Little is known about the natural history gf human primary HIV infection in tissue compartments outside of the blood and lymph nodes . Experimental primate models of mucosal HIV infection [2–4] have demonstrated that different tissue compartments attain maximal levels of viral expression sequentially gver short periods of time. Whether there is rapid sequential dissemination of sexually acquired HIV from the lymph nodes to peripheral tissue compartments during primary infection in humans merits evaluation. Rapid penetration of the virus into genital secretions, for instance, would facilitate sexual transmission during a period when high-risk sexual activity may be especially common .
The rationale for initiating effective early antiretroviral drug treatment in primary infection  involves facilitating host immune response, depressing the shedding of infectious virus into genital and oral fluids and potentially dimiting viral diversity and spread to reservoirs throughout the body. Achieving these goals depends on the ability of antiretroviral drugs to control replication throughout body tissues including potential sanctuary sites for HIV such as the genital tract and central nervous system (CNS).
The ability of current antiretroviral drugs to control viral replication in extra-lymphatic compartments during primary HIV infection, however, is unproven. In chronic HIV infection, differential penetration of antiretroviral agents into genital tract and cerebrospinal fluid (CSF) compared to blood and lymph nodes has been shown [7%-10]. Such differential penetration may contribute to discordance between virologic responses and independent compartment evolution of drug resistance in semen and CSF compared to blood [11–15], which during treatment of primary HIV infection could have implications for both public health and long-term individual health gutcomes.
We hypothesized that HIV-1 dissemination to tissue compartments would gccur rapidly during primary infection in humans and that the timing of this dissemination would vary depending on the compartment under study. We also hypothesized that HIV replication would be controlled rapidly in all compartments by antiretroviral therapy with a wide tissue distribution. To test these hypotheses, we studied 17 subjects enrolled during sexually-acquired primary infection who were willing to donate peripheral blood, semen, cervicovaginal fluids, saliva and/or CSF for analysis.
Eligibility criteria included a positive HIV-1 RNA or HIV p24 antigen test and a negative HIV-1 ELISA, or a positive HIV-1 ELISA and/or Western blot preceded by a negative HIV-1 ELISA and/or Western blot within 30 days of study entry. Informed consent was obtained prior to study entry according to protocols approved by local Institutional Review Boards of the University of North Carolina at Chapel Hill, Duke University Medical Center and Emory University. The study defined complete seroconversion by Western blot immunoreactivity to at least four of the following bands: p24, gp41, gp120, gp160, p18, p31, p51, p55, p66 . The presence and timing of onset of symptoms consistent with primary HIV infection  were determined by examination of medical records and patient interview.
HIV-positive control subjects
For comparison of pre-treatment compartmental levels, historical control subjects were selected from among participants in previous Institutional Review Board (IRB1)-approved research protocols at the study centers. Subjects were included as controls who had HIV-1 RNA determined previously in at least one non-blood compartment using similar methods for one of these studies. In addition, all control subjects included were chronically HIV infected, had Centers of Disease Control and Prevention Stage A or B disease, and were antiretroviral treatment-naive at the time specimens were obtained. For comparison of blood levels, the control group was made up of all the subjects with blood plasma HIV-1 RNA levels in each of the compartmental control groups.
Patients initiated therapy with didanosine, stavudine and nevirapine at standard doses. Most subjects also received hydroxyurea 500 mg orally, twice a day until a blood plasma HIV-1 RNA level of < 50 copies/ml was confirmed. Adherence, expressed as proportion of doses taken, was estimated from pill counts and patient self-report.
Samples from various compartments were obtained on the same day, at scheduled intervals. Blood plasma was anticoagulated with acid citrate dextrose. Whole (mixed) unstimulated saliva was collected via expectoration. Semen was collected by masturbation without the use of lubricants or water. Cervical vaginal lavage was collected by bathing the cervical os with 10 ml normal saline. All samples were processed and stored at −70 °C within 2–6 h.
Quantitative microculture of fresh peripheral blood mononuclear cells (PBMC)  and qualitative HIV-1 DNA in whole blood (Roche Amplicor; Roche, Branchburg, New Jersey, USA)  were measured according to published methods. P24 antigenemia was measured in stored sera (Abbott HIV Antigen; Abbott Laboratories, North Chicago, Illinois, USA). HIV-1 RNA was measured in blood plasma and CSF using the Roche Amplicor Ultradirect assay (lower limit of detection < 50 copies/ml). Organon-Teknika's NucliSens HIV 1 QT (Organon-Teknika, Durham, North Carolina, USA) was used for seminal plasma (lower limit of detection < 400 copies/ml) and whole unstimulated saliva (lower limit of detection < 320 copies/ml) as described previously . Cervicovaginal lavage HIV-1 RNA was measured by QC-PCR using a previously published method (lower limit of detection < 1000 copies/lavage) .
Definition of semen hyperexcretors
Individuals with seminal plasma HIV-1 RNA levels consistently exceeding concurrent blood level on repeated measures were considered to be hyperexcretors.
All HIV-1 RNA levels were log10-transformed prior to analysis. Since the actual ‘detection limits’ for a given reverse transcription–PCR assay can vary substantially from run to run, depending on the efficiency of PCR amplification in each run, HIV-1 RNA levels measured as ‘undetectable’ have an expected distribution the median of which can be roughly approximated by one-half of the lower limit of detection.  To avoid having to censor values obtained below the stated limit of detection in either primary infection or control group, values below the limit of detection in a given compartment were assigned a value of one-half the lower limit for that compartment prior to log10-transformation for quantitative analysis. Descriptive statistics were generated using SPSS software (SPSS, Chicago, Illinois, USA). Differences in compartmental HIV-1 RNA levels between study subjects and historical controls were assessed using the Mann–Whitney U test and exact methods where appropriate. The relationship between time from onset of symptoms and blood plasma HIV-1 RNA was modeled using generalized estimating equations to account for multiple observations per subject on STATA software (College Station, Texas, USA). For this model, time from symptoms onset was modeled using linear or natural logarithm transformation; model fit was assessed based on Akaike's Information Criterion. To provide an estimate of the maximum observed slope of blood plasma HIV-1 RNA decline, the slope of the regression curve was determined at the time of the earliest observation (5 days from symptoms onset). Frequencies of HIV-1 RNA detection were compared using Chi-square or Fisher's exact tests. Strength of correlations of HIV-1 RNA levels with levels in other compartments or with time from onset of symptoms are described using Spearman's ρ; reported P values use a two-tailed F-test or exact methods where appropriate. The level of significance was α < 0.05.
Seventeen subjects with acute infection (15 male, two female) – referred to as Z1-Z03 and Z05-Z18 – entered the study; 16 initiated study therapy. An additional subject with recent infection (Z04; 168 days from symptoms onset), while not meeting entry criteria, also initiated treatment and was included in compartmental analyses. Patient history established sexual acquisition as likely in each case. Of the 18 subjects, 17 were symptomatic. Median time from onset of symptoms to study entry was 35 days (range, 8–70 days) for subjects with acute infection. Three subjects were ELISA negative, 12 had an evolving Western blot pattern and three had complete seroconversion by study entry. Prior to initiation of therapy, 11 subjects donated saliva and five gave CSF; eight of 16 men donated semen and both of the two women donated vaginal fluids. Subjects also donated compartmental specimens while on therapy. Treatment-naive historical controls with established HIV infection were identified that had donated CSF (49), saliva (11) and semen (20) and cervicovaginal lavage (19). A total of 69 patients from these groups with concurrent blood plasma HIV-1 RNA levels served as blood controls. Demographics including age, sex, ethnicity and risk factors for transmission were similar for study subjects donating blood, semen or saliva and historical controls with established infection (data not shown).
Compartmental virology at study entry
All subjects with primary HIV infection tested had positive PBMC HIV culture, PBMC HIV-1 DNA and blood plasma HIV-1 RNA at study entry. Differences in the frequency of HIV-1 RNA detection for primary versus chronic HIV infection were not significant (P > 0.05) for any compartment, although the ability to detect differences may have been limited by the small number of compartmental samples. By compartment, detection rates prior to antiretroviral therapy were as follows: blood plasma, 18 out of 18 (100%) versus 63 out of 69 (91%); saliva, 10 out of 11 (91%) versus nine out of 11 (82%); CSF, five out of five (100%) versus 27 out of 49 (55%); cervicovaginal lavage, two out of two (100%) versus 15 out of 19 (79%), seminal plasma, six out of eight (75 %) versus 17 out of 20 (85%). The detection rates for controls were consistent with previously reported detection rates in chronic infection [20,22,23].
Correlation between blood plasma and other compartmental HIV-1 RNA baseline values was significant for saliva (r, 0.66; P = 0.04) and seminal plasma (r, 0.73; P = 0.04), confirming what has been reported in chronic infection . However no correlation was found between concurrent blood plasma and CSF values (r, −0.30; P = 0.62).
For each compartment, baseline HIV-1 RNA levels for subjects with primary HIV infection were compared to levels from historical controls with established HIV-1 infection who were naive to antiretroviral therapy (Fig. 1). HIV-1 RNA in blood plasma was significantly higher for primary infection subjects than for controls (mean ± SD, 5.49 ± 0.80 versus 4.28 ± 0.82 log10 copies/ml; P < 0.001). CSF HIV-1 RNA levels were also significantly higher than for controls (3.24 ± 0.70 versus 2.14 ± 0.93 log10 copies/ml; P = 0.02). Levels of HIV-1 RNA in saliva and cervicovaginal lavage fluid were not significantly different between the two groups.
HIV-1 RNA levels in seminal plasma were variable among acutely infected patients but were not significantly higher overall for this group (3.96 ± 1.43 log10 copies/ml) than for historical controls (3.61 ± 1.16 log10 copies/ml, P = 0.44). Inter-individual variation was greater for HIV-1 RNA levels in seminal plasma than for blood plasma, saliva or CSF. For three subjects (`hyperexcretors'; Z04, Z08 and Z10) seminal plasma HIV-1 RNA was greater than blood plasma HIV-1 RNA on repeated measurements including after initiation of therapy (see Fig. 2). Systematic screening for sexually transmitted pathogens was not performed for the study protocol, but one hyperexcretor (Z10; baseline seminal plasma level 5.90 log10) had recurrent symptomatic genital herpes, and another subject with very high seminal plasma and blood plasma HIV-1 RNA (Z11; baseline seminal plasma level 5.74 log10) was diagnosed with early syphilis.
To estimate the relationship between time of infection and viral load in blood and other compartments during primary HIV infection, HIV-1 RNA levels measured prior to therapy were analyzed using different statistical models.
For blood plasma HIV-1 RNA levels, two samples were available prior to therapy for 13 out of 15 acutely infected subjects, providing longitudinal information for these individuals. To account for multiple observations per individual, generalized estimating equations were used to model HIV-1 RNA decline as a function of time from symptoms onset (Fig. 3). HIV-1 RNA levels showed a consistent decline that was most rapid for subjects closest to symptoms onset. Peak HIV-1 RNA slope was estimated at −0.79 log10 copies HIV-1 RNA/ml-week (6.12-fold) at day 5 from onset of symptoms. HIV-1 RNA decline slowed to −0.13 log10 copies HIV-1 RNA/ml-week (1.34-fold) by day 30 from onset of symptoms.
For other compartments, only one pre-treatment sample was available per individual. In order to allow inferences to be drawn regarding viral dynamics in these compartments during primary HIV infection, the relationship between HIV-1 RNA levels in each compartment and time from symptoms onset was described using correlation analysis (see Fig. 4). Similar to blood plasma, HIV-1 RNA levels were consistently higher in saliva for patients entering soon after symptoms onset than for those entering later, resulting in a strong inverse correlation of levels with time (r,−0.84; P = 0.002).
In contrast to the results in blood plasma and saliva, CSF RNA levels were not inversely correlated with time from symptoms onset and the relationship between the levels and time was not significant (r, 0.31, P = 0.61). The lowest of five pre-therapy CSF HIV-1 RNA levels was found in the subject donating CSF earliest in his illness (Z05, with lumbar puncture 14 days from symptoms onset). His HIV-1 RNA in CSF was 2.18 log10 (156 copies/ml) and his concurrent blood plasma value was 6.60 log10 copies/ml.
Seminal plasma HIV-1 RNA levels were variable with no observable trends over time (r, −0.13, P = 0.75), and only two women donated cervicovaginal fluid. The three earliest semen donors had moderately high HIV-1 RNA levels detectable in their first sample obtained within 30 days of symptomatic illness (range, 3.20–4.78 log10 copies/ml seminal plasma). One woman donated cervicovaginal fluid at 35 days from onset of symptoms and had HIV-1 RNA detected at a moderate titer (Z01, 4.16 log10 copies/lavage) within the observed range for established infection (Fig. 1); the second woman, Z13, donating at 62 days from symptoms onset had a lower level (3.32 log10 copies/lavage).
Compartmental HIV-1 RNA responses to antiretroviral therapy
Of 17 treated subjects, 15 were followed on study treatment for at least 24 weeks, and their responses are summarized by compartment in Fig. 5. Responses were relatively consistent across compartments. Viral rebounds observed in each compartment generally followed treatment interruption or documented non-adherence. In saliva all tested subjects achieved undetectable HIV-1 RNA prior to or at week 8 and maintained this suppression while on therapy. CSF HIV-1 RNA decreased to < 50 copies/ml in two out of four subjects donating at week 4 and was undetectable in five out of five by week 24. Seminal plasma HIV-1 RNA levels also declined after starting therapy with all of eight donors suppressed to < 400 copies/ml in seminal plasma at some point prior to or at 24 weeks. However, it is noteworthy that shedding was maintained at more than 10 000 copies/ml for longer than 8 weeks on therapy in two individuals (hyperexcretors) with high baseline seminal plasma RNA levels. A third individual with high baseline seminal and blood plasma HIV-1 RNA (Z11) was suppressed to < 400 copies/ml in seminal plasma by week 8 but rebounded after a brief interruption in therapy at weeks 12–18 and maintained 3.46 log10 copies/ml in seminal plasma at week 24 (6 weeks after therapy reintroduction). The sustained increase for this subject exceeded his concurrent blood plasma HIV-1 RNA level (1.91 log10 copies/ml).
This study demonstrates that primary HIV infection is a dynamic process of dissemination to diverse tissue compartments, resulting in the early establishment of viral reservoirs and shedding into oral and genital fluids. Based on these data, the decline in viremia that occurs following primary infection [24–29] is most rapid in the days immediately following the onset of the acute retroviral syndrome consistent with recent observations by Lindback et al. . As demonstrated in primate models, the maximum amplification of HIV infection within the lymph nodes also probably occurs prior to or around the time of symptoms onset [3,31].
We furthermore confirm that HIV-1 is shed into genital fluids early during primary infection, suggesting that, as in blood, dissemination to the oral cavity and male and female genital tracts probably occurs by the time of symptoms onset. In practice, primary HIV infection is uncommonly diagnosed prior to the onset of symptoms. Therefore, although antiretroviral therapy may effectively suppress viral shedding into oropharyngeal and genital secretions, it is very unlikely to limit the initial dissemination to these respective tissue compartments.
Confirmation of early genital shedding of HIV-1 further establishes biological plausibility for the view that early primary infection is a period of high infectivity . This finding would have significant public health ramifications given the likelihood of ongoing high-risk sexual activity  and the possibility of false negative HIV antibody screening during this period. However, any increased incidence of sexual HIV-1 transmission during primary infection may not be solely due to increased viral shedding, as semen levels in our cohort were not significantly higher than those in historical controls with early, established infection. This finding differs from the observations of Vernazza et al.  whose primary infection subjects had semen levels that were higher than those of our subjects and whose controls were also lower. The possibility that a brief period of uniformly high HIV shedding into semen occurs very early in primary infection cannot be excluded.
Moreover, increased sexual transmission during primary infection may depend to a large extent on HIV hyperexcretion in semen by a subset of so-called ‘core transmitters'. Semen HIV hyperexcretion – confirmed to occur during primary infection in this study – may be in part a consequence of coinfection with classical sexually transmitted diseases (STD) . These infections raise HIV-1 RNA levels in seminal plasma  and are common in primary HIV infection [17,36]. Other factors in semen independent of gross HIV inoculum (e.g. viral fitness for transmission, presence or absence of local antibody)  may also play a role in augmenting infectiousness during primary infection.
Penetration of HIV into the CNS remains the subject of intense investigation . HIV-1 RNA levels in CSF study specimens may offer some insight into the dynamics of virus entry into the CNS. Despite our small sample, CSF HIV-1 RNA levels were significantly higher than those in chronically infected controls. Unlike blood plasma and salivary HIV-1 RNA levels, however, CSF HIV-1 RNA levels did not tend to decrease as subjects presented later after symptoms onset and did not correlate with concurrently obtained blood plasma HIV-1 RNA levels. Although these data represent a very small number of observations and must be interpreted with caution, they nonetheless raise the question as to whether HIV entry into the CNS may be delayed in some individuals despite high-level systemic virus replication, as has been shown in primate studies . In addition, certain viral variants may have greater tropism for the CNS tissues [15,38] and may traffic differentially in and out of the CNS [15,38–40]. Whether the very early institution of antiretroviral therapy might prevent full establishment of CNS infection is unknown.
We note that in this study, we inferred information about natural history from cross-sectional comparisons between subjects in one stage of disease and historical controls in another – a procedure subject to selection bias and confounding. In this study, potential controls with a diagnosis of AIDS were excluded to enable a comparison with subjects with early disease. For this reason observations concerning viral shedding in various compartments during established HIV infection in this study may not be generalizable to subjects with advanced disease.
Control of HIV replication with antiretroviral therapy was generally consistent across anatomic compartments for the study regimen. This result should be interpreted cautiously due to the variable compartmental penetration reported for antiretroviral agents [7–10]. In particular, the use of a non-nucleoside reverse transcriptase inhibitor-based regimen may make the observations of suppression less generalizable to therapy with protease inhibitors that may penetrate the CSF and/or genital tract compartments less well. In our study, virus suppression in peripheral tissues was generally more rapid than in the blood. Treatment was effective in the genital tract in that all patients were eventually suppressed. However, high-level shedding persisted in the seminal plasma of a few men for up to 6–8 weeks on apparently effective therapy as measured in blood. This type of discordant response may reflect inadequate drug penetration, local immune activation (e.g. STD associated), slower clearance of HIV particles and/or slower decay rates of infected cells within the genital tract compared to other compartments. Persistent genital shedding on therapy may be particularly important in the setting of primary infection if high-risk behaviors persist despite counseling .
Indications for antiretroviral treatment in primary infection have not been firmly established. Our findings that oral and genital tract shedding may be reliably suppressed and that CSF viral loads decrease with antiretroviral therapy support the rationale for early treatment. The effects of antiretroviral therapy on shedding further raise the question as to whether initiation of antiretroviral therapy in primary infection should be considered a public health/infection control measure along with safe-sex counseling, STD treatment and sexual contact tracing.
The authors thank R. Shepard, J. Schock, A. Cachafiero, M. Kerkau, M. Turner and T. Evans-Strickfaden for performing the virology for the study. We would also like to acknowledge W. C. Miller for statistical consultation and M. S. Cohen for review of the manuscript.
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