Automated Extraction and Quantification of Human Cytomegalovirus DNA in Whole Blood by Real-Time PCR Assay

The measurement of human cytomegalovirus (HCMV) DNA in blood is becoming the standard method for monitoring HCMV infection in immune-suppressed and unsuppressed patients. As various blood compartments can be used, we have compared the HCMV DNA measured in whole blood (WB), peripheral blood leukocytes (PBL), and plasma by real-time PCR. We tested 286 samples: HCMV DNA was extracted automatically from WB and PBL with the MagNA Pure instrument (Roche Molecular Biochemicals) and manually from plasma samples. The HCMV DNA from WB, PBL, and plasma was measured by real-time Light Cycler PCR. Primers and probe were located in the UL 83 region. HCMV DNA was detected more frequently in WB (88.5%) than in the PBL (65.7%) (P < 0.0001) or the plasma (55.2%) (P < 0.0001). There was a good correlation between the positive results in WB and in PBL (r = 0.68; P < 0.0001), and 3.15 log(10) genome copies in 200,000 PBL, equivalent to the threshold value of 50 pp65-positive polymorphonuclear cells per 200,000 leukocytes, was equivalent to 3.4 log(10) genome copies in 200 μl of WB. WB was shown to be suitable for automated extraction and the quantitation of HCMV DNA by real-time Light Cycler PCR by analysis of serial samples from representative patients of various populations. This system may be very useful for monitoring of immune-suppressed and unsuppressed patients

HIV AND HCV COINFECTION: PREVALENCE, ASSOCIATED FACTORS AND GENOTYPE CHARACTERIZATION IN THE MIDWEST REGION OF BRAZIL

Estudo transversal sobre a prevalência, fatores associados e distribuição dos genótipos do HCV foi realizado em 848 pacientes infectados pelo HIV, recrutados em centros de referência na Região Centro-Oeste do Brasil. A taxa de prevalência de coinfecção HIV-HCV foi de 6,9% (IC 95%: 5,2-8,6). Na análise multivariada, o aumento da idade, o uso de drogas ilícitas (injetáveis e não injetáveis), história de transfusão de sangue antes de 1994, e ausência de companheiro constante foram fatores associados independentes e significativos para a coinfecção HIV-HCV. A análise filogenética baseada na região NS5B revelou a presença de dois principais genótipos do HCV em circulação: genótipos 1 (58,3%) e 3 (41,7%). A prevalência da coinfecção HIV-HCV foi menor do que as relatadas em estudos realizados com pacientes infectados pelo HIV em diferentes regiões do Brasil, devido ao fato de que o uso de drogas ilícitas não é modo frequente de transmissão do HIV neste Estado do Brasil. Triagem sorológica de pacientes HIV-positivos para HCV antes de iniciar o tratamento antirretroviral, identificação completa dos fatores associados e a implementação de programas eficazes de redução de danos são altamente recomendados para fornecer informações úteis, para o tratamento e para evitar a coinfecção com HCV nestes pacientes.A cross-sectional study on prevalence, associated factors and genotype distribution of HCV infection was conducted among 848 HIV-infected patients recruited at reference centers in the Midwest Region of Brazil. The prevalence rate of HIV-HCV coinfection was 6.9% (95% CI: 5.2 to 8.6). In multivariable analysis, increasing age, use of illicit drugs (injection and non-injection), a history of blood transfusion before 1994, and the absence of a steady partnership were significant independent associated factors for HIV-HCV coinfection. The phylogenetic analysis based on the NS5B region revealed the presence of two major circulating genotypes of HCV: genotypes 1 (58.3%) and 3 (41.7%). The prevalence of HIV-HCV coinfection was lower than those reported in studies conducted with HIV-infected patients in different regions of Brazil, due to the fact that illicit drug use is not a frequent mode of HIV transmission in this region of Brazil. Serologic screening of HIV-patients for HCV before initiating antiretroviral treatment, a comprehensive identification of associated factors, and the implementation of effective harm reduction programs are highly recommended to provide useful information for treatment and to prevent HCV coinfection in these patients