Evidence of genotypic resistance diversity of archived and circulating viral strains in blood and semen of pre-treated HIV-infected men
Ghosn, Jadea,c; Viard, Jean-Paulb; Katlama, Christinec; de Almeida, Martad; Tubiana, Rolandc; Letourneur, Francke; Aaron, Laurentb; Goujard, Cécilef; Salmon, Dominiqueg; Leruez-Ville, Mariannea; Rouzioux, Christinea; Chaix, Marie-Laurea
From the aLaboratoire de Virologie, EA MRT 3620 Université R. Descartes, CHU Necker, Paris, the bService d'Immunologie Clinique, CHU Necker, Paris, the cService de Maladies Infectieuses, CHU Pitié-Salpêtrière, Paris, the dCECOS, CHU Cochin, Paris, the eInstitut Cochin, Paris, the fService de Médecine Interne, CHU Bicêtre, Le Kremlin-Bicêtre and the gService de Médecine Interne, CHU Cochin, Paris, France.
Correspondence to Jade Ghosn, MD, Laboratoire de Virologie, CHU Necker Enfants Malades, 149, rue de Sèvres, 75015 Paris, France.
Tel: + 33 1 44 49 49 61; fax: +33 1 44 49 49 60; e-mail: firstname.lastname@example.org
Received: 8 May 2003; revised: 10 July 2003; accepted: 12 August 2003.
Objective: To study the genetic diversity of drug-resistant HIV strains present in blood and in semen, especially those archived in peripheral blood mononuclear cells (PBMC) and non-sperm cells (NSC).
Methods: Paired blood and semen samples were collected from twenty heavily pre-treated HIV-infected men. HIV RNA in blood plasma (BP) and seminal plasma (SP), as well as proviral DNA in PBMC and NSC were quantified and used for resistance genotyping. Phylogenetic analysis of protease gene clones was used to explore the diversity of the viral quasi-species.
Results: Median BP HIV RNA, PBMC proviral DNA, SP HIV RNA and non-sperm cell proviral DNA loads were respectively: 4.77, 3.65, 3.16 and 1.77 log10 copies per ml or per 106 cells. Resistant HIV strains were found in the BP and PBMC of all the patients, in the SP of 14 patients, and in the NSC of five patients. Overall, the blood and genital compartments exhibited different genotypic resistance patterns in six patients (30%), with additional resistance mutations in the semen of four patients. Phylogenetic analysis of clones of HIV protease gene showed that viral strains in SP originated not only from passive diffusion from BP, but also from local production in semen. The storage of archived proviruses differed according to the anatomic reservoir.
Conclusion: HIV resistant strains are frequent (70%) in the semen of heavily pre-treated men, and the diversity of genotypic resistance pattern confirms HIV compartmentalization. Thus, the risk of sexual transmission of resistant strains can only be partly predicted by standard tests applied to BP.
Although human immunodeficiency virus (HIV) infection leads to a systemic disease, several lines of evidence suggest HIV compartmentalization both at the cellular level [1–6] and in anatomic compartments, such as the central nervous system [7–9] and the kidney [10–12]. The male genital tract is a potential reservoir for HIV partly because of its unique vascular features (e.g. the blood–testes barrier), and also because T lymphocytes and macrophages isolated from semen of HIV-infected men harbour provirus [13,14]. Several comparative studies of viral populations in blood and semen indicate that the male genital tract may constitute a distinct compartment in some patients, based on the isolation of phylogenetically distinct viral quasi-species from semen and blood [15–17]. Although antiretroviral combination therapy appears to reduce viral shedding in semen , the rate and pattern of emergence of resistance may differ between the blood compartment and the male genital tract [19–22]. Furthermore, as HIV may evolve in a compartment-specific manner, and given the role of semen in sexual transmission, it is important to study drug-resistant viruses in the male genital tract. Distinct resistance patterns may arise from the compartmentalization of viral replication [18,21, 23,24], a phenomenon possibly enhanced by suboptimal drug concentrations in semen [25–28]. The presence of resistant strains of HIV-1 in the male genital tract increases the risk of sexual transmission of resistant strains [19,21,22,25]. Recent data suggesting the spread of sexually transmitted drug-resistant HIV-1 strains in Europe and the United States underline the major public health implications of this issue [29–32].
Increasing numbers of HIV-1-infected patients have a history of multiple treatment failure. Yet, little is known about viral resistance in the male genital tract of patients who have resistant HIV-1 strains in their blood compartment. We therefore evaluated the frequency of HIV-1 resistant strains in the genital compartment of heavily pre-treated men with a history of therapeutic failure. We first quantified HIV RNA in blood plasma (BP) and seminal plasma (SP), and HIV DNA in peripheral blood mononuclear cells (PBMC) and non-sperm cells (NSC). We then used the genotypic viral mutational pattern as a marker to study and compare cell-free and archived cell-associated strains in blood and semen.
Patients and methods
Patients and study design
HIV-1 infected men were eligible for this cross-sectional multicentre study if they met the following criteria: age > 18 years, blood plasma HIV RNA > 1000 copies/ml (3 log10), previous failure of at least three antiretroviral regimens, no clinical acute genital infection, willingness to participate to the study and to sign a written informed consent. The study was approved by the Cochin Hospital ethics committee.
Sample collection and processing
Single paired samples of blood and semen were collected on the same day for each patient. Semen was obtained by masturbation at the Necker Clinical Investigations Center after a recommended 2-day period of sexual abstinence. Samples were collected in sterile containers and processed within 1 h after collection according to WHO recommendations. Spermatozoa and NSC were counted and spermatozoa motility was evaluated using standard methods. Two-millilitre aliquots diluted 1 : 1 in RPMI culture medium (Gibco-BRL, Cergy-Pontoise, France) containing 5 mg/ml of bromeline (Sigma-Aldrich Chimie, Saint-Quentin, France) were then centrifuged for 20 min at 300 g on a two-layer discontinuous gradient of 47.5 and 95% Percoll (Sigma-Aldrich Chimie). SP, NSC and spermatozoa were recovered separately. Selected spermatozoa and NSC were washed twice in RPMI medium at 600 g and 200 g respectively, then counted and kept as dry pellets at −80°C. SP fractions were centrifuged for 10 min at 6000 g and supernatants were aliquoted and stored at −80°C.
PBMC were isolated from whole blood by centrifugation on a one-layer Ficoll Hypaque gradient. The PBMC were washed three times in RPMI medium, then counted and kept as dry pellets at −80°C.
HIV RNA in blood plasma
Free HIV virions (HIV RNA) in BP were quantified by using the HIV-1 Monitor 1.5 HIV RNA assay kit (Roche SA, Meylan, France) according to the manufacturer's instructions: the detection limit was 200 copies/ml.
HIV RNA in seminal plasma
Free HIV virions were extracted from 250 μl of SP by the Nuclisens kit (Organon Teknika, Fresnes, France). HIV RNA was amplified with the HIV-1 Monitor 1.5 assay (Roche SA). The internal control provided with the kit was routinely added to the Nuclisens lysis buffer prior to extraction in order to validate both the extraction and amplification steps. Each batch included one positive and one negative control, consisting of seminal plasma from HIV-seronegative subjects, spiked or unspiked with a predetermined number of HIV RNA copies. The detection limit was 200 copies/ml.
HIV DNA assay in non-sperm cells and in peripheral blood mononuclear cells
HIV proviral DNA was quantified in 5 × 106 PBMC and 0.2 to 3 × 106 NSC. Four hundred microlitres of lysis buffer from the Whole Blood Specimen Preparation Kit (Roche SA) were added to the cellular fractions. DNA was extracted using the QIAamp DNA Blood Mini kit (Qiagen, Courtaboeuf, France) and quantified by spectrophotometry. Five hundred nanograms of extracted DNA was amplified with an ‘in-house’ real-time polymerase chain reaction (PCR) method on the ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystem, Courtaboeuf, France), targeting a conserved region of the HIV-1 LTR gene, with a sensitivity of 10 copies per reaction .
Genotypic resistance tests and sequences alignment
The HIV-1 reverse transcriptase (RT) and protease genes were amplified from cell-associated proviral DNA and from cell-free HIV RNA by a first-round PCR followed by nested PCR using published primers . PCR final products were visualized on gels then purified using the QIAquick PCR Purification HIV kit (Qiagen, Valencia, California, USA). After purification, PCR products were sequenced using the fluorescent dideoxy-terminator method (Big Dye Terminator kit; Applied Biosystem, Perkin Elmer, Foster City, California, USA) on an Applied Biosystem 377 automated DNA sequencer (Applied Biosystem, Perkin Elmer). Sequences were aligned using Sequence Navigator software. The amino acids at codons associated with resistance to nucleoside analogue reverse transcriptase inhibitors (NRTI), non-nucleoside analogue reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI) are reported according to the 2002 IAS list except for mutation 63P in the protease gene (http://www.iasusa.org). HIV drug resistance was defined according to the HIV-1 genotypic resistance interpretation algorithm of the French National Agency for Research on AIDS (http://www.hivfrenchresistance.org).
Clones of the HIV-1 protease gene
Protease gene PCR products from three patients were cloned into the pCR Topo 2-1 plasmid (TOPO TA Cloning kits; Invitrogen BV, The Netherlands) as recommended by the manufacturer. DNA was purified with the Mini-Prep kit (Qiagen) and then sequenced as described above.
Direct sequences of the RT and protease genes from the twenty patients, and sequences of protease gene clones from three patients were aligned with Clustal W 1.6 software. Pairwise evolutionary distances were estimated using Kimura's two-parameter method, then the trees were constructed by a neighbour-joining method (neighbour program implemented in the Phylip package) . The reliability of each tree topology was estimated from 100 bootstrap replicates . Trees were also inferred by using the maximum likelihood model.
Spearman's test was used to calculate correlations. Viral loads were compared using the Mann–Whitney test.
Patients’ characteristics at inclusion
Twenty HIV-infected men were enrolled between December 2001 and May 2002 (Table 1). Their median age was 42.5 years (range, 36–51 years), the median CD4 cell count at inclusion was 258 × 106/l (range, 72–560), the median nadir CD4 cell count was 40 × 106/l (range, 1–218). Thirteen patients (65%) had a history of AIDS-defining events (1993 CDC definition). The median time since the initiation of antiretroviral treatment was 84 months (range, 68–170). All patients had been heavily pre-treated, with a median of seven previous antiretroviral regimens (range, 3–15). At the time of sampling, five patients had been in a ‘wash-out’ phase for a median of 6 weeks (range, 4–12 weeks), while the other 15 were receiving a failing antiretroviral regimen containing a median of four drugs (range, 3–6).
The semen characteristics of the 20 patients were as follows: median ejaculate volume 2 ml (range, 0.1–6; normal value > 2 ml), median spermatozoa count 44 million/ml (range, 0–380; normal value > 30 × 106/ml), median percentage of spermatozoa with rapid motility 10% (range, 0–35%; normal value > 25%), median NSC count 8.5 million/ml (range, 0–32; normal value < 1 × 106/ml).
HIV RNA values in blood plasma and seminal plasma
HIV RNA was detectable in the BP of all 20 patients, with a median of 4.77 log10 or 59 150 copies/ml (range, 1680–474 000 copies/ml) (Table 1). The median HIV RNA value in the BP of patients without treatment (n = 5; 113 000 copies/ml, 5.05 log10) did not differ from that of patients on treatment (n = 15; 36 000 copies/ml, 4.55 log10) (P = 0.17, Mann–Whitney test).
The median HIV RNA value in SP was 1450 copies/ml or 3.16 log10 (range, < 200–260 000 copies/ml). The median HIV RNA value in the SP of men without treatment (n = 5; 13 750 copies/ml, 4.13 log10) was higher than that of men on treatment (n = 15; 590 copies/ml, 2.77 log10) (P = 0.07, Mann–Whitney test).
The median HIV RNA value was significantly higher in BP (60 000 copies/ml, 4.77 log10) than in SP (1450 copies/ml, 3.16 log10) (P < 0.05, Mann–Whitney test). In one of the patients off treatment (VN 20), HIV RNA load was higher in SP (4.92 log10) than in BP (4.19 log10).
HIV DNA values in PBMC and non-sperm cells
HIV DNA was detected in PBMC of all the patients, with a median value of 3.65 log10 or 4500 copies/106 PBMC (range, 173–100 000 copies/106 cells) (Table 1). A trend towards a correlation was found between HIV DNA load in PBMC and the nadir CD4 cell count (P = 0.0787; r2 = −0.4; Spearman's test). HIV DNA was detected in NSC of nine of 20 patients (45%). The median HIV DNA value was 1.77 log10 or 60 copies/106 NSC (range, 32–416 copies/106 cells) in these nine patients. The other 11 patients had undetectable HIV DNA in NSC. Patients with undetectable HIV RNA in SP had undetectable HIV DNA in NSC. All patients with detectable HIV DNA in NSC had detectable HIV RNA in SP. HIV RNA load in SP correlated strongly with HIV DNA load in NSC (P = 0.0016; r2 = 0.724, Spearman's test).
Mutations at positions conferring resistance to antiretroviral drugs are reported in Tables 2 and 3.
The RT and protease genes were amplified in BP from 20 patients. Mutations conferring resistance to at least one antiretroviral drug were observed in all but one of the patients (VN 18). The RT and protease genes were amplified in PBMC from all patients, except for the RT gene in patient VN 07. All patients harboured viruses archived in PBMC which bore at least one resistance mutation in the RT and/or protease gene.
HIV-1 was amplified from the SP of 15 patients, 14 of whom harboured resistant strains. The RT and/or protease gene could not be amplified in SP from five patients with low SP HIV RNA load (below 500 copies/ml). HIV DNA was amplified from NSC of seven patients, five of whom harboured resistant archived strains. Among the 13 patients in whom amplification was unsuccessful, the HIV DNA load in NSC was below the detection limit in 10, and between 40 and 60 copies/106 cells in the other three.
The genotypic resistance patterns differed between HIV RNA in BP and HIV DNA in PBMC in 16 of 20 patients (80%) (VN 01, VN 03–06, VN 08, VN 10–15, VN 17–20). Only patients VN 05 and VN 18 were off treatment. Overall, the blood compartment and the genital compartment exhibited different genotypic resistance patterns in six of 20 patients (30%) (VN 07, VN 09, VN 11, VN 14, VN 15, and VN 18). In four patients (VN 07, VN 09, VN 11, VN 18), viral strains present in the genital compartment harboured more resistance mutations than those in the blood compartment (in either HIV RNA or proviral DNA) (Table 4).
Phylogenetic analysis of direct sequences in the 20 patients
To explore the inter- and intra-individual genetic diversity of HIV-1, we analysed all available sequences [55 RT (Fig. 1a) and 57 protease gene sequences (Fig. 1b)]. All sequences showed the expected patient-specific clustering. Nineteen patients were infected by subtype B strains, while patient VN 12 was infected by a CRF02 strain (data not shown). In most cases, we observed genetic diversity not only between the blood compartment and the genital compartment, but also between the cell-free viruses and the cell-associated archived proviruses in a given compartment. However, strains present in SP were more likely to be closely related to BP viruses.
Phylogenetic analysis of HIV protease gene clones in three patients
In order to determine the origin of viral strains in the male genital compartment, we analyzed the sequences of protease gene clones in three patients: VN 09, VN 14, and VN 15 (Fig. 2). In all three cases, the most striking finding was the homogeneity of viral quasi-species in BP and SP, and the greater genetic diversity of archived proviruses. The homology of the SP and BP viral quasi-species of patients VN 09 and VN 14 suggested that the cell-free viral quasi-species in the genital compartment of these two patients probably arose by passive diffusion from BP.
Further analysis of resistance mutations showed the persistence of wild-type HIV-1 archived in PBMC and NSC. Some primary major resistance mutations were only detected in the clones, and not by direct sequence analysis (mutations 30N and 90M for PBMC1 and PBMC7, respectively, from patient VN 09). Moreover, the clones from the genital compartment bore some resistance mutations that were only present in this compartment (mutation 30N in clone SC5 from patient VN 15).
Understanding HIV-1 compartmentalization and the nature of archived proviruses has important implications in patient management. In this study, we compared the genetic diversity and the mutational resistance patterns of the cell-free HIV quasi-species and cell-associated archived proviruses, in the blood and genital compartments of HIV-infected men in whom several antiretroviral regimens had failed.
We have confirmed the compartmentalization between blood and the male genital tract. We also found evidence of local viral production, a phylogenetically distinct viral population and a distinct mutational pattern in the male genital tract.
Local virus production is suggested by the significantly higher HIV RNA load in the SP of patient VN 20 relative to his HIV RNA load in BP, and particularly by the correlation between HIV RNA load in SP and HIV DNA load in NSC. Only 55% patients had detectable levels of HIV proviral DNA in NSC, which limits any definitive conclusion to rule out between local viral production versus phramacological compartmentalization. However, the homology between clone NSC4 and clones from the SP of patient VN 15 is suggestive of local virus production in the male genital tract. Given the low levels of HIV RNA and HIV DNA in SP and NSC respectively, we are conscious that variants identified by our cloning method might not be representative of all quasi-species present in the genital compartment.
Differences in the resistance patterns between blood and genital tract viruses observed in six patients also point to HIV compartmentalization. Moreover, we observed some primary mutations only in the genital tract, emphasizing the fact that the storage of archived proviruses differ according to the anatomic reservoir. Moreover, it is particularly interesting that M184V persisted in the genital tract in three (VN 07, VN 09, VN 18) out of the five patients off treatment despite a presumable reversion in the blood compartment, suggesting a different dynamics of the reversion of resistance mutations according to the compartment. The most striking feature was the intra-individual diversity of archived proviruses in blood and male genital tract, with the persistence of both wild-type and multi-resistant viruses, confirming findings from Ruff et al. . The presence of wild-type viruses was particularly noteworthy in these 20 extensively pre-treated patients, 15 of whom were on a failing antiretroviral combination at the time of the study, and harboured resistant viruses in BP. The trend towards a correlation between HIV DNA load in PBMC and the nadir CD4 cell count suggests that the archived viral pool increases with the progression of HIV disease. These findings indicate that wild-type viral sequences archived in the two reservoirs early in infection had not been completely replaced by the dynamic processes affecting the pool of latently infected cells, despite of the presence of resistant circulating virions in BP and SP. In other words, the production of resistant circulating viral particles in BP and SP appears to reflect the predominant viral population emerging under drug-selection pressure.
The resistance patterns of clones of archived HIV DNA exhibited wide intra-individual diversity, and revealed the existence of some resistance mutations that were not seen during routine genotypic resistance testing of BP, suggesting that all previously circulating wild-type and drug-resistant forms of the virus in a given patient can be archived in PBMC as well as in NSC. Archived viruses in latently infected seminal cells are likely to be replication-competent  and may re-emerge in favourable conditions. Such conditions could be created by exposure of productive cells in the male genital tract to suboptimal antiretroviral concentrations, permitting ongoing HIV-1 production and infectiousness [25,38]. Indeed, although all NRTI and NNRTI are known to reach suppressive concentrations in the male genital tract [39–43], PI diffusion in this compartment is variable [27,28,38,43–48]. Thus, with the widespread administration of combined antiretroviral regimens, it will be then critical to deliver adequate drug concentrations to all compartments in which the virus can replicate.
As regards sexual transmission of resistant HIV strains, we show that, at a given time, routine HIV RNA quantification and genotypic resistance tests applied to BP can only partially predict this risk, as recently confirmed by mathematical models . We also show, with regard to sub-compartmentalization in the male genital tract, that semen cells  and free viral particles present in seminal plasma might be differently involved in the spread of resistant HIV-1 strains. As sexual intercourse is the main route of HIV transmission, our findings suggest that people living with HIV should themselves be a target of prevention campaigns. Given the increasing prevalence of HIV-1 strains with reduced drug susceptibility, further studies are needed to monitor the role of sexual transmission in the spread of drug-resistant virus.
We thank all the patients who agreed to participate in this study. We would also like to thank Dr Agnès Mogenet and Professor Jean-Louis Bresson for the clinical monitoring. This work was supported by a scholarship from the Fondation pour la Recherche Médicale, and by grants from the French National AIDS Research Agency (Agence Nationale de Recherche sur le Sida).
1. Wong JK, Hezareh M, Gunthard HF, Havlir DV, Ignacio CC, Spina CA, et al. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 1997, 278:1291–1295.
2. Perno CF, Newcomb FM, Davis DA, Aquaro S, Humphrey RW, Calio R, et al. Relative potency of protease inhibitors in monocytes/macrophages acutely and chronically infected with human immunodeficiency virus. J Infect Dis 1998, 178:413–422.
3. Finzi D, Hermankova M, Pierson T, Carruth LM, Buck C, Chaisson RE, et al. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 1997, 278:1295–1300.
4. Lambotte O, Taoufik Y, de Goer MG, Wallon C, Goujard C, Delfraissy JF. Detection of infectious HIV in circulating monocytes from patients on prolonged highly active antiretroviral therapy. J Acquir Immune Defic Syndr 2000, 23:114–119.
5. Smith BA, Gartner S, Liu Y, Perelson AS, Stilianakis NI, Keele BF, et al. Persistence of infectious HIV on follicular dendritic cells. J Immunol 2001, 166:690–696.
6. Blankson JN, Persaud D, Siliciano RF. The challenge of viral reservoirs in HIV-1 infection. Annu Rev Med 2002, 53: 557–593.
7. Venturi G, Catucci M, Romano L, Corsi P, Leoncini F, Valensin PE, et al. Antiretroviral resistance mutations in human immunodeficiency virus type 1 reverse transcriptase and protease from paired cerebrospinal fluid and plasma samples. J Infect Dis 2000, 181:740–745.
8. Wang TH, Donaldson YK, Brettle RP, Bell JE, Simmonds P. Identification of shared populations of human immunodeficiency virus type 1 infecting microglia and tissue macrophages outside the central nervous system. J Virol 2001, 75:11686–11699.
9. Ellis RJ, Gamst AC, Capparelli E, Spector SA, Hsia K, Wolfson T, et al. Cerebrospinal fluid HIV RNA originates from both local CNS and systemic sources. Neurology 2000, 54:927–936.
10. Winston JA, Bruggeman LA, Ross MD, Jacobson J, Ross L, D'Agati VD, et al. Nephropathy and establishment of a renal reservoir of HIV type 1 during primary infection. N Engl J Med 2001, 344:1979–1984.
11. Marras D, Bruggeman LA, Gao F, Tanji N, Mansukhani MM, Cara A, et al. Replication and compartmentalization of HIV-1 in kidney epithelium of patients with HIV-associated nephropathy. Nat Med 2002, 8:522–526.
12. Bruggeman LA, Ross MD, Tanji N, Cara A, Dikman S, Gordon RE, et al. Renal epithelium is a previously unrecognized site of HIV-1 infection. J Am Soc Nephrol 2000, 11:2079–2087.
13. Coombs RW, Reichelderfer PS, Landay AL. Recent observations on HIV type-1 infection in the genital tract of men and women. AIDS 2003, 17:455–480.
14. Quayle AJ, Xu C, Mayer KH, Anderson DJ. T lymphocytes and macrophages, but not motile spermatozoa, are a significant source of human immunodeficiency virus in semen. J Infect Dis 1997, 176:960–968.
15. Zhu T, Wang N, Carr A, Nam DS, Moor-Jankowski R, Cooper DA, et al. Genetic characterization of human immunodeficiency virus type 1 in blood and genital secretions: evidence for viral compartmentalization and selection during sexual transmission. J Virol 1996, 70:3098–3107.
16. Byrn RA, Zhang D, Eyre R, McGowan K, Kiessling AA. HIV-1 in semen: an isolated virus reservoir. Lancet 1997, 350:1141.
17. Kiessling AA, Fitzgerald LM, Zhang D, Chhay H, Brettler D, Eyre RC, et al. Human immunodeficiency virus in semen arises from a genetically distinct virus reservoir. AIDS Res Hum Retroviruses 1998, 14 Suppl 1:S33–41.
18. Leruez-Ville M, Dulioust E, Costagliola D, Salmon D, Tachet A, Finkielsztejn L, et al. Decrease in HIV-1 seminal shedding in men receiving highly active antiretroviral therapy: an 18 month longitudinal study (ANRS EP012). AIDS 2002, 16: 486–488.
19. Hecht FM, Grant RM, Petropoulos CJ, Dillon B, Chesney MA, Tian H, et al. Sexual transmission of an HIV-1 variant resistant to multiple reverse- transcriptase and protease inhibitors. N Engl J Med 1998, 339:307–311.
20. Kroodsma KL, Kozal MJ, Hamed KA, Winters MA, Merigan TC. Detection of drug resistance mutations in the human immunodeficiency virus type 1 (HIV-1) pol gene: differences in semen and blood HIV-1 RNA and proviral DNA. J Infect Dis 1994, 170:1292–1295.
21. Eron JJ, Vernazza PL, Johnston DM, Seillier-Moiseiwitsch F, Alcorn TM, Fiscus SA, et al. Resistance of HIV-1 to antiretroviral agents in blood and seminal plasma: implications for transmission. AIDS 1998, 12:F181–189.
22. Eyre RC, Zheng G, Kiessling AA. Multiple drug resistance mutations in human immunodeficiency virus in semen but not blood of a man on antiretroviral therapy. Urology 2000, 55:591.
23. Tachet A, Dulioust E, Salmon D, De Almeida M, Rivalland S, Finkielsztejn L, et al. Detection and quantification of HIV-1 in semen: identification of a subpopulation of men at high potential risk of viral sexual transmission. AIDS 1999, 13:823–831.
24. Liuzzi G, D'Offizi G, Topino S, Zaccarelli M, Amendola A, Capobianchi MR, et al. Dynamics of viral load rebound in plasma and semen after stopping effective antiretroviral therapy. AIDS 2003, 17:1089–1092.
25. Kashuba AD, Dyer JR, Kramer LM, Raasch RH, Eron JJ, Cohen MS. Antiretroviral-drug concentrations in semen: implications for sexual transmission of human immunodeficiency virus type 1. Antimicrob Agents Chemother 1999, 43:1817–1826.
26. Hoetelmans RM. Sanctuary sites in HIV-1 infection. Antivir Ther 1998, 3:13–17.
27. Lafeuillade A, Solas C, Halfon P, Chadapaud S, Hittinger G, Lacarelle B. Differences in the detection of three HIV-1 protease inhibitors in non- blood compartments: clinical correlations. HIV Clin Trials 2002, 3:27–35.
28. Solas C, Lafeuillade A, Halfon P, Chadapaud S, Hittinger G, Lacarelle B. Discrepancies between protease inhibitor concentrations and viral load in reservoirs and sanctuary sites in human immunodeficiency virus-infected patients. Antimicrob Agents Chemother 2003, 47:238–243.
29. Little SJ. Transmission and prevalence of HIV resistance among treatment-naive subjects. Antivir Ther 2000, 5:33–40.
30. Little SJ. Is transmitted drug resistance in HIV on the rise? It seems so. BMJ 2001, 322:1074–1075.
31. Little SJ, Holte S, Routy JP, Daar ES, Markowitz M, Collier AC, et al. Antiretroviral-drug resistance among patients recently infected with HIV. N Engl J Med 2002, 347:385–394.
32. Chaix ML, Descamps D, Harzic M, Schneider V, Deveau C, Tamalet C, et al. Stable prevalence of genotypic drug resistance mutations but increase in non-B virus among patients with HIV-1 primary infection in France. AIDS 2003, 17:2635–2643.
33. Leruez-Ville M, de Almeida M, Tachet A, Dulioust E, Guibert J, Mandelbrot L, et al. Assisted reproduction in HIV-1-serodifferent couples: the need for viral validation of processed semen. AIDS 2002, 16:2267–2273.
34. Pasquier C, Millot N, Njouomb R, Sandres K, Cazabat M, Puel J, et al. HIV-1 subtyping using phylogenetic analysis of pol gene sequences. J Virol Methods 2001, 94:45–54.
35. Felsenstein J. PHYLIP, Phylogeny Inference Package, version 3.6 (alpha). Seattle: Department of Genetics, University of Washington; 2001.
36. Ruff CT, Ray SC, Kwon P, Zinn R, Pendleton A, Hutton N, et al. Persistence of wild-type virus and lack of temporal structure in the latent reservoir for human immunodeficiency virus type 1 in pediatric patients with extensive antiretroviral exposure. J Virol 2002, 76:9481–9492.
37. Zhang H, Dornadula G, Beumont M, Livornese L, Jr., Van Uitert B, Henning K, et al. Human immunodeficiency virus type 1 in the semen of men receiving highly active antiretroviral therapy. N Engl J Med 1998, 339:1803–1809.
38. Taylor S, Back DJ, Drake SM, Workman J, Reynolds H, Gibbons SE, et al. Antiretroviral drug concentrations in semen of HIV-infected men: differential penetration of indinavir, ritonavir and saquinavir. J Antimicrob Chemother 2001, 48:351–354.
39. Pereira AS, Kashuba AD, Fiscus SA, Hall JE, Tidwell RR, Troiani L, et al. Nucleoside analogues achieve high concentrations in seminal plasma: relationship between drug concentration and virus burden. J Infect Dis 1999, 180:2039–2043.
40. van Praag RM, van Heeswijk RP, Jurriaans S, Lange JM, Hoetelmans RM, Prins JM. Penetration of the nucleoside analogue abacavir into the genital tract of men infected with human immunodeficiency virus type 1. Clin Infect Dis 2001, 33: e91–92.
41. Taylor S, Reynolds H, Sabin CA, Drake SM, White DJ, Back DJ, et al. Penetration of efavirenz into the male genital tract: drug concentrations and antiviral activity in semen and blood of HIV-1- infected men. AIDS 2001, 15:2051–2053.
42. Reddy YS, Gotzkowsky SK, Eron JJ, Kim JY, Fiske WD, Fiscus SA, et al. Pharmacokinetic and pharmacodynamic investigation of efavirenz in the semen and blood of human immunodeficiency virus type 1-infected men. J Infect Dis 2002, 186:1339–1343.
43. van Praag RM, Repping S, de Vries JW, Lange JM, Hoetelmans RM, Prins JM. Pharmacokinetic profiles of nevirapine and indinavir in various fractions of seminal plasma. Antimicrob Agents Chemother 2001, 45:2902–2907.
44. Eron JJ, Jr., Smeaton LM, Fiscus SA, Gulick RM, Currier JS, Lennox JL, et al. The effects of protease inhibitor therapy on human immunodeficiency virus type 1 levels in semen (AIDS clinical trials group protocol 850). J Infect Dis 2000, 181:1622–1628.
45. Pereira AS, Smeaton LM, Gerber JG, Acosta EP, Snyder S, Fiscus SA, et al. The pharmacokinetics of amprenavir, zidovudine, and lamivudine in the genital tracts of men infected with human immunodeficiency virus type 1 (AIDS clinical trials group study 850). J Infect Dis 2002, 186:198–204.
46. Sankatsing SU, Droste J, Burger D, Van Praag RM, Jurriaans S, Lange JM, et al. Limited penetration of lopinavir into seminal plasma of HIV-1-infected men. AIDS 2002, 16:1698–1700.
47. Lafeuillade A, Solas C, Chadapaud S, Hittinger G, Poggi C, Lacarelle B. HIV-1 RNA levels, resistance, and drug diffusion in semen versus blood in patients receiving a lopinavir-containing regimen. J Acquir Immune Defic Syndr 2003, 32:462–464.
48. Taylor S, Pereira AS. Antiretroviral drug concentrations in semen of HIV-1 infected men. Sex Transm Infect 2001, 77:4–11.
49. Chakraborty H, Helms RW, Sen PK, Cohen MS. Estimating correlation by using a general linear mixed model: evaluation of the relationship between the concentration of HIV-1 in blood and semen. Statis. Med. 2003, 22:1457–1464.
50. Miller CJ, Vogel P, Alexander NJ, Dandekar S, Hendrickx AG, Marx PA. Pathology and localization of simian immunodeficiency virus in the reproductive tract of chronically infected male rhesus macaques. Lab Invest 1994, 70:255–262.
This article has been cited 38 time(s).
Current Hiv Research
Standing in the way of eradication: HIV-1 infection and treatment in the male genital tract
Current Hiv Research, 3(4):
Antimicrobial Agents and ChemotherapyPenetration of atazanavir in seminal plasma of men infected with human immunodeficiency virus type 1Antimicrobial Agents and Chemotherapy
International Journal of Infectious DiseasesUnderstanding transmitted HIV resistance through the experience in the USAInternational Journal of Infectious Diseases
Penetration of enfuvirtide, tenofovir, efavirenz, and protease inhibitors in the genital tract of HIV-1-infected men
Acute hepatitis C in M-infected men who have sex with men
Hiv Medicine, 5(4):
Presse MedicaleAssisted procreation technology and people with HIVPresse Medicale
Plos OneCompartmentalization of HIV-1 within the Female Genital Tract Is Due to Monotypic and Low-Diversity Variants Not Distinct Viral PopulationsPlos One
Journal of AndrologyDecreased semen volume and spermatozoa motility in HIV-1-infected patients under antiretroviral treatmentJournal of Andrology
VirologieRole of NNRTIs in the prevention of HIV-1 mother to child transmissionVirologie
Gynecologie Obstetrique & FertiliteThe male genital tract: A host for HIVGynecologie Obstetrique & Fertilite
HIV-1 drug resistance surveillance using dried whole blood spots
Antiviral Therapy, 12(1):
Journal of Pharmacy and PharmacologyQuantification of antiretroviral drugs for HIV-1 in the male genital tract: current data, limitations and implications for laboratory analysisJournal of Pharmacy and Pharmacology
Ritonavir-boosted protease inhibitor monotherapy for the treatment of HIV-1 infection
AIDS Reviews, 10(1):
No benefit of a structured treatment interruption based on genotypic resistance in heavily pretreated HIV-infected patients
Journal of Antimicrobial ChemotherapyAbsence of HIV-1 shedding in male genital tract after 1 year of first-line lopinavir/ritonavir alone or in combination with zidovudine/lamivudineJournal of Antimicrobial Chemotherapy
AIDS Research and Human RetrovirusesComparison of HIV Type 1 Sequences from Plasma, Cell-Free Breast Milk, and Cell-Associated Breast Milk Viral Populations in Treated and Untreated Women in MozambiqueAIDS Research and Human Retroviruses
International Journal of AndrologyHIV infection of the male genital tract - consequences for sexual transmission and reproductionInternational Journal of Andrology
AntibiotiquesHIV infection: Negative effects of viral replication during treatmentAntibiotiques
American Journal of PathologySusceptibility of human testis to human immunodeficiency virus-1 infection in situ and in vitroAmerican Journal of Pathology
Gynecologie Obstetrique & FertiliteAssisted reproductive techniques in the context of HIV: the infectiologist's point of viewGynecologie Obstetrique & Fertilite
Current Hiv Research
Pathophysiology of HIV-1 in semen: Current evidence for compartmentalisation and penetration by antiretroviral drugs
Current Hiv Research, 3(3):
Future VirologyPersistence of drug-resistant HIV-1 and possible implications for antiretroviral therapyFuture Virology
VirologyConstruction and tropism characterisation of recombinant viruses exhibiting HIV-1 env gene from seminal strainsVirology
Sexually Transmitted InfectionsIncrease in hepatitis C virus incidence in HIV-1-infected patients followed up since primary infectionSexually Transmitted Infections
Sequential transmission and long-term persistence of an HIV strain partially resistant to protease inhibitors
New Microbiologica, 32(2):
Journal of Clinical MicrobiologyDetermining Seminal Plasma Human Immunodeficiency Virus Type 1 Load in the Context of Efficient Highly Active Antiretroviral TherapyJournal of Clinical Microbiology
Journal of Antimicrobial ChemotherapyRapid selection and archiving of mutation E157Q in HIV-1 DNA during short-term low-level replication on a raltegravir-containing regimenJournal of Antimicrobial Chemotherapy
Journal of VirologyQuantification of the effects on viral DNA synthesis of reverse transcriptase mutations conferring human immunodeficiency virus type 1 resistance to nucleoside analoguesJournal of Virology
Current Hiv Research
Factors affecting sexual transmission of HIV-1: Current evidence and implications for prevention
Current Hiv Research, 3(3):
Plos OneInfection of Semen-Producing Organs by SIV during the Acute and Chronic Stages of the DiseasePlos One
Reservoirs and immunitary responses during the natural history of HIV-1 infection
HIV; resistance; semen; provirus; compartmentalization; diversity; therapeutic failure
© 2004 Lippincott Williams & Wilkins, Inc.
Highlight selected keywords in the article text.