ABSTRACT Malaria is an infectious vector-borne disease with other important routes of transmission, such as blood transfusion and organ/tissue transplantation, due to asymptomatic reservoirs of Plasmodium presenting with low parasitemia. Reports of transfusion-transmitted malaria have shown that in immunosuppressed recipients, infections can be fatal if they are not diagnosed and timely treated. All Plasmodium species can survive on blood components at temperatures from 2 to 6 °C for some days or even weeks. This report describes two candidates for blood donation harboring Plasmodium, infected in an area considered non-endemic. Blood samples were collected from donors who attended a blood bank in Sao Paulo and tested by microscopy, qPCR for Plasmodium genus-specific amplification, targeting the parasite 18S ribosomal subunit gene and a multiplex qPCR based on mtDNA of the five species. Under microscopy, only structures resembling Plasmodium were observed. The qPCR whose standard curve tested parasites varying from 2 to 0.1 parasites/ µL, showed the presence of Plasmodium DNA in the two blood donors, as did the multiplex qPCR that revealed the presence of P. malariae. The prevalence of positive donors varies according to the level of transmission, ranging from 0.7 to 55% in endemic areas. In non-endemic regions, prevalences are lower, however, transfusion malaria can evolve to severe cases, due to the lack of suspicion of this transmission route. Asymptomatic donors from low transmission regions pose a risk to blood banks, with particular emphasis on those located in areas with malaria elimination goals.

ABSTRACT Zika virus (ZIKV) clinical presentation and frequency/duration of shedding need further clarification. Symptomatic ZIKV-infected individuals identified in two hospitals in Sao Paulo State, Brazil, were investigated regarding clinical characteristics, shedding in body fluids, and serodynamics. Ninety-four of 235 symptomatic patients (Site A: 58%; Site B: 16%) had Real-Time PCR-confirmed ZIKV infection; fever, headache and gastrointestinal symptoms were less frequent, and rash was more frequent compared to ZIKV-negative patients. Real-Time PCR in serum had worse performance compared to plasma, while urine had the highest sensitivity. Shedding in genital fluids and saliva was rare. IgM positivity was the highest <14 days after the symptoms onset (86%), decreasing >28 days (24%); IgG positivity increased >14 days (96%) remaining positive in 94% of patients >28 days. ZIKV prevalence varied importantly in two neighboring cities during the same transmission season. Urine Real-Time PCR can improve diagnostic sensitivity; serum testing is less useful. Accurate serological tests are needed to improve diagnosis and surveillance.

ABSTRACT Introduction: Cervical cancer remains an important burden for HIV-infected women in the era of combination antiretroviral therapy. Recommendations for cervical screening in these women diverge and may include high-risk HPV (HRHPV) testing. We aimed to evaluate the clinical usefulness of a single HRHPV testing for cervical screening of HIV-infected women. Methods: 723 HIV-infected women from a Brazilian prospective cohort were included between 1996 and 2012. Inclusion criteria were: normal cervical cytology at baseline and having a HRHPV-test at baseline. We calculated incidence rates of any squamous intraepithelial lesion (SIL) and high grade SIL+ (HSIL+) and negative predictive values (NPV) within 12 and 36 months. Hazard Ratios were obtained using Cox proportional hazards regression models. Results: Incidence rate for both outcomes was low (9.9 cases per 100 PY [95% CI 8.8–11.0] for any SIL and 1.3 cases per 100 PY [95% IC 0.9–1.8] for HSIL+). Women with a HRHPV positive status at baseline had 1.7-fold (95% CI 1.3–2.2) and 3.2-fold (95% CI 1.5–7.1) increased risk of presenting any SIL and HSIL+, respectively, during follow-up. Negative-HRHPV test presented high NPV for both periods and outcomes (any SIL: 92.4% [95% CI 89.7–94.6] for 12 months and 80.9% [95% CI 77.2–84.3] for 36 months; and HSIL+: 99.8% [95% CI 98.9–100.0] for 12 months and 99.0 [95% CI 97.6–99.7] for 36 months). Conclusions: Incidence of any and high grade cytological abnormality was significantly higher among HIV-infected women with positive-HRHPV test. A single negative-HRHPV test helped reassure follow-up free of cytological abnormalities through three years of follow-up in HIV-infected women with negative cytology.

OBJECTIVES: Quantitative fluorescence polymerase chain reaction (QF-PCR) is a rapid and reliable method for screening aneuploidies, but in Brazil, it is not used in public services. We investigated the accuracy of QF-PCR for the prenatal recognition of common aneuploidies and compared these results with cytogenetic results in our laboratory. METHOD: A ChromoQuant QF-PCR kit containing 24 primer pairs targeting loci on chromosomes 21, 13, 18, X and Y was employed to identify aneuploidies of the referred chromosomes. RESULTS: A total of 162 amniotic fluid samples analyzed using multiplex QF-PCR were compared with karyotyping analysis. The QF-PCR results were consistent with the results of cytogenetic analysis in 95.4% of all samples. CONCLUSION: QF-PCR was demonstrated to be efficient and reliable for prenatal aneuploidy screening. This study suggests that QF-PCR can be used as a rapid diagnostic method. However, rearrangements and some mosaic samples cannot be detected with this test; thus, those exceptions must undergo cytogenetic analysis.

RESUMO OBJETIVO Estimar a prevalência de infecção do colo do útero pelo HPV entre mulheres assistidas pela Estratégia Saúde da Família e identificar os fatores relacionados à infecção. MÉTODOS Trata-se de estudo transversal, no qual participaram 2.076 mulheres de 20 a 59 anos, residentes em Juiz de Fora, MG, convocadas para rastreamento organizado, realizado em unidades com a Estratégia Saúde da Família implantada. As participantes responderam ao questionário padronizado, realizando exame citológico cervical convencional e teste para HPV de alto risco oncogênico. Foram calculadas estimativas de prevalência de infecção pelo HPV segundo características selecionadas, referenciadas na literatura, relacionadas ao status socioeconômico, saúde reprodutiva e estilo de vida. RESULTADOS A prevalência global de infecção pelo HPV foi 12,6% (IC95% 11,16–14,05). A prevalência para o pooled primer contendo 12 tipos de HPV oncogênicos (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 e 68) foi 8,6% (IC95% 7,3–9,77). Na análise multivariada, observou-se que as seguintes variáveis estavam significativamente associadas a uma maior prevalência de infecção por HPV: estado conjugal (solteira: RP ajustada = 1,40; IC95% 1,07–1,8), consumo de bebidas alcoólicas (qualquer frequência durante a vida: RP ajustada = 1,44; IC95% 1,11–1,86) e número de parceiros sexuais ao longo da vida (≥ 3: RP ajustada = 1,35; IC95% 1,04–1,74). CONCLUSÕES A prevalência de infecção pelo HPV na população estudada varia de média a particularmente alta entre as mulheres jovens. A prevalência de infecção por HPV16 e HPV18 se assemelha às mundiais. Uma distribuição homogênea entre os tipos do pooled primer precederia a infecção isolada pelo HPV18 em magnitude, podendo ser a diferença maior que a observada. A identificação da prevalência de HPV de alto risco oncogênico pode auxiliar na identificação de mulheres sob maior risco de evolução para lesão preneoplásica.

ABSTRACT OBJECTIVE Estimate the prevalence of cervical HPV infection among women assisted by the Family Health Strategy and identify the factors related to the infection. METHODS A cross-sectional study involving 2,076 women aged 20–59 years old residing in Juiz de Fora, State of Minas Gerais, who were asked to participate in an organized screening carried out in units were the Family Health Strategy had been implemented. Participants answered the standardized questionnaire and underwent a conventional cervical cytology test and HPV test for high oncogenic risk. Estimates of HPV infection prevalence were calculated according to selected characteristics referenced in the literature and related to socioeconomic status, reproductive health and lifestyle. RESULTS The overall prevalence of HPV infection was 12.6% (95%CI 11.16–14.05). The prevalence for the pooled primer contained 12 oncogenic HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) was 8.6% (95%CI 7.3–9.77). In the multivariate analysis, it was observed that the following variables were significantly associated with a higher prevalence of HPV infection: marital status (single: adjusted PR = 1.40, 95%CI 1.07–1.8), alcohol consumption (any lifetime frequency: adjusted PR = 1.44, 95%CI 1.11–1.86) and number of lifetime sexual partners (≥ 3: adjusted PR = 1.35, 95%CI 1.04–1.74). CONCLUSIONS The prevalence of HPV infection in the study population ranges from average to particularly high among young women. The prevalence of HPV16 and HPV18 infection is similar to the worldwide prevalence. Homogeneous distribution among the pooled primer types would precede the isolated infection by HPV18 in magnitude, which may be a difference greater than the one observed. The identification of high-risk oncogenic HPV prevalence may help identify women at higher risk of developing preneoplastic lesions.

RESUMO OBJETIVO Estimar a prevalência de infecção do colo do útero pelo HPV entre mulheres assistidas pela Estratégia Saúde da Família e identificar os fatores relacionados à infecção. MÉTODOS Trata-se de estudo transversal, no qual participaram 2.076 mulheres de 20 a 59 anos, residentes em Juiz de Fora, MG, convocadas para rastreamento organizado, realizado em unidades com a Estratégia Saúde da Família implantada. As participantes responderam ao questionário padronizado, realizando exame citológico cervical convencional e teste para HPV de alto risco oncogênico. Foram calculadas estimativas de prevalência de infecção pelo HPV segundo características selecionadas, referenciadas na literatura, relacionadas ao status socioeconômico, saúde reprodutiva e estilo de vida. RESULTADOS A prevalência global de infecção pelo HPV foi 12,6% (IC95% 11,16–14,05). A prevalência para o pooled primer contendo 12 tipos de HPV oncogênicos (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 e 68) foi 8,6% (IC95% 7,3–9,77). Na análise multivariada, observou-se que as seguintes variáveis estavam significativamente associadas a uma maior prevalência de infecção por HPV: estado conjugal (solteira: RP ajustada = 1,40; IC95% 1,07–1,8), consumo de bebidas alcoólicas (qualquer frequência durante a vida: RP ajustada = 1,44; IC95% 1,11–1,86) e número de parceiros sexuais ao longo da vida (≥ 3: RP ajustada = 1,35; IC95% 1,04–1,74). CONCLUSÕES A prevalência de infecção pelo HPV na população estudada varia de média a particularmente alta entre as mulheres jovens. A prevalência de infecção por HPV16 e HPV18 se assemelha às mundiais. Uma distribuição homogênea entre os tipos do pooled primer precederia a infecção isolada pelo HPV18 em magnitude, podendo ser a diferença maior que a observada. A identificação da prevalência de HPV de alto risco oncogênico pode auxiliar na identificação de mulheres sob maior risco de evolução para lesão preneoplásica.

ABSTRACT OBJECTIVE Estimate the prevalence of cervical HPV infection among women assisted by the Family Health Strategy and identify the factors related to the infection. METHODS A cross-sectional study involving 2,076 women aged 20–59 years old residing in Juiz de Fora, State of Minas Gerais, who were asked to participate in an organized screening carried out in units were the Family Health Strategy had been implemented. Participants answered the standardized questionnaire and underwent a conventional cervical cytology test and HPV test for high oncogenic risk. Estimates of HPV infection prevalence were calculated according to selected characteristics referenced in the literature and related to socioeconomic status, reproductive health and lifestyle. RESULTS The overall prevalence of HPV infection was 12.6% (95%CI 11.16–14.05). The prevalence for the pooled primer contained 12 oncogenic HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) was 8.6% (95%CI 7.3–9.77). In the multivariate analysis, it was observed that the following variables were significantly associated with a higher prevalence of HPV infection: marital status (single: adjusted PR = 1.40, 95%CI 1.07–1.8), alcohol consumption (any lifetime frequency: adjusted PR = 1.44, 95%CI 1.11–1.86) and number of lifetime sexual partners (≥ 3: adjusted PR = 1.35, 95%CI 1.04–1.74). CONCLUSIONS The prevalence of HPV infection in the study population ranges from average to particularly high among young women. The prevalence of HPV16 and HPV18 infection is similar to the worldwide prevalence. Homogeneous distribution among the pooled primer types would precede the isolated infection by HPV18 in magnitude, which may be a difference greater than the one observed. The identification of high-risk oncogenic HPV prevalence may help identify women at higher risk of developing preneoplastic lesions.

Abstract: INTRODUCTION: This study aimed to investigate Flavivirus infection in birds captured in green areas of São Paulo. METHODS: Oropharyngeal swabs, cloacal swabs, and blood samples from 170 birds captured in two green areas in São Paulo, Brazil were subjected to real time-polymerase chain reaction (RT-qPCR) analysis of Flavivirus specific NS5 gene fragment. RESULTS: All samples were negative for the presence of viral ribonucleic acid (RNA). CONCLUSIONS: Despite the negative results, Flavivirus surveillance must be performed regularly due to favorable ecological conditions for virus circulation and transmission among birds in these areas and their close proximity to humans.

This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy.

ABSTRACTBACKGROUND: It is recognized that hepatitis C virus subtypes (1a, 1b, 2a, 2b, 2c and 3a) originated in Africa and Asia and spread worldwide exponentially during the Second World War (1940) through the transfusion of contaminated blood products, invasive medical and dental procedures, and intravenous drug use. The entry of hepatitis C virus subtypes into different regions occurred at distinct times, presenting exponential growth rates of larger or smaller spread. Our study estimated the growth and spread of the most prevalent subtypes currently circulating in São Paulo.METHODS:A total of 465 non-structural region 5B sequences of hepatitis C virus covering a 14-year time-span were used to reconstruct the population history and estimate the population dynamics and Time to Most Recent Common Ancestor of genotypes using the Bayesian Markov Chain Monte Carlo approach implemented in BEAST (Bayesian evolutionary analysis by sampling tree software/program).RESULTS:Evolutionary analysis demonstrated that the different hepatitis C virus subtypes had distinct growth patterns. The introduction of hepatitis C virus-1a and -3a were estimated to be circa 1979 and 1967, respectively, whereas hepatitis C virus-1b appears to have a more ancient entry, circa 1923. Hepatitis C virus-1b phylogenies suggest that different lineages circulate in São Paulo, and four well-supported groups (i.e., G1, G2, G3 and G4) were identified. Hepatitis C virus-1a presented the highest growth rate (r = 0.4), but its spread became less marked after the 2000s. Hepatitis C virus-3a grew exponentially until the 1990s and had an intermediate growth rate (r = 0.32). An evident exponential growth (r = 0.26) was found for hepatitis C virus-1b between 1980 and the mid-1990s.CONCLUSIONS:After an initial period of exponential growth, the expansion of the three main subtypes began to decrease. Hepatitis C virus-1b presented inflated genetic diversity, and its transmission may have been sustained by different generations and transmission routes other than blood transfusion. Hepatitis C virus-1a and -3a showed no group stratification, most likely due to their recent entry.

OBJETIVO: Comparar o desempenho de duas técnicas de genotipagem de papilomavírus humano (HPV), Linear Array e PapilloCheck, em mulheres com lesão intra-epitelial de alto grau (LIAG).MÉTODOS: Foram selecionadas 88 mulheres com diagnóstico citopatológico de LIAG em 2 centros de referência em patologia cervical em Salvador, Bahia, no período de julho de 2006 a janeiro de 2009. Após o diagnóstico citopatológico de LIAG, foram realizadas a coleta de células do colo uterino para a genotipagem do HPV e a biópsia sob visão colposcópica para análise histopatológica do fragmento retirado. Posteriormente à confirmação de NIC2+ pelo exame histopatológico, foi realizada a genotipagem do HPV em 41 mulheres pelas técnicas Linear Array e PapilloCheck.RESULTADOS: Os dois testes apresentaram taxa de concordância geral para detecção do vírus HPV de 97,2% (35/36). Das 36 amostras válidas, 35 (97,2%) foram consideradas positivas em ambos os testes e apenas uma amostra (2,8%) foi considerada discordante. Os genótipos do HPV mais prevalentes detectados através da técnica do Linear Array foram: HPV 16, HPV 56, HPV 35, HPV 45 e HPV 70; e pela técnica PapilloCheck foram: HPV 16, HPV 56, HPV 11, HPV 35 e HPV 42. Foi observado índice semelhante de infecção por múltiplos tipos do HPV nos dois testes analisados (72,5% no Linear Array e 75,0% no PapilloCheck).CONCLUSÕES: Os testes de genotipagem Linear Array e PapilloCheck apresentaram um desempenho equivalente na detecção dos tipos de HPV oncogênicos em mulheres com LIAG, tendo o PapilloCheck a vantagem de ser um método que evita a subjetividade da leitura dos genótipos de HPV.

PURPOSE: The aim of this study was to compare the performance of two human papillomavirus (HPV) genotyping techniques, Linear Array and PapilloCheck, in women with high-grade squamous intraepithelial lesion (HSIL).METHODS: A total of 88 women with cytological diagnosis of HSIL were recruited at 2 reference centers in cervical pathology in Salvador, Bahia, Brazil, from July 2006 to January 2009. After the cytological diagnosis of HSIL, cervix cells were collected to determine the HPV genotype and a biopsy was obtained under colposcopic vision for histopathological analysis. After the confirmation of CIN2+ by histopathology, HPV genotyping was performed on 41 women by the Linear Array and PapilloCheck methods.RESULTS: Both tests showed an overall concordance rate for HPV detection of 97.2% (35/36). Of the 36 valid samples, 35 (97.2%) were positive in both tests and 1 (2.8%) was discordant, with the Linear Array indicating the presence of multiple types. The most prevalent HPV genotypes detected by the Linear Array technique were HPV 16, HPV 56, HPV 35, HPV 45, and HPV 70; and those detected by the PapilloCheck technique were HPV 16, HPV 56, HPV 11, HPV 35, and HPV 42. A similar rate of infection with multiple HPV types was observed with the two tests (72.5% with the Linear Array and 75.0% with the PapilloCheck).CONCLUSIONS: Linear Array genotyping assay and PapilloCheck showed equivalent performance for the detection of oncogenic HPV types in women with HSIL, with PapilloCheck having the advantage of being a method that avoids subjectivity when reading the HPV genotypes.

Objetivo: o objetivo deste estudo foi identificar os genótipos do HBV nas amostras de soros recebidas pelo Laboratório de Hepatites do Centro de Virologia do Instituto Adolfo Lutz. Métodos: foram analisadas aleatoriamente 94 amostras de soropositivas, provenientes de diversos municípios do Estado de São Paulo. Para determinação dos genótipos, foi realizada Nested-PCR das regiões S e Pol do HBV. Os genótipos foram identificados comparando os resultados amplificados com as sequências depositadas no GenBank. Resultados: foi possível determinar o genótipo de 91 (97%) amostras do total analisado e os genótipos identificados foram: genótipos A (55,3%), D (32%), F (5,3%), C (3,2%) e G (1%). Há poucos dados a respeito da epidemiologia do genótipo G. Esse genótipo tem sido detectado em áreas restritas do mundo. Geralmente, a infecção pelo genótipo G ocorre em indivíduos HIV positivos do sexo masculino. Neste trabalho, a amostra identificada como G foi também positiva para HIV e era de uma paciente do sexo feminino, dado raro na literatura científica. Conclusão: neste trabalho, foram identificados os genótipos mais frequentes, assim como uma cepa do genótipo G, rara entre os encontrados em nosso meio.

Objective: the aim of this study was to identify HBV genotypes in serum samples from patients from the state of São Paulo, received by the viral hepatitis laboratory, at the Virology Centre of Instituto Adolfo Lutz, from various municipalities. Methods: a total of 94 serum samples were randomly analyzed. Genotyping was performed using nested PCR for amplification of S and Pol regions from viral genome. Genotypes were identified comparing the sequences obtained with the sequences deposited in GenBank. Results: we were able to determine the genotype of 91 (97%) samples, as follows: genotype A (55.3%), D (32%), F (5.3%), C (3.2%) and G (1%). There are few data on the epidemiology of genotype G. This genotype has been detected in restricted areas around the world. Frequently, the genotype G infection occurs in HIV-positive male patients. In our case, the sample identified as G was also positive for HIV but in a female patient, which is an uncommon finding in the scientific literature. Conclusion: in this work, we identified the most frequent genotypes in São Paulo as well as the genotype G, rare among the genotypes found in our environment.

A descoberta de ácidos nucleicos fetais livres no plasma de gestantes possibilitou o desenvolvimento de novos testes de diagnóstico pré-natal não invasivo para a determinação do sexo e do Rh fetal. Esses testes foram implantados no sistema de saúde pública de diversos países da Europa há mais de cinco anos. As novas possibilidades de aplicação diagnóstica dessas tecnologias são a detecção de aneuploidias cromossômicas fetais, de doenças monogênicas fetais e de distúrbios relacionados com a placenta, temas pesquisados intensivamente por diversos grupos ao redor do mundo. O objetivo deste estudo é expor a situação brasileira no âmbito de pesquisa e utilização clínica dos testes disponíveis comercialmente que utilizam esses marcadores moleculares plasmáticos, ressaltando as vantagens, tanto econômicas quanto de segurança, que os testes não invasivos têm em relação aos atualmente utilizados em nosso sistema de saúde pública.

The discovery of cell-free fetal nucleic acids in the plasma of pregnant women has allowed the development of new, noninvasive prenatal diagnostic tests for the determination of fetal gender and Rh. These tests have been implemented in the public health system in several countries of Europe for over five years. The new possibilities for diagnostic use of these technologies are the detection of fetal chromosomal aneuploidies, monogenic fetal disorders, and placental-related disorders, subjects that have been intensively studied by several groups around the world. The aim of this review was to assess the Brazilian research and clinical scenarios regarding the utilization of commercially available tests that use these plasma markers, stressing the advantages, both economic and safety-related, that non-invasive tests have when compared to those currently used in the Brazilian public health system.

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%), enquanto 57 amostras foram concordantemente RNA positivas pelo nested-PCR e PCR em tempo real (22,5%), perfazendo um índice total de exposição de 48% (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.

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% of the patients whereas the antibody was identified in 25.3% 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.