Not required, meaning that HMPL-013 web sample-taking will not affect the natural history of HPV infection as there is no risk of micro-lesions being produced, nor will inflammatory reactions occur [15]. Despite of multiple studies available in the literature that have evaluated HPV-DNA detection from urine sample [15], a few number of these have been described the diagnostic performance of this sample in HIV-positive women population. Furthermore those who have done it had included a limited number of individuals [9,17]. In Colombia high prevalence of HPV infection and co-infection in healthy women population have been reported, using cervical samples [18,19]. However haven’t be evaluated HPV DNA detection from urine samples neither in HIV-positive women population. This study aimed at identifying the infection, coinfection (defined here as being infection by more than one type of HPV simultaneously) and type-specific distribution profile of six highrisk HPV (HR-HPV) types and two low-risk (LR-HPV) types, from paired cervical and urine samples of women diagnosed with HIV/ AIDS, confirmed by Western blot. Finally, we evaluated the diagnostic performance of urine samples compared to cervical samples for detecting HPV infection.Sample size was calculated assuming an estimated 80 HPV infection rate in HIV-positive women [4,17,20], according to data reported in the literature. Estimators were calculated using 0.05 precision along with 95 confidence intervals (95 CI) using STATA9 software sampsi command.Collecting and processing cervical and urine samplesAll the women enrolled in the study were informed about the research objective; they signed an informed consent form and filled in a questionnaire to facilitate collecting socio-demographic data and information regarding their GDC-0941 sexual habits and other risk factors related to acquiring HPV infection. Each woman’s urine and cervical samples were taken on the same day; the first sample from a midstream urine specimen was self-collected, kept at 4uC and processed within 72 hours after being collected. The second sample taken from cervical cells was obtained during Papanicolau test, following Colombian obligatory health plan guidelines regarding cervical cancer detection and control programs in Colombia [21]; these cells were preserved in 95 ethanol [22,23] and kept at 4uC until being processed. The histological findings were reported following the Bethesda classification [13]. The cells were precipitated by spinning at 2,3006 g for 20 minutes at 4uC for urine samples and at 15,0006 g for 10 minutes at 4uC for cervical samples. DNA was extracted from cell pellets of paired samples using a QuickExtract DNA extraction kit (Epicentre, Madison, WI), following the manufacturer’s instructions. Two PCR amplifications were made with specific primers directed at a segment of the human b-globin constitutive gene (GH20/PC04 and PC03/PC04) for evaluating DNA integrity [18,22,24].Detecting human papillomavirus DNA by PCR amplificationSamples yielding a positive result for the human b-globin gene were amplified for detecting HPV using three consensus primer sets (for detecting more infected women) as it has been reported that using a single set might lead to underestimating viral prevalence compared to studies using more than one generic detection system [25]. Two of the primers sets were directed to the region encoding late viral protein L1: GP5+/6+ and MY09/11 [26,27]; PCR conditions have been described previously [22].Not required, meaning that sample-taking will not affect the natural history of HPV infection as there is no risk of micro-lesions being produced, nor will inflammatory reactions occur [15]. Despite of multiple studies available in the literature that have evaluated HPV-DNA detection from urine sample [15], a few number of these have been described the diagnostic performance of this sample in HIV-positive women population. Furthermore those who have done it had included a limited number of individuals [9,17]. In Colombia high prevalence of HPV infection and co-infection in healthy women population have been reported, using cervical samples [18,19]. However haven’t be evaluated HPV DNA detection from urine samples neither in HIV-positive women population. This study aimed at identifying the infection, coinfection (defined here as being infection by more than one type of HPV simultaneously) and type-specific distribution profile of six highrisk HPV (HR-HPV) types and two low-risk (LR-HPV) types, from paired cervical and urine samples of women diagnosed with HIV/ AIDS, confirmed by Western blot. Finally, we evaluated the diagnostic performance of urine samples compared to cervical samples for detecting HPV infection.Sample size was calculated assuming an estimated 80 HPV infection rate in HIV-positive women [4,17,20], according to data reported in the literature. Estimators were calculated using 0.05 precision along with 95 confidence intervals (95 CI) using STATA9 software sampsi command.Collecting and processing cervical and urine samplesAll the women enrolled in the study were informed about the research objective; they signed an informed consent form and filled in a questionnaire to facilitate collecting socio-demographic data and information regarding their sexual habits and other risk factors related to acquiring HPV infection. Each woman’s urine and cervical samples were taken on the same day; the first sample from a midstream urine specimen was self-collected, kept at 4uC and processed within 72 hours after being collected. The second sample taken from cervical cells was obtained during Papanicolau test, following Colombian obligatory health plan guidelines regarding cervical cancer detection and control programs in Colombia [21]; these cells were preserved in 95 ethanol [22,23] and kept at 4uC until being processed. The histological findings were reported following the Bethesda classification [13]. The cells were precipitated by spinning at 2,3006 g for 20 minutes at 4uC for urine samples and at 15,0006 g for 10 minutes at 4uC for cervical samples. DNA was extracted from cell pellets of paired samples using a QuickExtract DNA extraction kit (Epicentre, Madison, WI), following the manufacturer’s instructions. Two PCR amplifications were made with specific primers directed at a segment of the human b-globin constitutive gene (GH20/PC04 and PC03/PC04) for evaluating DNA integrity [18,22,24].Detecting human papillomavirus DNA by PCR amplificationSamples yielding a positive result for the human b-globin gene were amplified for detecting HPV using three consensus primer sets (for detecting more infected women) as it has been reported that using a single set might lead to underestimating viral prevalence compared to studies using more than one generic detection system [25]. Two of the primers sets were directed to the region encoding late viral protein L1: GP5+/6+ and MY09/11 [26,27]; PCR conditions have been described previously [22].
