral se detectó mediante RPC en tiempo real. Para el análisis estadístico se utilizó las pruebas chi-cuadrado de Pearson y Mann-Whitney, considerando significativo un valor de p < 0,05. Resultados: 33% de las muestras resultaron positivas para BKPyV y se encontró una correlación significativa entre la presencia de genoma de BKPyV y la ausencia de carga viral de VIH. Se evidenció la necesidad de considerar más de un blanco de amplificación del genoma de BKPyV. Todas las muestras fueron negativas para JCPyV. Discusión: La prevalencia de BKPyV en pacientes chilenos con infección por VIH es superior a la informada en la mayoría de los reportes internacionales. Se requiere estudios que evalúen la interacción entre ambos virus. Estos pacientes deberían ser sometidos a controles urológicos y nefrológicos periódicos.

Introduction: Polyomavirus BK (BKPyV) and JC (JCPyV) are persistent pathogens able to reactivate in im-munocompromised patients, involving mostly urinary and central nervous system. There are no Chilean studies in HIV positive patients. Objective: To detect BKPyV and JCPyV in blood of Chilean HIV positive adult patients and to correlate these results with clinical-related variables. Materials and Methods: 96 stored blood samples from HIV patients belonging to the north area of Santiago were analyzed. Viral genomes of both viruses were detected by real-time PCR. For statistical analysis, chi-square (Pearson) and Mann-Whitney tests were used and p-values < 0.05 were considered significant. Results: 33% of the samples were positive for BKPyV and a significant correlation was found between the presence of BKPyV genome and the absence of detectable HIV viral load. We demonstrated the need to consider more than one amplification target to detect the BKPyV genome. All the samples were negative for JCPyV genome. Discussion: BKPyV prevalence in Chilean HIV patients is higher than most of international studies. New studies regarding the interaction between both viruses are required. These patients should undergo periodic evaluations by urologist and nephrologist.

JC polyomavirus infection in candidates for kidney transplantation living in the Brazilian Amazon Region

This study evaluated the relative occurrences of BK virus (BKV) and JC virus (JCV) infections in patients with chronic kidney disease (CKD). Urine samples were analysed from CKD patients and from 99 patients without CKD as a control. A total of 100 urine samples were analysed from the experimental (CKD patients) group and 99 from the control group. Following DNA extraction, polymerase chain reaction (PCR) was used to amplify a 173 bp region of the gene encoding the T antigen of the BKV and JCV. JCV and BKV infections were differentiated based on the enzymatic digestion of the amplified products using BamHI endonuclease. The results indicated that none of the patients in either group was infected with the BKV, whereas 11.1% (11/99) of the control group subjects and 4% (4/100) of the kidney patients were infected with the JCV. High levels of urea in the excreted urine, low urinary cellularity, reduced bladder washout and a delay in analysing the samples may have contributed to the low prevalence of infection. The results indicate that there is a need to increase the sensitivity of assays used to detect viruses in patients with CDK, especially given that polyomavirus infections, especially BKV, can lead to a loss of kidney function following transplantation.

High prevalence of the simultaneous excretion of polyomaviruses JC and BK in the urine of HIV-infected patients without neurological symptoms in São Paulo, Brazil / Alta prevalência de excreção simultânea de poliomavírus JC e BK na urina de pacientes HIV+ sem sintomas neurológicos em São Paulo, Brasil

OBJECTIVE: To evaluate the prevalence of the urinary excretion of BKV and JCV in HIV-infected patients without neurological symptoms. METHODS: Urine samples from HIV-infected patients without neurological symptoms were tested for JC virus and BK virus by PCR. Samples were screened for the presence of polyomavirus with sets of primers complementary to the early region of JCV and BKV genome (AgT). The presence of JC virus or BK virus were confirmed by two other PCR assays using sets of primers complementary to the VP1 gene of each virus. Analysis of the data was performed by the Kruskal-Wallis test for numerical data and Pearson or Yates for categorical variables. RESULTS: A total of 75 patients were included in the study. The overall prevalence of polyomavirus DNA urinary shedding was 67/75 (89.3%). Only BKV DNA was detected in 14/75 (18.7%) urine samples, and only JCV DNA was detected in 11/75 (14.7%) samples. Both BKV and JCV DNA were present in 42/75 (56.0%) samples. CONCLUSION: In this study we found high rates of excretion of JCV, BKV, and simultaneous excretion in HIV+ patients. Also these results differ from the others available on the literature.

OBJETIVO: Avaliar a prevalência de excreção urinaria de vírus JC (VJC) e vírus BK (VBK) em pacientes HIV+ sem sintomas neurológicos. MÉTODOS: Amostras de urina de pacientes HIV+ sem sintomas neurológicos foram testados para a presença de VJC e VBK através da técnica de PCR. As amostras foram triadas para a presença de poliomavírus com par de primers complementares a região precoce do genoma do VBK e do VJC (AgT). A presença foi confirmada através de dois outros ensaios de PCR dirigidos a região do gene VP1 de ambos os vírus. A análise estatística foi realizada com auxílio do teste de Kruskal-Wallis para dados numéricos e Pearson ou Yater para variáveis categóricas. RESULTADOS: Ao todo foram inclusos no estudo 75 pacientes. A prevalência geral de excreção de poliomavírus na urina foi de 67/75 (89,3%). O DNA do vírus VBK foi detectado em 14/75 (18,7%) das amostras de urina, e o DNA do VJC foi detectado em 11/75 (14,7%) das amostras testadas. Ambos os vírus estavam presentes simultaneamente em 42/75 (56%) das amostras de urina. CONCLUSÃO: Encontramos, no presente estudo, uma alta taxa de excreção de VJC, VBK e excreção simultânea em pacientes HIV+. Ainda, esses resultados diferem de outros disponíveis na literatura.

Use of John Cunningham virus polymerase chain reaction in the diagnosis of progressive multifocal leucoencephalopathy - A rare presenting manifestation in an HIV-positive patient.

John Cunningham virus infection is an important cause of progressive multifocal leucoencephalopathy (PML) in the context of advanced human immunodeficiency virus infection. Limited data are available regarding the true incidence of PML as a presenting manifestation of HIV. We report one such case and also highlight the effective use of polymerase chain reaction in confirming its diagnosis.

Human polyomaviruses JC and BK in the urine of Brazilian children and adolescents vertically infected by HIV

The aim of this study was to characterize the urinary excretion of the BK (BKV) and JC (JCV) human polyomaviruses in a cohort of human immunodeficiency virus (HIV)-infected children and adolescents. One hundred and fifty-six patients were enrolled: Group I included 116 HIV-infected children and adolescents [median age = 11.4 years (y); range 1-22 y]; Group II included 40 non-HIV-infected healthy controls (median age = 11.37 y; range 7-16 y). Single urine samples from both groups were screened for the presence of JCV and BKV DNA by polymerase chain reaction at enrolment. The overall rate of JCV and BKV urinary excretion was found to be 24.4 percent and 40.4 percent, respectively (n = 156). Group I had urinary excretion of JCV and BKV in 27.6 percent and 54.3 percent of subjects, respectively. In contrast, Group II showed positive results for JCV in 17.5 percent of subjects and for BKV in 12.5 percent of subjects (p Pearson JCV = 0.20; p Pearson BKV < 0.0001). In Group I, there was no association between JCV/BKV shedding and age, gender or CD4 values. Patients with an HIV viral load < 50 copies/mL had a lower excretion of BKV (p < 0.001) and a trend of lower JCV excretion (p = 0.07). One patient in Group I (1/116, 0.9 percent) showed clinical and radiological features consistent with progressive multifocal leukoencephalopathy, suggesting that children with HIV/polyomavirus coinfection should be kept under surveillance.

Comparison of a conventional polymerase chain reaction with real-time polymerase chain reaction for the detection of neurotropic viruses in cerebrospinal fluid samples.

Purpose : To compare a conventional polymerase chain reaction (PCR) and real-time PCR for the detection of neurotropic DNA viruses. Materials and Methods : A total of 147 cerebrospinal fluid (CSF) samples was collected from patients attending a tertiary care hospital in South India for a period from 2005 to 2008. All these samples were tested using a conventional multiplex/uniplex PCR and a real-time multiplex/uniplex PCR. This technique was used to detect a large number of herpes viruses responsible for central nervous system infections, including HSV-1, HSV-2, VZV, CMV and EBV and the polyoma virus JCV. Results : Overall, in the entire set of samples, the real-time PCR yielded 88 (59.9%) positives and conventional PCR had six (4.1%) positives. Conclusion : Our results suggest that the real-time PCR assay was more sensitive compared with the conventional PCR. The advantage of real-time PCR is that it can be performed much faster than conventional PCR. Real-time PCR is less time-consuming, less labour-intensive and also reduces the chance of contamination as there is no post-amplification procedure. In the entire study population, the major viruses detected using real-time PCR were EBV (34%), HSV-2 (10.8%) and VZV (6.8%).

Leucoencefalopatia multifocal progressiva confirmada por PCR para vírus JC no líquor: relato de caso / Progressive multifocal leukoencephalopathy confirmed by PCR for JC virus in cerebrospinal fluid: case report

Relatamos um caso de leucoencefalopatia multifocal progressiva, doença infecciosa desmielinizante do sistema nervoso central, seguido de revisão da literatura. Comenta-se a importância diagnóstica dos métodos de imagem, bem como da análise do líquor e da técnica de reação em cadeia da polimerase (PCR). Até o presente momento, não há terapia efetiva para a condição, com sobrevida média de 1-6 meses nos pacientes não tratados; evolução favorável pode ocorrer no subgrupo de pacientes que obtém melhora da imunidade com o uso de terapia anti-retroviral.

Detection of JC virus infection in progressive multifocal leukoencephalopathy: the first documented case in Thailand.

Progressive Multifocal Leukoencephalopathy (PML) is a demyelinating brain disease caused by human polyoma JC virus (JCV). This disease is an important cause of morbidity and mortality in AIDS patients. Definite diagnosis currently requires a brain biopsy. PCR for JCV of CSF, an emerging diagnostic tool, has a high specificity for the diagnosis of PML in patients with characteristics on clinical and neuroradiological findings. The authors report a 36-year-old woman who presented with prolonged fever, progressive weakness, and slow speech for 2 months. Clinical features and MRI findings were compatible with PML. Qualitative PCR for JCV of CSF showed a positive result. This report emphasizes the yield of PCR, the CSF for JCV in a diagnosis of PML, which may reduce the need for a brain biopsy in such cases.

Co-infección encefálica por papovavirus (virus JC) y varicela Zoster en un paciente con SIDA / Encephalic co-infection by Papovavirus (JC Virus) and Varicella Zoster in an AIDS patient

Se presenta el caso clínico de una paciente infectada por el virus VIH, en etapa Sida, que desarrolló una co-infección por Popovavirus (Virus JC) y Varicella Zoster (V-Z) a nivel encefálico. Las manifestaciones clínicas e imagenológicas de ambos oportunistas son similares y a veces indistinguibles, tal como sucedió en esta paciente. La realización de la técnica denominada reacción en cadena de la polimerasa (PCR) aplicada al LCR; permitió establecer el diagnóstico. Se resalta la posibilidad de que coexistan estos dos oportunistas a nivel encefálico, posibilidad no reportada, en la literatura revisada, y la necesidad, dadas las similitudes existentes de discriminar ambas entidades. La detección del V-Z abre posibilidades terapéuticas mejorando el pronóstico de la intercurrencia infecciosa