Cell-free HIV-1 RNA Research Articles

The effect of vaginal candidiasis on the shedding of human immunodeficiency virus in cervicovaginal secretions

The purpose of this study was to evaluate the influence of symptomatic vulvovaginal candidiasis on the shedding of HIV-1 in cervicovaginal secretions of HIV-1-infected women. We obtained paired blood and cervicovaginal lavage samples from 66 HIV-infected women with symptomatic vulvovaginal candidiasis, and 249 HIV-infected control patients without genital infection. HIV-1 RNA in plasma, proviral HIV-1 DNA, HIV-1 RNA transcripts, and cell-free HIV-1 RNA in cervicovaginal secretions were quantitatively evaluated by competitive polymerase chain reaction (cPCR) and reverse transcriptase PCR (cRT-PCR). We used logistic regression on ordered data to assess the influence of vulvovaginal candidiasis on the HIV-1 load in cervicovaginal secretions adjusting for potential confounders. Overall, the amount of HIV-1 RNA in plasma was significantly correlated with HIV-1 DNA (Spearman rank 0.153 +/- 0.059, P = .006), HIV-1 RNA transcripts (Spearman rank 0.169 +/- 0.058, P = .003), and cell free HIV-1 RNA (Spearman rank 0.185 +/- 0.059, P = .001) load in cervicovaginal secretion. Forty-eight out of 182 (26.4%) patients who tested negative for HIV-1 RNA in plasma were positive for HIV-DNA in their cervicovaginal secretions. In logistic regression analysis vulvovaginal candidiasis was significantly associated with increasing loads of HIV-1 RNA transcripts (Odds ratio [OR] 1.97, 95% CI 1.09-3.57, P = .025) and cell free HIV-1 RNA (OR 2.03, 95% CI 1.10-3.73, P = .02) in cervicovaginal secretions. In HIV-infected women, vulvovaginal candidiasis is associated with an increased number of copies of cell-associated and cell-free HIV-1 RNA in cervicovaginal secretions.

Markers of local immunity in cervico-vaginal secretions of HIV infected women: implications for HIV shedding

Objectives: To link local proinflammatory cytokines with HIV related nucleic acids in cervico-vaginal secretions and the factors associated with them. Methods: An observational study on 60 HIV positive women attending...

Human Immunodeficiency Virus Type 1-Related Nucleic Acids and Papillomavirus DNA in Cervicovaginal Secretions of Immunodeficiency Virus-Infected Women

In Brief Objective To evaluate simultaneous human immunodeficiency virus (HIV)-related nucleic acids and human papillomavirus (HPV)-DNA in cervicovaginal secretions of HIV-seropositive women. Methods We collected 47 paired blood and cervicovaginal lavage samples from 124 known HIV-1–seropositive women. Proviral HIV-1 DNA, cell-associated, and cell-free HIV-1 RNA in cervicovaginal secretions were quantitatively evaluated by competitive polymerase chain reaction (PCR) and reverse transcription PCR. Polymerase chain reaction and subsequent restriction fragment length polymorphism analysis of PCR products were used to detect HPV types 6, 11, 16, 18, 31, 33, 35, and 56. Results Proviral HIV-1 DNA, cell-associated, and cell-free HIV-1 RNA were detected in 52.4% (65 of 124), 38.7% (48 of 124), and 33.9% (42 of 124) of lavage samples, respectively. Human papillomavirus-DNA in cervicovaginal secretions was detected in 64% (79 of 124) of participants. The rate of detection of HPV types of intermediate to high oncogenic risk was higher in HIV-positive women who tested positive for cell-associated (odds ratio [OR] 3.57, 95% confidence interval [CI] 1.17, 11.12) or cell-free (OR 4.63, 95% CI 1.42, 15.51) HIV-1 RNA in cervicovaginal secretions than their counterparts who tested negative. Logistic regression analysis showed that the association between HPV infection and the detection of HIV-1 RNA in cervicovaginal secretions persisted after adjustment for potential confounders such as CD4+ cell counts, HIV-1 RNA in plasma, use of antiretroviral drugs, vaginal infection, and regular condom use. In univariable and multivariable analysis, HPV-DNA detection was associated with amounts of cell-free and cell-associated HIV-1 RNA in cervicovaginal secretions (χ2 for trend 10.35, and 9.84, P = .001 and .002, respectively). Conclusions The rate of HPV detection in the genital tract of HIV-1–seropositive women is associated with the amount of cell-associated and cell-free HIV-1 RNA present in cervicovaginal secretions. The association does not appear to be attributable entirely to the effect of HIV-related immunodepression. In human immunodeficiency virus-infected women, papillomavirus shedding is associated significantly with the amount of cell-associated and cell-free immunodeficiency-virus RNA in cervicovaginal secretions.

Factors associated with nucleic acids related to human immunodeficiency virus type 1 in cervico-vaginal secretions.

To assess HIV-related nucleic acids in cervico-vaginal secretions and the factors associated with them. Observational study. Department of Obstetrics and Gynaecology, University of Pavia, Italy. HIV-positive patients attending a cytology service. Paired blood and cervico-vaginal lavage samples were obtained from 122 known HIV-seropositive patients during periodic visits for cytologic screening for lower genital tract neoplasia. Vaginal specimens for the diagnosis of bacterial vaginosis, trichomonas vaginalis and candida infection were also obtained. HIV-1-RNA in plasma, proviral HIV-1-DNA, cell associated and cell-free HIV-1 RNA in cervico-vaginal secretions were quantitatively evaluated by competitive polymerase chain reaction (c-PCR) and reverse transcriptase PCR (cRT-PCR). Prevalences of HIV related nucleic acids in cervico-vaginal secretions and their univariate and multivariate associations with clinical variables. Proviral HIV-1 DNA, cell-associated and cell-free HIV-1 RNA were detected in 50% (61/122), 37.7% (46/122) and 32.8% (40/122) of the patients, respectively. In logistic regression analysis, the presence of HIV-1 RNA in blood was the factor which correlated best with the detection of HIV-1 DNA (OR = 5.48, 95% CI = 2.28-13.20), cell-associated (OR = 4.85; 95% CI = 1.89-12.45) and cell-free HIV-1 RNA (OR = 4.63, 95% CI = 1.74-12.33) in cervico-vaginal samples. However, between 20% and to 35% of patients who tested negative for blood HIV-1 RNA were positive for either HIV-1 DNA or HIV-1 RNA detection in cervico-vaginal lavages. Bacterial vaginosis was associated with an increased prevalence of cell-associated (OR = 3.58, 95% CI = 1.22-10.54) and cell-free HIV-1 RNA (OR = 2.94, 95% CI = 1.0-8.7) detection in cervico-vaginal secretions. Additional factors associated with increased prevalence of HIV-1 RNA detection were advanced stage of HIV disease and vulvovaginal candidiasis. Although the presence of HIV-1 RNA in blood is the factor which correlates best with the detection of HIV-related nucleic acids in cervico-vaginal secretions, the shedding of HIV in the genital tract can occur in 20-30% of non-viremic subjects. Bacterial vaginosis and candida infection could have a facilitating role in local HIV viral replication and shedding.

Factors associated with nucleic acids related to human immunodeficiency virus type 1 in cervico-vaginal secretions

Objective To assess HIV-related nucleic acids in cervico-vaginal secretions and the factors associated with them. Design Observational study. Setting Department of Obstetrics and Gynaecology, University of Pavia, Italy. Population HIV-positive patients attending a cytology service. Methods Paired blood and cervico-vaginal lavage samples were obtained from 122 known HIV-seropositive patients during periodic visits for cytologic screening for lower genital tract neoplasia. Vaginal specimens for the diagnosis of bacterial vaginosis, trichomonas vaginalis and candida infection were also obtained. HIV-1-RNA in plasma, proviral HIV-1-DNA, cell associated and cell-free HIV-1 RNA in cervico-vaginal secretions were quantitatively evaluated by competitive polymerase chain reaction (c-PCR) and reverse transcriptase PCR (cRT-PCR). Main outcome measure Prevalences of HIV related nucleic acids in cervico-vaginal secretions and their univariate and multivariate associations with clinical variables. Results Proviral HIV-1 DNA, cell-associated and cell-free HIV-1 RNA were detected in 50% (61/122), 37.7% (46/122) and 32.8% (40/122) of the patients, respectively. In logistic regression analysis, the presence of HIV-1 RNA in blood was the factor which correlated best with the detection of HIV-1 DNA (OR = 5.48, 95% CI=2.28-13.20), cell-associated (OR = 4.85; 95% CI = 1.89–12.45) and cell-free HIV-1 RNA (OR = 4.63, 95% CI = 1.74–12.33) in cervico-vaginal samples. However, between 20% and to 35% of patients who tested negative for blood HIV-1 RNA were positive for either HIV-1 DNA or HIV-1 RNA detection in cervico-vaginal lavages. Bacterial vaginosis was associated with an increased prevalence of cell-associated (OR=3.58, 95% CI = 1.22–10.54) and cell-free HIV-1 RNA (OR = 2.94, 95% CI = 1.0–8.7) detection in cervico-vaginal secretions. Additional factors associated with increased prevalence of HIV-1 RNA detection were advanced stage of HIV disease and vulvovaginal candidiasis. Conclusions Although the presence of HIV-1 RNA in blood is the factor which correlates best with the detection of HIV-related nucleic acids in cervico-vaginal secretions, the shedding of HIV in the genital tract can occur in 20-30% of non-viremic subjects. Bacterial vaginosis and candida infection could have a facilitating role in local HIV viral replication and shedding.

Human immunodeficiency virus type 1-related nucleic acids and papillomavirus DNA in cervicovaginal secretions of immunodeficiency virus-infected women

Objective: To evaluate simultaneous human immunodeficiency virus (HIV)-related nucleic acids and human papillomavirus (HPV)-DNA in cervicovaginal secretions of HIV-seropositive women. Methods: We collected 47 paired blood and cervicovaginal lavage samples from 124 known HIV-1–seropositive women. Proviral HIV-1 DNA, cell-associated, and cell-free HIV-1 RNA in cervicovaginal secretions were quantitatively evaluated by competitive polymerase chain reaction (PCR) and reverse transcription PCR. Polymerase chain reaction and subsequent restriction fragment length polymorphism analysis of PCR products were used to detect HPV types 6, 11, 16, 18, 31, 33, 35, and 56. Results: Proviral HIV-1 DNA, cell-associated, and cell-free HIV-1 RNA were detected in 52.4% (65 of 124), 38.7% (48 of 124), and 33.9% (42 of 124) of lavage samples, respectively. Human papillomavirus-DNA in cervicovaginal secretions was detected in 64% (79 of 124) of participants. The rate of detection of HPV types of intermediate to high oncogenic risk was higher in HIV-positive women who tested positive for cell-associated (odds ratio [OR] 3.57, 95% confidence interval [CI] 1.17, 11.12) or cell-free (OR 4.63, 95% CI 1.42, 15.51) HIV-1 RNA in cervicovaginal secretions than their counterparts who tested negative. Logistic regression analysis showed that the association between HPV infection and the detection of HIV-1 RNA in cervicovaginal secretions persisted after adjustment for potential confounders such as CD4+ cell counts, HIV-1 RNA in plasma, use of antiretroviral drugs, vaginal infection, and regular condom use. In univariable and multivariable analysis, HPV-DNA detection was associated with amounts of cell-free and cell-associated HIV-1 RNA in cervicovaginal secretions (χ 2 for trend 10.35, and 9.84, P = .001 and .002, respectively). Conclusions: The rate of HPV detection in the genital tract of HIV-1–seropositive women is associated with the amount of cell-associated and cell-free HIV-1 RNA present in cervicovaginal secretions. The association does not appear to be attributable entirely to the effect of HIV-related immunodepression.

Treatment of cervicitis is associated with decreased cervical shedding of HIV-1.

To determine whether cervical mucosal shedding of HIV-1 RNA and HIV-1 infected cells decreases following successful treatment of cervicitis. Prospective interventional study. Sexually Transmitted Infections Clinic, Coast Provincial General Hospital, Mombasa, Kenya. Thirty-six HIV-1 seropositive women with cervicitis: 16 with Neisseria gonorrhoeae, seven with Chlamydia trachomatis, and 13 with non-specific cervicitis. Treatment of cervicitis. Levels of total (cell-free and cell-associated) HIV-1 RNA and presence of HIV-1 DNA (a marker for infected cells) in cervical secretions before and after resolution of cervicitis. After treatment of cervicitis, the median HIV-1 RNA concentration in cervical secretions was reduced from 4.05 to 3.24 log10 copies/swab (P = 0.001). Significant decreases in cervical HIV-1 RNA occurred in the subgroups with N. gonorrhoeae (3.94 to 3.28 log10 copies/swab; P = 0.02) and C. trachomatis (4.21 to 3.19 log10 copies/swab; P = 0.02). Overall, the prevalence of HIV-1 infected cells in cervical secretions also decreased after treatment, from 67% to 42% (odds ratio, 2.8; 95% confidence interval, 1.3-6.0; P = 0.009). Detection of infected cells was associated with higher mean HIV-1 RNA levels (4.04 versus 2.99 log10 copies/swab; P< 0.0001). Effective treatment of cervicitis resulted in significant decreases in shedding of HIV-1 virus and infected cells in cervical secretions. Treatment of sexually transmitted diseases may be an important means of decreasing the infectivity of HIV-1 seropositive women by reducing exposure to HIV-1 in genital secretions.

Quantitative assessment of cell-associated and cell-free virus in cervicovaginal samples of HIV-1-infected women.

To examine the amount of cell-free and cell-associated virus in cervicovaginal secretions (CVS) of HIV-infected women. Paired cervicovaginal and blood samples from 61 seropositive women were quantitatively evaluated by competitive polymerase chain reaction (cPCR) and reverse transcription-PCR (cRT-PCR) for: (1) genomic RNA from plasma and cell-free CVS, and (2) unspliced (u/s) RNA transcripts and proviral DNA in cells from secretions. HIV DNA was detected in 42.6%, u/s transcripts in 32.7% and cell-free HIV RNA in 31.1% of 61 cervicovaginal samples. The median copy numbers of HIV DNA, u/s transcripts, and cell-free RNA were 125 copies/10(5) cells, 40 copies/10(5) cells, and 300 copies/mL of secretion, respectively. Nineteen of 26 (73.1%) and 17 of 26 (65.3%) women positive for DNA were also positive for RNA transcripts and cell-free RNA, respectively (P<0.001). A significant correlation between the amounts of cell-free and u/s transcripts was also found (Spearman Rho 0.618, P=0.014). The prevalences of u/s transcripts and cell-free RNA were 42.6% and 53.8% respectively among patients with detectable blood RNA, and 22.9% (P=0.09) and 14.3% (P=0.0017) among patients with undetectable blood RNA. In stepwise logistic regression, cell-free RNA was independently associated with the presence of detectable blood viremia. The amount of HIV DNA was lower among subiects currently under treatment (50 copies/10(5) cells) than in untreated subjects (250 copies/10(5) cells) (P=0.037). Both cell-free and cell-associated HIV could be detected and quantitated in CVS, providing a means to examine the level of viral activity in the female genital tract.

Asymptomatic urethritis and detection of HIV-1 RNA in seminal plasma.

OBJECTIVE: To define risk factors for detection of HIV-1 RNA in semen in men attending the two largest HIV clinics in the West Midlands. METHODS: 94 HIV-1 seropositive men at...

A Combination of Nucleoside Analogues and a Protease Inhibitor Reduces HIV-1 RNA Levels in Semen: Implications for Sexual Transmission of HIV Infection

Direct contact with semen is the major route of sexual acquisition of human immunodeficiency virus (HIV) in homosexual and heterosexual partners of seropositive men. In this study, we show that concentrations of HIV-1 RNA molecules in plasma and semen of seropositive patients are related to the duration and type of antiretroviral agents used in treatment. In patients treated with zidovudine alone, 1, 3 and 6 months after the start of therapy, the mean HIV-1 load in plasma was reduced by 0.57, 0.38 and 0.21 log10 and in semen by 0.66, 0.50 and 0.15 log10, respectively. In patients treated with zidovudine plus didanosine at months 1, 3 and 6, the mean decrease in plasma HIV-1 RNA was 1.40, 1.25 and 1.12 log10 and in semen 1.10, 1.41 and 1.32 log10, respectively. In patients treated with a combination of a protease inhibitor and two nucleoside analogues the mean log10 decrease was 1.77, 1.83, 1.71 and 2.38 log10 in plasma and 1.17, 1.74, 2.19 and 3.02 log10 in semen at 1, 2, 3 and 4 months, respectively. Treatment with a combination of a protease inhibitor and two nucleoside analogues caused a dramatic decrease in cell-free HIV-1 RNA in semen, which is a reliable measure of viral load. These findings could have implications for the sexual transmission of HIV-1.

Detection of HIV-1 in seminal plasma and seminal cells of HIV-1 seropositive men

It is of critical importance to precisely understand the modalities of HIV presence in semen, especially with regard to procreation. In this study, paired blood and semen samples from 31 human immunodeficiency virus type 1 (HIV-1)-positive men were assessed for cell-free HIV-RNA load in blood plasma (BP) and seminal plasma (SP), and for detection of HIV by culture, PCR and RT-PCR in semen cellular fractions separated by centrifugation on Percoll gradient. HIV-RNA was detected in 94% of BP and 84% of SP samples. For 11 men (35%), HIV-RNA load in SP was equal or superior to that observed in blood. HIV-DNA presence was demonstrated (either by PCR or culture positivity) in 39% of the non-spermatozoal cells (NSC)-enriched fractions, and in one Percoll-selected sperm pellet. HIV-RNA was detected in 17% (4/23) of the sperm pellets. This positivity was associated with an HIV-RNA load in SP equal or superior to the HIV-RNA load in blood, a high rate of HIV-DNA detection in the NSC fraction, and a low CD4+ cell count. In such conditions, a significant viral production inside the genital tract is more likely to be present, and a close association between HIV and gametes might occur. Assisted procreation with selected spermatozoa should be preceded by accurate assessments of viral presence in blood, SP and semen cellular fractions.

Dynamics of cell-free viral burden in HIV-1-infected patients.

Whereas previous cross-sectional studies have shown a 1-2 log increase in cell-free HIV-1 RNA viral burden associated with disease progression, recent longitudinal studies reveal a less dynamic association. We addressed this conundrum in a cohort of 17 rapid and 20 slow progressors. Initial levels of serum viral RNA from the rapid progressors were 1 log unit higher than those from the slow progressors. Serum RNA increased by >0.5 log in 10 of the rapid progressors (increase group), but the remaining seven showed a <0.5 log increase (static group). Patients whose serum RNA increased had lower amounts of serum RNA at study entry but not at follow-up compared with the static group. There was a trend toward lower CD4 counts in the static group at follow-up. These data suggest that the timing of specimen evaluation can influence interpretation of viral RNA dynamics in HIV disease.

Reduction in plasma human immunodeficiency virus ribonucleic acid after dideoxynucleoside therapy as determined by the polymerase chain reaction.

Cell-free HIV RNA in plasma was detected and quantitated after antiviral therapy by the polymerase chain reaction. RNA was extracted from plasma, reverse transcribed to cDNA, amplified by polymerase chain reaction, and quantitated by absorbance based on an enzyme-linked affinity assay. 72 HIV antibody-positive subjects had one plasma sample taken. 39 who were not receiving antiretroviral therapy at the time had a mean plasma HIV RNA copy number of 690 +/- 360 (mean +/- SEM) per 200 microliters of plasma, while 33 subjects who had been receiving zidovudine therapy for a minimum of 3 mo had a mean copy number of 134 +/- 219 (P less than 0.05). 27 additional HIV antibody-positive patients had two plasma samples taken before and 1 mo after initiating dideoxynucleoside therapy. Plasma HIV RNA copy number fell from 540 +/- 175 to 77 +/- 35 (P less than 0.05). Finally, nine of these subjects had two baseline samples obtained before initiating therapy and two posttreatment samples 1 and 2 mo after therapy was begun. Mean plasma RNA copy number declined from 794 +/- 274 to less than 40 (below the lower limit of sensitivity) after 1 mo of therapy, with suppression maintained after 2 mo of therapy. These results suggest that gene amplification can be used to detect and quantitate changes in plasma HIV RNA after dideoxynucleoside therapy. Plasma HIV polymerase chain reaction may be a more sensitive marker to monitor antiviral therapy, particularly in asymptomatic patients where measurement of p24 antigen or quantitative plasma cultures are negative.

Detection, quantification and sequencing of HIV-1 from the plasma of seropositive individuals and from factor VIII concentrates

A highly sensitive and reliable RNA polymerase chain reaction method has been developed which has been used to detect, quantify and sequence cell-free HIV RNA directly from the plasma of seropositive individuals. Plasma from 10 out of 12 haemophiliacs tested was found to contain detectable levels of HIV-1 RNA [log mean value: 1.2 x 10(3) copies for Centers for Disease Control (CDC) group II patients, 5.5 x 10(3) copies for CDC group IV patients]. The presence of cell-free circulating virus in both symptomatic and asymptomatic individuals suggests that viral replication continues throughout the course of infection. The same procedure has been used to detect and sequence HIV-1 RNA in two batches of unheated commercial factor VIII concentrate distributed in 1981 and 1983. The sequences obtained revealed a closer relationship to North American than to African strains of HIV-1.