Longitudinal comparison between plasma and seminal HIV-1 viral loads during antiretroviral treatment

This study was designed to investigate the impact of anti-retroviral therapy on both plasma and seminal HIV-1 viral loads and the correlation between viral loads in these compartments after treatment. Viral load, CD4+ and CD8+ T-cell counts were evaluated in paired plasma and semen samples from 36 antiretroviral therapy-naïve patients at baseline and on days 45, 90, and 180 of treatment. Slopes for blood and seminal viral loads in all treated patients were similar (p = 0.21). Median HIV-1 RNA titers in plasma and semen at baseline were 4.95 log10 and 4.48 log10 copies/ml, respectively. After 180 days of therapy, the median viral load declined to 3.15 log10 copies/ml (plasma) and 3.2 log10 copies/ml (semen). At this timepoint 22 patients presented HIV-1 viral load below 400 copies/ml in either plasma or semen, but only 9 had viral loads below 400 copies/ml in both compartments. Key-words: Viral load. Semen. HIV-1. Antiretroviral therapy. Plasma. Resumo Este estudo foi desenhado para investigar o impacto do tratamento com anti-retrovirais na evolução das cargas virais plasmáticas e seminais do HIV-1. A carga viral do HIV-1 e a contagem de linfócitos T CD4+ e CD8+ foi determinada em amostras pareadas de sangue e sêmen de 36 pacientes virgem de tratamento nos dias 0, 45, 90 e 180 após o início da terapia. As curvas de declínio das cargas virais plasmática e seminal foram semelhantes (p= 0.21). As medianas da carga viral plasmática e seminal no pré-tratamento (dia 0) foram 4.95 e 4.48 log10 cópias/ml, respectivamente. Seis meses após o início da terapia, a mediana da carga viral plasmática era 3.15 log10 cópias/ml e a seminal 3.2 log10 cópias/ml. Neste mesmo periodo, 22 pacientes apresentavam carga viral abaixo de 400 cópias/ml no plasma e/ou sêmen, enquanto apenas 9 pacientes apresentavam carga viral abaixo do limite de detecção nos dois compartimentos. Palavras-chaves: Carga viral. Sêmen. HIV-. Terapia antiretroviral. Plasma. 1. Cellular and Molecular Immunology Laboratory, Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória, ES, Brazil. 2. Santa Casa de Misericórdia de Vitória, Vitória, ES, Brazil. Address to: Dr. Rodrigo Ribeiro-Rodrigues. Núcleo de Doenças Infecciosas/CBM/UFES. Av. Marechal Campos 1468, Maruípe 29040-091 Vitória, ES, Brazil. Tel: 55 27 335-7207, Fax: 55 27 335-7204 e-mail: rodrigrr@ndi.ufes.br Recebido para publicação em 15/4/2002 Aceito em 10/11/2003 Despite the fact that HIV-1 was first recovered from semen samples in 1984, and sexual activity continues to be the most common form of HIV transmission, little is known about the impact of seminal viral load on HIV-1 infection1 8. A wide variety of biological and behavioral factors have been associated with the risk of sexual transmission of HIV-114. Recently, a strong association between HIV-1 plasma viral load and the risk of heterosexual transmission was reported13. It has been suggested that high levels of HIV-1 replication probably contribute to both rapid disease progression and enhanced sexual transmission3 5 18. Small shortterm studies have demonstrated that antiretroviral therapy reduces both blood and seminal viral load and may diminish HIV transmission6 7 18. We have shown that the correlation between plasma and seminal viral loads in therapy-naïve patients decreases in parallel with declining CD4 cell counts. The current study was conducted to prospectively evaluate the impact of antiretroviral therapy on HIV viral loads in plasma and semen of antiretroviral-naïve HIV-1+ patients; and, to compare viral loads in both semen and plasma compartments, with peripheral blood T-cell counts at baseline and during treatment.

Despite the fact that HIV-1 was first recovered from semen samples in 1984, and sexual activity continues to be the most common form of HIV transmission, little is known about the impact of seminal viral load on HIV-1 infection 1 8 .A wide variety of biological and behavioral factors have been associated with the risk of sexual transmission of HIV-1 14 .Recently, a strong association between HIV-1 plasma viral load and the risk of heterosexual transmission was reported 13 .It has been suggested that high levels of HIV-1 replication probably contribute to both rapid disease progression and enhanced sexual transmission 3 5 18 .Small short-term studies have demonstrated that antiretroviral therapy reduces both blood and seminal viral load and may diminish HIV transmission 6 7 18 .We have shown that the correlation between plasma and seminal viral loads in therapy-naïve patients decreases in parallel with declining CD4 cell counts 13 .
The current study was conducted to prospectively evaluate the impact of antiretroviral therapy on HIV viral loads in plasma and semen of antiretroviral-naïve HIV-1 + patients; and, to compare viral loads in both semen and plasma compartments, with peripheral blood T-cell counts at baseline and during treatment.
Pinto Neto LFS et al

MATERIAL AND METHODS
Institutional Review Board.Prior to its implementation, this study was submitted and approved by the Biomedical Center Internal Review Board.A signed informed consent was obtained from all enrolled patients before their inclusion in the study.
Patients.The study population consisted of 36 consecutive adult anti-retroviral therapy-naïve HIV-1 + men attending the HIV clinic at Santa Casa de Misericórdia, Vitória.ES, Brazil, between March 1998, and July 1999.All patients were free of signs or symptoms of sexually transmitted diseases.Clinical staging was performed according to the CDC classification system 2 .These patients were treated with either two-drug (two nucleoside analogs reverse transcriptase inhibitors) or three-drug (two nucleoside analogs reverse transcriptase inhibitors plus one protease inhibitor or one non-nucleoside reverse transcriptase inhibitor regimens) schemes according to Brazilian Consensus on Antiretroviral Therapy, current at the time of the study 10 .After informed consent, all 36 patients were requested to donate blood and semen samples at baseline and 45, 90, and 180 days after beginning therapy.
Quantitation of CD4 + lymphocytes in blood samples.Blood was collected by venipuncture in Vacuntainer tubes (Becton Dickinson, Franklin Lakes, New Jersey, USA) using K 3 -EDTA as an anticoagulant and processed within 4 hours of collection.Peripheral blood CD4 and CD8 cell counts per microliter were determined by flow cytometry (FACScount, Becton & Dickinson, Mountain View, CA, USA) using standardized protocol recommended by the manufacturer.Results were expressed as mean ± standard deviation.
Quantitation of HIV-1 RNA in blood and seminal plasma samples.Plasma was obtained from K 3 -EDTA-treated whole blood samples centrifuged at 600 x g for 7 minutes and stored in aliquots at -70°C within 4 hours of collection.In order to ensure the quality of semen samples, patients were asked to comply with a 72-hour sexual abstinence prior to sample collection.Semen samples were processed within 4h of ejaculation as described by Vernazza et al 18 .Briefly, the total volume of semen was measured and the sample diluted 1:2 with viral transpor t medium (plain RPMI 1640 + 1000U/ml penicillin + 1000mg/ ml streptomycin).Diluted sample was centrifuged at 1,600 x g for 5 min, and the supernatant was collected.Aliquots were stored at -70°C until used for the quantification of HIV-1 RNA.Semen samples are referred to as seminal plasma.HIV-1 viral load in either blood plasma (PVL) or seminal plasma (SVL) were quantified by the NASBA method (Organon Teknika, Durham, North Carolina, USA) 4 16 .The detection limit of the assay was 400 HIV-1 RNA copies/ml.Both plasma and seminal viral load results were expressed as medians.In order to evaluate intersample variability in PVL and SVL, 2 consecutive paired blood and semen samples were collected and analyzed (interval of 11.2 ±7.5 weeks between the two collection points) from 17 therapy-naïve patients before the treatment began.
Treatment.Antiretroviral regimen for each patient was selected by the physician in charge following the guidelines of the Brazilian Consensus on Antiretroviral Therapy in place at the time of the study.Twelve patients were treated with two nucleoside analog reverse transcriptase inhibitors (9 used AZT + ddI and 3 used AZT + ddC).Whereas, 24 patients were treated with three drugs, 2/24 patients were treated with nucleoside analogs (AZT + 3TC) and a non-nucleoside reverse transcriptase inhibitors (Efavirenz) and the remaining 22 patients were treated with two nucleoside analogs (AZT + 3TC) and a protease inhibitor (10 with Indinavir, 8 with Ritonavir and 4 with Nelfinavir).For further analysis and discussion of results, these patients were divided into those receiving two-drug regimens (12 patients) and those receiving three-drug regimens (24 patients).Patient compliance was encouraged and followed by the clinical team throughout the protocol.
Statistical analysis.Considering the non-normal distribution of RNA concentrations, confirmed by Kolmogorov-Smirnov tests, continuous variables were analyzed following log transformation and linear regression analysis by the Wilcoxon's test.Correlations between non-parametric variables (HIV-1 RNA titers on blood and semen, CD4 and CD8 counts) were evaluated using Spearman's rank correlation test.The decline of viral load slopes on blood plasma and semen samples were compared by multivariate analysis.All statistical analyses were carried out using SPSS 8.0 software (SPSS Inc) and by SAS (SAS institute Inc.SAS/STAT guide for personal computers, version 6, Cary, NC, USA).
HIV-1 RNA was detected and quantified at baseline on all blood and semen samples.Inter-sample variability in PVL and SVL was evaluated using 2 consecutive paired blood and semen samples collected from 17 treatment-naïve patients.Albeit PVL and SVL in these 17 patients were not significantly different (p = 0.463 and p = 0.906, respectively), SVL samples presented a broader variation when compared to their paired PVL samples (data not shown).

DISCUSSION
In the present work, we investigated the impact of anti-retroviral therapy on CD4 counts, PVL and SVL in 36 patients with HIV infection during a 6-month period after treatment initiation.In our study, the mean increase in CD4 counts after 6 months of treatment was lower than that previously reported 12 15 .This difference was probably due to the fact that a subset of patients had received a 2-drug regimen during followup.In fact, when only patients taking 3-drug (24/36) were considered, an increase of 161 CD4 + cells/µl was observed, which is similar to the improvement (150 CD4 + cells/µl) previously reported by Schooley et al 15 .After 6 months of antiretroviral therapy, 25% of patients (9/36) had undetectable PVL and SVL, 2 treated with 2-drug regimens and 7 with a 3-drug combination (Table 2).Some patients presented discordant HIV-1 viral loads, 6/36 (17%) had non-detectable PVL but detectable SVL and 7 (19%) presented non-detectable SVL but a detectable PVL.Fifteen out of 36 treated patients (42%) had PVL below the detection limit after 6 months of therapy.Again, if separated according to their therapeutic schemes, 25% (3/12) of the patients taking 2 drugs and 50% (12/24) of the patients taking 3 drugs had non-detectable PVL.The difference between the two groups was not significant (p = 0.27), probably due to the size of our cohort.
At baseline, when patients were grouped according to their CD4 cell counts, correlation between PVL and SVL was dependent on the CD4 cell counts.No correlation between PVL and SVL was observed among patients with CD4 counts below 200 CD4 + cells/µl.However, after 6 months of treatment, the correlation between PVL and SVL was no longer dependent on CD4 + cell counts (data not shown).During treatment, a similar decrease was observed in both PVL and SVL (p = 0.21), confirming previously published results indicating an overall correlation between PVL and SVL 4 17 .Although discordant HIV-1 viral load titers were observed in plasma and semen samples from several patient samples after 6 months of therapy, VL was below the detection limit in plasma but detectable in semen samples from 6 patients (patients # 14, 18, 19, 26, 27 and 30) and VL was below the detection limit in semen but detectable in plasma in another 7 patients (patients # 2, 7, 12, 21, 23, 25 and 36).The discordance observed in these 13 patients, corroborates the idea of HIV compartmentalization, which may be characterized by an imperfect correlation between blood and seminal compartments.Blood and seminal compartmentalization of HIV infection, may explain, in part, the differences in sexual transmission rates observed among different populations.Recently, Quinn et al. reported that an important correlation between plasma viral load and heterosexual transmission of HIV was observed among seropositive men in Sub-Saharan Africa 14 .
Our study confirms the decline in SVL after antiretroviral therapy as described by other authors 1 6 11 18 .However, the lack of correlation between PVL and SVL in a significant number of patients in our cohort suggests that, in a clinical setting, when inhibition of HIV-1 + replication in blood plasma is achieved it may not be the case in another compartment, such as germinal tissues.Clearly, it may amplify the risk of development of drug-resistant mutants as described before 9 .Therefore, more studies are needed to fully investigate the efficacy of antiretroviral therapy on HIV load in semen from HIV-1 + patients and its impact on viral shedding.
CD8 cell counts are expressed as mean ± standard deviation of cell counts/µl.PVL and SVL are expressed as medians of log 10 copies/ml.

Figure 1 -
Figure 1 -A) Changes in CD4 and CD8 T cell counts before and during antiretroviral treatment with different drug schemes, and B) Changes in seminal and plasma HIV-1 viral load on patients receiving 2 -and 3 -drugs combination therapy.

Figure 2 -
Figure 2 -Median HIV-1 viral load on both plasma and seminal samples from all 36 patients receiving antiretroviral therapy.PVL = plasma viral load and SVL = seminal viral load.Median±SEM (standard of the mean).