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Simplified regimens containing protease-inhibitors (PI)-sparing combinations were used in patients with virological suppression after prolonged highly active antiretroviral therapy. This study evaluated the total HIV-1 DNA quantitation as a predictor of long-term success for PI-sparing simplified therapy. Sixty-two patients were enrolled in a prospective non-randomized cohort. All patients have been receiving a triple-therapy regimen, two nucleoside reverse transcriptase inhibitors (NRTIs) plus one PI, for at least 9 months and were characterized by undetectable plasma HIV-1 RNA levels (

Background: Since the introduction of highly active antiretroviral therapy (HAART), morbidity and mortality rates have sharply decreased among HIV-infected patients. Studies of possible differences between men and women in the course of HIV infection give conflicting results. The objective of this study was to assess sex differences during HAART. Methods: A literature search by using the MEDLINE database between March 2002 and February 2007 was performed to identify all published studies on the sex-specific differences on the impact of HAART. All articles with measures of effect (preferably adjusted odds ratio, relative risk or hazard ratio with 95% CI) of sex on viroimmunological and clinical parameters during HAART were included. Five different topics of interest in our research were selected: time of initiation of HAART, adherence, viroimmunological response, clinical response and adverse reactions during HAART. Results: US data report an initiation of HAART at an earlier disease stage in men compared with women. After initiation of HAART, most authors do not report any viroimmunological difference, although a few clinical studies showed a significantly better virological response in women compared with men. Nevertheless, women were more likely to be less adherent to antiretrovirals and to have non-structured treatment interruptions than men. This is likely to be related to the higher number of adverse reactions they experience during HAART. Finally, discordant opinions with regard to clinical benefits during HAART exist, but recent clinical and observational trials suggest a better clinical outcome for women. Conclusions: We found little evidence of sex differences during antiretroviral treatment. Nevertheless, most of these studies were underpowered to detect sex differences and had limited follow-up at 6 or 12 months. Design of new gender-sensitive clinical trials with both prolonged follow-up and sample size representative of the current HIV prevalence among women are strongly needed to detect the likely sex differences of antiretroviral agents during HIV infection. © The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

In a longitudinal study on 181 naïve patients who responded to therapy (mean follow-up 4 years), high baseline human immunodeficiency virus (HIV)-RNA values correlated with high levels of cellular HIV-DNA at all time points (p

Patients and Methods. At the Rome Transplant Network, a JACIE accredited metropolitan transplant program, a matched-pair analysis has been conducted on 116 of 255 patients transplanted between January 2008 and December 2012. The patients transplanted from Id Sib (n=58) or Haplo related donors (n=58) were completely matched for the following features: patient age, gender, diagnosis (7 subgroups), disease phase (early: CR1+CR2; advanced: >CR2+active disease), myeloablative or reduced intensity conditioning regimen consisting of Thiotepa, i.v. Busulphan and Fludarabine (TBF) association, donor age and donor/recipient sex, AB0 and CMV combinations. As GVHD prophylaxis, all patients received the standard CSA and MTX combination with the addition of ATG, MMF and Basiliximab in Haplo bone marrow recipients. The transfusion policy, supportive care and antinfectious prophylaxis were identical for all patients. Results. By comparingId Sib to Haplo recipients, the cumulative incidence (CI) of grade II-IV and III-IV acute-GVHD was 18±5% vs 42±7% (p=0.002) and 7±3% vs 14±5% (p=ns), respectively. The CI of extensive chronic GVHD was 23±6% for both patient series. The 5-year CI of TRM was 30±6% vs 36±6% (p=ns), respectively. The main causes of TRM were infections and the 6-month CI of Infection Related Mortality (IRM) was 26±6% in Haplo transplant and 10±4% in Id Sib (p=0.04). Although not statistically significant, the 5-year CI of relapse was higher in Id Sib (40±7%) than in Haplo recipients (28±6%). With a median follow-up of 3.5 years (range, 1 - 6), the 1- and 5-year disease free survival (DFS) was respectively 50±7% and 37±6% for Id Sibs and 45±7% and 36±6% for Haplo (Figure 1). Since 2 years after transplant, DFS curves of Id Sib and Haplo patients remained at plateau and were overlapping. Conclusions. This analysis considered a high number of factors for matching patients, who were grafted according to an identical transplant program. We can conclude that as consequence of a more intensive GVHD prophylaxis and therapy ensuing from higher incidence of >II grade acute GVHD, Haplo recipients are mainly exposed to a risk of early infection mortality. On the other hand, Id Sib patients seem to express less graft-versus-tumor activity with increasing risk of relapse after transplant. The identical long-term DFS justifies to consider the unmanipulated bone marrow transplant from haploidentical donor a valid alternative for patients lacking an HLA identical sib. Finally, it can appear at present provocative, but certainly realistic in perspective, the hypothesis that the donor choice in a familiar setting will mainly take into account other favourable donor/recipient combined characteristics rather than HLA compatibility. Figure 1 - Disease Free Survival Figure 1. - Disease Free Survival Disclosures No relevant conflicts of interest to declare.

The association between human immunodeficiency virus (HIV) DNA load and immunologic parameters was investigated in 163 HIV-infected patients with undetectable plasma viremia during highly active antiretroviral therapy (HAART). Patients with HIV DNA below the 25th percentile (133 copies/10 6 peripheral blood mononuclear cells) had higher pre-HAART ( P = 0.001) and current ( P = 0.005) CD4 counts and a prolonged duration of treatment ( P = 0.001). At adjusted analysis, prolonged duration of treatment was independently associated with lower ( P = 0.006) and undetectable ( P < 0.001) HIV DNA values.

The duration of the clinical, virologic, and immunologic response to HAART, is not well defined. In this observational multi-center study 2,143 patients were enrolled classified according to virologic suppression (500 cp/ml) and immune recovery (100 CD4+ cells/mul from baseline) at month 12 of HAART as complete responders, virologic only responders, immunologic only responders and non-responders. Kaplan Meyer curves, multivariate and politomous regression analysis were used. Complete responders patients were 781 (36.4%), immunologic only responders 441 (20.6%), virologic only responders 336 (15.7%), and non-responders 585 (27.3%). Using multivariate analysis, being antiretroviral-naive increased the probability of having both a virologic only or a complete response and reduced the probability of an immunologic only response (P0.001 for all tests). Older age was associated directly with a virologic only response and inversely associated with an immunologic only response (P = 0.027 and P = 0.035, respectively). Using politomous analysis, patients baseline HIV-RNA level more than 5 log cp/ml had a 1.9-fold higher probability of an immunologic response than of a complete response (P = 0.001). After 4 years, the clinical progression rate was six times greater in non-responders, 1.9 times greater in virologic only responders, and 2.3 times greater in immunologic only responders than for responders. However, patients with virologic only response or with immunologic only response had a significantly reduced risk for clinical progression than non-responders (P0.001). After 4 years of HAART, the risk of clinical progression in patients with immunologic only or virologic only response is low but still higher than in complete responder patients.

Objective: To assess gender differences in the long-term clinical, virological and immunological outcomes during highly active antiretroviral therapy (HAART). Methods: This longitudinal observational multicentre study followed 2460 HIV-infected patients who had begun a protease inhibitor-based regimen for a median period of 43 months. Outcome measures were virological suppression (< 500 copies/ml), confirmed virological rebound after suppression, and death or new AIDS-defining illness (ADl). Results: At baseline, 690 female patients (28.0%) had significantly lower age, higher prevalence of heterosexual contact and lower prevalence of intravenous drug use as risk factors for HIV infection compared with males. Furthermore, females had a lower number of AIDS-defining illnesses, higher CD4 cell counts and lower viral loads. No gender differences were reported in terms of proportion of patients achieving viral suppression or exhibiting rebound after achieving viral suppression. Female patients experienced reduced clinical progression during follow-up compared with males (P = 0.008) by Kaplan-Meier analysis; however this difference was not significant in an adjusted analysis. In a multivariate model, the interaction between gender and risk factor for HIV or viral load showed that female drug users and female patients with a baseline HIV RNA viral load of 10(4)-10(5) copies/ml had a favourable clinical outcome compared with males (P = 0.035 and P = 0.015, respectively). Conclusion: No differences were found between genders in terms of virological and immunological outcomes during long-term HAART. Nevertheless, a lower risk of clinical progression was reported among female patients with intermediate baseline viral load than in males. (c) 2005 Lippincott Williams C Wilkins.

A cross-sectional study was undertaken on the correlates of infection for the human immunodeficiency virus (HIV) and hepatitis viruses B and C (HBV and HCV) in a sample of inmates from eight Italian prisons. A total of 973 inmates were enrolled [87.0% males, median age of 36 years, 30.4% intravenous drug users (IDUs), 0.6% men who have sex with men (MSWM)]. In this sample, high seroprevalence rates were found (HIV: 7.5%; HCV: 38.0%; anti-HBc: 52.7%; HBsAg: 6.7%). HIV and HCV seropositivity were associated strongly with intravenous drug use (OR: 5.9 for HIV; 10.5 for HCV); after excluding IDUs and male homosexuals, the HIV prevalence remained nonetheless relatively high (2.6%). HIV prevalence was higher for persons from Northern Italy and Sardinia. The age effect was U-shaped for HIV and HCV infections; HBV prevalence increased with age. Tattoos were associated with HCV positivity (OR: 2.9). The number of imprisonments was associated with HIV infection, whereas the duration of imprisonment was only associated with anti-HBc. The probability of being HIV-seropositive was higher for HCV-seropositive individuals, especially if IDUs. In conclusion, a high prevalence of HIV, HCV, and HBV infections among inmates was observed: these high rates are in part attributable to the high proportion of IDUs. Frequency of imprisonment and tattoos were associated, respectively, with HIV and HCV positivity. Although it is possible that the study population is not representative of Italy's prison inmate population, the results stress the need to improve infection control measures users was prisons.

A longitudinal study of the replication capacity of HIV strains isolated from 18 patients failing highly active antiretroviral therapy (HAART) was undertaken at the time of genotypic guided change of therapy and after 12 months. Patients were divided in two groups according to the response to therapy: immune responders (12 patients with immune recovery defined as having more than 100 CD4 cells compared to baseline value), and failing patients (six patients without immune recovery). At enrollment no significant difference in terms of CD4 cell count and HIV plasma viremia was detected between the two groups. One year after change of therapy, all patients experienced a decrease in the replication capacity of HIV strains. The HIV replication capacity of the failing and of immune-responder patients decreased from 60% (range 14-96%) to 26.4% (range 0.4-74.5) and from 46.8% (range 15-98%) to 3.6% (range 0.1-26.8%), respectively. At month 12, the difference of HIV replication capacity between the two groups reached a statistical significance (P

To investigate whether vertical transmission of the human herpesvirus 8 (HHV-8) may occur during pregnancy or at delivery, we enrolled 295 women recruited attending the Division of Obstetrics and Gynecology of a University Teaching of Rome Tor Vergata, S. Eugenio Hospital. The study population was divided in two groups: 245 pregnant women who underwent amniocentesis for genetic screening at 16-18 weeks gestation (group 1) and 50 women at the childbirth (group 2). Maternal blood was obtained from all women. Amniotic fluid (group 1) and cord blood (group 2) were obtained at midtrimester and at delivery, respectively. The presence of anti-HHV-8 antibodies in serum samples was investigated by an immunfluorescence assay. All amniotic fluids, maternal blood, and cord blood samples from HHV-8 seropositive women were tested for the presence of HHV-8 DNA sequences by the polymerase chain reaction. Thirty women, 27 of the group 1 and three of the group 2, were found to have anti-HHV-8 antibodies. Two neonates of the three seropositive mothers of the group 2 had anti-HHV-8 antibodies in cord blood. HHV-8 DNA sequences were detected in the blood of one woman of the group 2. None of the amniotic fluid and cord blood samples had detectable HHV-8 DNA sequences. This study suggests that vertical transmission of HHV-8 is unlikely or, at least, very rare.

The replicative capacity of HIV is studied by carrying out replication-competition experiments with the insertion of the gene of interest. These assays cannot capture the complicated patterns of mutations of different genes.A cross sectional study was carried out on 10 HIV-infected nai;ve patients and on 15 patients failing HAART. The CD8-depleted PBMCs, with known proviral DNA and cellular HIV-RNA copy numbers, were cultured. A reference curve was determined using the data obtained from 10 nai;ve patients. The replicative capacity was calculated as the ratio multiplied by 100 of the p24 antigen level of isolates over the p24 antigen level determined on the reference curve.A linear correlation between p24 antigen level and the infectious doses of HIV-DNA alone or plus cellular RNA copy number of PBMCs was found in naive patients (r=0.63, P

Sturmer et al. presented an interesting paper (1) on the influence of the classic zidovudine (ZDV) mutation patterns and the role of the additional mutations at positions 208, 211, 214, and 333 on phenotypic ZDV resistance. The presence of the 211/214 mutation combination in the background of genotypic highly ZDV-resistant viruses increased ZDV resistance 2.4-fold, and the 208/211/214 combination increased ZDV resistance 8-fold. The authors suspect a stabilization mechanism of the three-dimensional structure of the enzyme that could be explained by the vicinity of the mutations at positions 208, 211, and 214 to the ZDV-associated mutations L210W and T215Y/F.These mutations are likely involved in the compensation of the possible loss of fitness caused by the T215Y/F mutation. We investigated the drug susceptibility of human immunodeficiency virus (HIV) strains of 19 HIV-infected patients failing double nucleoside reverse transcriptase inhibitor (NRTI) therapy by using the genotypic and the phenotypic assays. We identified four patterns of genotypic resistance (Table ​(Table1),1), namely, pattern 1, in patients with the 184V mutation alone; pattern 2, in patients with the 184V mutation combined with the 208Y, 211K, and/or 214F mutations; pattern 3, in patients with the 184V mutation and thymidine analogue mutations (TAMs) (including 41L, 210W, 215Y/F, and/or 219Q/E); and pattern 4, in patients with TAMs and the 184V mutation combined with the 208Y, 211K, and/or 214F mutations. As expected, genotypic patterns 1 and 3 showed a phenotypic resistant pattern to lamivudine (3TC) alone and to both 3TC and ZDV, respectively. The presence of 208/211/214 polymorphism combined with the 184V mutation (genotypic pattern 2) was related to a high phenotypic cross-resistance within all NRTIs analyzed. The presence of TAMs in addition to this pattern (genotypic pattern 4) showed a reduced phenotypic resistance for stavudine (d4T) and, to a smaller extent, for didanosine, ZDV, and abacavir. TABLE 1. Pattern of genotypic and phenotypic resistance of 19 HIV strains of patients failing double NRTI therapy The impact of the 208/211/214 polymorphism in terms of NRTI drug susceptibility on a background of a resistant virus appears to be related to the presence of the 184V mutation alone. High cross-resistance to all NRTIs has been detected only in HIV strains with this polymorphism combined with the 184V mutation in absence of TAMs. This particular genotypic-phenotypic pattern was detected among patients treated with d4T and 3TC. This phenomenon is likely to be related to the presence of different pathways in the emergence of NRTI resistance mutations in HIV-infected patients failing a d4T-3TC or ZDV-3TC regimen. Longitudinal studies are needed to evaluate the significance of this polymorphism in the clinical outcome of patients treated with highly active antiretroviral therapy.

We compared the efficacy and the toxicity of zidovudine (AZT) versus stavudine (d4T), in combination with lamivudine (3TC) and indinavir, in AZT-, dideoxyinosine (ddI)-, and/or dideoxycytosine (ddC)-experienced patients in a randomized comparative multicenter trial. One hundred seventy human immunodeficiency virus type 1 (HIV-1)-infected patients, who had received AZT, ddI, and/or ddC for at least 6 months but were naive for d4T, 3TC, and protease inhibitors, were randomized to AZT at 250 to 300 mg twice daily, 3TC at 150 mg twice daily, and indinavir at 800 mg every 8 h or to d4T at 40 mg twice daily, 3TC at 150 mg twice daily, and indinavir at 800 mg every 8 h. The primary endpoint was time to virological failure, defined as plasma HIV-1 RNA levels of5,000 copies/ml after at least 8 weeks of antiretroviral therapy. Additional endpoints were change from baseline in CD4 cell counts, AIDS-defining events and adverse events, and proportion of patients with HIV-1 RNA levels of500 copies/ml and HIV-1 RNA levels of50 copies/ml. At week 80, 15 patients in the AZT arm and 14 patients in the d4T arm had reached the primary endpoint, and time to virological failure did not differ between the two arms (P = 0.98). In the d4T and in the AZT arms, 67 and 73% of patients, respectively, had HIV-1 RNA levels of500 copies/ml (P = 0.50). The median change from baseline in CD4 cell count was 195 x 10(6) and 175 x 10(6)/liter for the d4T- and AZT-containing arms, respectively. The proportions of patients with HIV-1 RNA levels of50 copies/ml at weeks 8, 16, and 24 were similar in the two arms. The occurrence of serious adverse events was not significantly different between arms. In conclusion, in these patients heavily pretreated with AZT, switching from AZT to d4T when initiating indinavir and 3TC did not bring any additional benefit compared to maintaining AZT.

Whether highly active antiretroviral therapy (HAART) should be modified in patients with persistent increases in CD4 + T cells despite detectable viral loads is an unresolved question. Forty-three heavily pretreated human immunodeficiency virus (HIV)-infected patients with virologic failure during HAART were studied before a change of therapy guided by genotypic analysis and during follow-up. Patients with an increase in CD4 + cell count (>100 cells/ml) over pre-HAART values were considered to be discordant patients (20 individuals), whereas patients with a lower increase or no increase in CD4 + cell count were considered failing patients (23 individuals). Based on univariate analysis, a high CD4 + cell count before antiretroviral treatment, homosexual behavior as a risk factor for HIV infection, reduced drug exposure to nonnucleoside reverse transcriptase inhibitors, low replicative capacity of HIV isolates, and more frequent detection of HIV isolates with a non-B subtype, an R5 biological phenotype, and M184V and T215Y/F mutations were factors associated with a discordant response to HAART. Based on multivariate analysis, only the M184V mutation remained significantly associated with a viroimmunologic discordant response (odds ratio, 25.48; 95% confidence interval, 1.43 to 453.93). No difference in lamivudine exposure was found between discordant (95%) and failing (91%) patients. Twelve months after the genotypic analysis-guided change of therapy, 3 discordant (15%) and 6 failing patients (26%) achieved undetectable viral loads (500 copies/ml, discordant responses were observed in 5 out of 15 discordant patients and in 4 out of 16 failing patients. A relationship between the M184V mutation and a viroimmunologic discordant response to HAART was found. After the genotypic analysis-driven change of therapy, similar rates of virologic suppression were detected in the two groups.

In 32 patients for whom highly active antiretroviral therapy was failing, a good agreement between drug resistance-associated mutations in plasma and peripheral blood mononuclear cells (PBMCs) was found ( k = 0.85). The mutations with the lowest agreement were 20R, 63P, and 84V in the protease gene and 184V in the reverse transcriptase gene. In eight patients, primary drug resistance mutations were detected only in PBMCs.

ContextHuman herpesvirus 8 (HHV-8) infection causes Kaposi sarcoma and lymphoproliferative disorders in immunosuppressed adults. Its manifestations in immunocompetent hosts are unknown.ObjectivesTo determine whether HHV-8 primary infection is symptomatic in immunocompetent children and to identify the epidemiological and virological correlates of HHV-8 infection.Design and SettingProspective cohort study conducted in the pediatric emergency department of a hospital in Alexandria, Egypt, between December 1, 1999, and April 30, 2000.PatientsEighty-six children aged 1 to 4 years who were evaluated for a febrile syndrome of undetermined origin.Main Outcome MeasuresSerological assay and polymerase chain reaction of blood and saliva samples for HHV-8. Information on potential risk factors for HHV-8 infection was also collected.ResultsThirty-six children (41.9%) were seropositive; HHV-8 DNA sequences were detected in 14 (38.9%) of these 36 children (detected in saliva in 11 of 14). Significant associations were found between HHV-8 infection and close contact with at least 2 other children in the community (36 of 63 vs 6 of 23 for

To assess the circulation of human herpesvirus (HHV)-8 infection over the years, two seroprevalence surveys were conducted, which tested sera from HIV-infected individuals recruited 10 years apart (206 individuals from 1986 to 1988 and 177 individuals from 1997 to 1998). For all patients, antibodies to hepatitis C virus (HCV). hepatitis B virus (HBV), and HHV-8 lytic and latent antigens were evaluated. HHV-8 seroprevalence was higher among individuals recruited in the 1990s (31.6% for anti-lytic, 8.5% for anti-latent antibodies) compared with similar findings in those seen in the late 1980s (14.6% and 3.4% for anti-lytic and anti-latent antibodies, respectively), with a twofold increase of the risk of HHV-8 infection. However, the increase was observed only among injecting drug users, whereas seroprevalence tended to slightly increase among those infected by sexual contact. At univariate analysis, time of recruitment and being homosexual men were factors associated with HHV-8 infection, an association that remained after adjusting for age. HBV infection was significantly associated with HHV-8 infection (odds ratio [OR]. 2.2: 95% confidence interval [CI], 1.3-3.6), whereas those infected with HCV had a lower probability of having HHV-8 antibodies (OR, 0.3: 95% CI, 0.20-0.6). After controlling for age and gender, time of recruitment remained independently associated with HHV-8 infection among injecting drug users. In conclusion. HHV-8 seroprevalence appears. to be increased during 10 years among HIV-infected injection drug users but not among homosexual men. who remain those at the highest risk of infection.

Objective: To study virologic and immunologic factors associated with discordant treatment response in HIV-infected patients receiving highly active antiretroviral therapy (HAART). Design: Study participants included a total of 27 patients: (a) 10 discordant patients (mean CD4+ cell count, 396.1 A— 106 cells/l; mean HIV-RNA, 5.4 log10 copies/ml); (b) seven responder patients (mean CD4+ cell count, 997.5 A— 106 cells/l); and (c) 10 failing patients (mean CD4+ cell count 66.5 A— 106 cells/l; mean HIV-RNA, 5.4 log10 copies/ml). Methods: The HIV-1 isolation rate and biological phenotype, drug resistance genotypic mutations of HIV-1 strains, recall and HIV-1-specific antigen lymphocyte proliferation (LP), and interleukin (IL)-15 production were studied. Results: Virus isolation was obtained in 30% of discordant patients, and in 100% of failing patients. A higher replication constant was reported in discordant patients. No difference in the number of drug resistance mutations and biological phenotypes of HIV-1 was found in discordant patients with respect to failing patients. Discordant patients developed positive LP responses to Candida albicans and HIV-1 p24. LP in response to C. albicans, HIV-1 p24 and gp160 was positive in responder patients. No significant LP was found in failing patients. Increased levels of IL-15 after stimulation with lipopolysaccaride (LPS) and C. albicans were found in both discordant patients and responder patients. Conversely, a strong reduction of IL-15 levels was observed in failing patients. Conclusion: The present results suggest that decreased virus isolation rate, restoration of both lymphocyte proliferation and IL-15 production are factors involved in the discordant antiretroviral therapy response of HIV-infected patients. © 2002 Lippincott Williams & Wilkins.

The combination of interleukin 2 (IL-2) and antiretroviral therapy (ART) represents an emerging strategy in the treatment of patients infected with HIV. Aside from its immunomodulatory role, however, IL-2 may induce replication of human herpesvirus 8 (HHV-8)/Kaposi sarcoma (KS)-associated herpesvirus. We retrospectively evaluated HHV-8 plasma viremia and cellular load, as well as anti-HHV-8 antibody titers, in sequential samples from 84 patients receiving ART alone or in combination with IL-2. At baseline, HHV-8 plasma viremia was present only in 2 HHV-8-seropositive patients in whom KS subsequently developed during or immediately after termination of IL-2 therapy. The level of viremia increased during follow-up and peaked at the time of the clinical manifestation of KS. Moreover, transient peaks of HHV-8 viremia were temporally associated with administration of IL-2. HHV-8 plasma viremia was never detected in the other 47 patients receiving IL-2 nor in 35 controls treated only with ART. Thus, IL-2 therapy seems safe in most patients infected with both HIV and HHV-8, except for those with detectable HHV-8 viremia, who may not be eligible for IL-2 treatment. (C) 2002 by The American Society of Hematology.

Infection with human herpesvirus type 8 and with human T-cell leukaemia virus type-1 shows strong geographic variations. We conducted this study to assess prevalence and risk factors for human herpesvirus type 8 infection in Havana City, Cuba. Information and residual serum samples already collected for a hospital based case–control study were used. A total of 379 individuals (267 males and 112 females; median age=63 years) were evaluated. Antibodies to the lytic antigen of human herpesvirus type 8 were detected by using an immunofluorescence assay, while human T-cell leukaemia virus type-1 serology was performed by means of an ELISA test (alpha Biotech). Overall, 64 subjects (16.9%, 95% confidence interval: 13.1–20.0) were positive for human herpesvirus type 8 antibodies. Human herpesvirus type 8 seroprevalence significantly increased with age (odds ratio=1.9 for ⩾65 vs

Accurate laboratory tests for the diagnosis of active human herpesvirus 8 (HHV-8) infection are becoming essential to study the pathogenesis of HHV-8-associated tumors and for the clinical management of HHV-8-infected individuals. We have developed a highly sensitive, calibrated quantitative real-time PCR assay for the measurement of cell-free HHV-8 DNA in body fluids, based on the addition of a synthetic DNA calibrator prior to DNA extraction. The calibrator controls each sample for the presence of PCR inhibitors, determines a cutoff value of sensitivity for negative samples, and normalizes positive samples for the efficiency of DNA recovery. The assay shows a wide dynamic range of detection (between 1 and 10 6 viral genome equivalents/reaction) and a high degree of accuracy even in the presence of high amounts (up to 1 μg) of human genomic DNA. Moreover, the assay has a very high sensitivity (lower detection limit, 10 genome equivalents/ml) and a high degree of reproducibility and repeatability with a coefficient of variation (CV) of

The aim of the study was to analyze the relationship between genotypic and phenotypic drug resistance profiles of human immunodeficiency virus type 1 (HIV-1) strains isolated from patients during double-analogue nucleoside therapy. A drug-resistant HIV strain was isolated from 20 out of 25 patients, with 16 (64%) subjects carrying a virus with multiple drug resistance mutations. The most frequent resistance mutations were M184V (18 isolates) and M41L (7 isolates). Discordance between the genotypic and phenotypic profile for at least one drug was detected in 16 out of 25 strains. Particularly, eight isolates had a discordant genotypic-phenotypic resistance pattern for two drugs and one isolate had such a pattern for three drugs. A genotypic resistance pattern with a phenotypic sensitivity profile was detected in six isolates (four resistant to zidovudine and two resistant to lamivudine). On the other hand for several strains a genotypic pattern of sensitivity pattern to abacavir (10 strains), didanosine (7 strains), stavudine (3 strains), zidovudine (2 strains), and lamivudine (1 strain) with a phenotypic resistance profile was detected. After a follow-up period of 8 months, an impairment of virological and immunological parameters was detected only in subjects with an HIV-1 isolate with a phenotypic resistance profile in despite of the genotypic results. Predicting resistance phenotype from genotypic data has important limitations. Despite the low number of patients and the short follow-up period, this study suggests that during failing therapy with analogue nucleosides, a phenotypic analysis could be performed in spite of an HIV genotypic sensitivity pattern.

P681 HBsAg MUTATIONS WITH ENHANCED CAPABILITY TO EVADE IMMUNE RESPONSE ARE ASSOCIATED WITH HBV REACTIVATION DURING IMMUNOSUPPRESSION R. Salpini, C. Alteri, L. Colagrossi, M.-C. Bellocchi, D. Armenia, F. Di Santo, L. Carioti, F. Continenza, A. Bertoli, Y. Louzoun, M. Pollicita, A. Ricciardi, C. Mastroianni, M. Paoloni, A. Marrone, L. Sarmati, C. Sarrecchia, M. Andreoni, M. Angelico, C.-F. Perno, V. Svicher. University of Rome Tor Vergata, I.N.M.I. L. Spallanzani, Rome, Italy; Bar-Ilan University, Ramat Gan, Israel; Sapienza University of Rome, Rome, S.S. Filippo e Nicola Hospital, Avezzano, Second University of Naples, Naples, Tor Vergata University Hospital, Rome, Italy E-mail: valentina.svicher@uniroma2.it

The probability of HIV infection by sexual contact, although it varies greatly, appears to be lower than that of infection by other routes of exposure. The aim of this study was to evaluate immunological determinants involved in protection against HIV infection in subjects with multiple and repeated sexual exposures to the virus. Twenty-two subjects were studied for CD8+ cell anti-HIV suppression activity and serum neutralizing activity against the HIV strain of their own partners, beta-chemokine production, and natural killer cell activity. CD8+ cell anti-HIV activity and neutralizing activity of sera were found in 13 (76%) and 12 (70.5%) out of 17 HIV-1 negative subjects, respectively. Six individuals had a relevant immune response against HIV: three subjects with a high CD8+ cell antiviral suppression activity and three individuals with sera neutralizing activity titer >1:10. These last three subjects had the highest beta-chemokine levels, a very prolonged period of multiple sexual intercourse (>6 years) and a seropositive partner with a high viral load. A partial reduction of neutralizing activity titer was observed when pre-incubating the sera with anti-beta-chemokine neutralizing antibodies. A spontaneous natural killer cell activity was suppressed in the majority of HIV-1 negative subjects with sexual exposure in comparison with normal individuals. The protection from sexual HIV transmission appears to be the result of a network of different humoral and cellular factors.

Antiretroviral-treated human immunodeficiency virus (HIV) type 1-seropositive individuals can remain clinically stable for a long period of time with an increasing CD4 cell count irrespective of incomplete viral suppression. We evaluated the role of neutralizing antibody (NtAb) activity in the etiopathogenesis of this viro-immunological disconnection (defined as an increasing CD4 + -cell count despite a persistent, detectable viral load during antiretroviral therapy) in 33 patients failing therapy with two analogue nucleoside reverse transcriptase inhibitors. An HIV NtAb titer of ≥1:25 was detected in specimens from 16 out of 33 (48%) patients. A significant correlation was found between NtAb titers and CD4 + -cell counts ( P = 0.001; r = 0.546) but not with HIV RNA levels in plasma. Five patients with a viro-immunological disconnection had an NtAb titer of >1:125, statistically higher than the NtAb titers for the remaining 28 patients with both virologic and immunologic failure ( P < 0.0001). The HIV-specific humoral immune response could play a role during antiretroviral treatment to improve immunological function despite an incomplete suppression of viral load.

The modes of transmission of HHV-8 are still unclear.To evaluate the distribution and transmission of HHV-8 infection.Serosurvey conducted in a Cameroon hospital among 292 persons, including children (5-10 years), adolescents (15-20 years), and adults (30-40 years). Antibodies against lytic and latent antigens to HHV-8 were detected by immunofluorescence assay; antibodies against Epstein-Barr virus viral antigens were detected by enzyme-linked immunoabsorbent assay.The prevalence of HHV-8 antilytic antibodies remained stable and was 39.8% among children, 51.5% among adolescents, and 61.8% among adults. Epstein-Barr virus seroprevalence was high among children, and remained stable among adolescents and adults. A history of sexually transmitted diseases was an independent determinant of HHV-8 infection (adjusted odds ratio 2.47; 95% CI 1.09-4.91).The high prevalence of HHV-8 infection among children indicates nonsexual modes of transmission in Cameroon, with sexual transmission occurring among adolescents and adults.The modes of transmission of HHV-8 are still unclear. To evaluate the distribution and transmission of HHV-8 infection serosurvey was conducted in a Cameroon hospital among 292 persons, including children (5-10 years), adolescents (15-20 years), and adults (30-40 years). Antibodies against lytic and latent antigens to HHV-8 were detected by immunofluorescence assay; antibodies against Epstein-Barr virus viral antigens were detected by enzyme-linked immunoabsorbent assay. The prevalence of HHV-8 antilytic antibodies remained stable and was 39.8% among children, 51.5% among adolescents, and 61.8% among adults. Epstein-Barr virus seroprevalence was high among children, and remained stable among adolescents and adults. A history of sexually transmitted diseases was an independent determinant of HHV-8 infection (adjusted odds ratio, 2.47; 95% CI, 1.09-4.91). The high prevalence of HHV-8 infection among children indicates nonsexual modes of transmission in Cameroon, with sexual transmission occurring among adolescents and adults.

Objectives: The aim of the study was to investigate changes in virological characteristics of HIV strains isolated from 38 HIV-seropositive subjects during antiretroviral therapy. Design and methods: Patients with a CD4+ cell count ≤ 300 〈 10 6 /l were treated with zidovudine (12 individuals) and saquinavir (10 individuals) alone or in combination (16 individuals). CD4+ cell count, viral load, HIV biological phenotype and drug resistance were evaluated during the study period. Results: After 52 weeks, 28 subjects (74%) harboured drug-resistant strains. In patients with a syncytium-inducing (SI) strain, a decline of CD4+ cell count and an increase of viral load were observed aside from the emergence of drug resistance. Conversely, at the emergence of antiretroviral resistance, an immunological and virological deterioration was observed only in patients who had a non-syncytiuminducing (NSI) strain. During the study, a phenotype switching of HIV isolates was detected in eight (21%) patients and a temporal correspondence between the appearance of phenotype switching and the emergence of drug resistance was found in seven cases. Three patients harbouring saquinavir-resistant strains showed a switch from SI to NSI variants associated with a moderate increase in CD4+ cell count. Conclusions: The emergence of resistant strains during antiretroviral therapy may be associated with the selection of viral strains with less cytopathogenicity, while it could become a poor prognostic sign in patients with NSI isolates. AIDS 1997, 11:1211‐1217

BackgroundDual/mixed-tropic HIV-1 strains are predominant in a significant proportion of patients, though little information is available regarding their replication-capacity and susceptibility against CCR5-antagonists in-vitro. The aim of the study was to analyze the replication-capacity and susceptibility to maraviroc of HIV-1 clinical isolates with different tropism characteristics in primary monocyte-derived-macrophages (MDM), peripheral-blood-mononuclear-cells (PBMC), and CD4(+) T-lymphocytes.MethodsTwenty-three HIV-1 isolates were phenotipically and genotipically characterized as R5, X4 or dual (discriminated as R5(+)/X4, R5/X4, R5/X4(+)). Phenotypic-tropism was evaluated by multiple-cycles-assay on U87MG-CD4(+)-CCR5(+)-/CXCR4(+)-expressing cells. Genotypic-tropism prediction was obtained using Geno2Pheno-algorithm (false-positive-rate [FPR] = 10%). Replication-capacity and susceptibility to maraviroc were investigated in human-primary MDM, PBMC and CD4(+) T-cells. AMD3100 was used as CXCR4-inhibitor. Infectivity of R5/Dual/X4-viruses in presence/absence of maraviroc was assessed also by total HIV-DNA, quantified by real-time polymerase-chain-reaction.ResultsAmong 23 HIV-1 clinical isolates, phenotypic-tropism-assay distinguished 4, 17 and 2 viruses with R5-tropic, dual/mixed-, and X4-tropic characteristics, respectively. Overall, viruses defined as R5(+)/X4-tropic were found with the highest prevalence (10/23, 43.5%). The majority of isolates efficiently replicated in both PBMC and CD4(+) T-cells, regardless of their tropism, while MDM mainly sustained replication of R5- or R5(+)/X4-tropic isolates; strong correlation between viral-replication and genotypic-FPR-values was observed in MDM (rho = 0.710;p-value = 1.4e-4). In all primary cells, maraviroc inhibited viral-replication of isolates not only with pure R5- but also with dual/mixed tropism (mainly R5(+)/X4 and, to a lesser extent R5/X4 and R5/X4(+)). Finally, no main differences by comparing the total HIV-DNA with the p24-production in presence/absence of maraviroc were found.ConclusionsMaraviroc is effective in-vitro against viruses with dual-characteristics in both MDM and lymphocytes, despite the potential X4-mediated escape. This suggests that the concept of HIV-entry through one of the two coreceptors "separately" may require revision, and that the use of CCR5-antagonists in patients with dual/mixed-tropic viruses may be a therapeutic-option that deserves further investigations in different clinical settings.

Background: In western countries, human herpesvirus-8 (HHV-8) appears to be transmitted mainly by sexual contact. To evaluate the role of other transmission routes, especially in developing countries, we estimated the seroprevalence of HHV-8 in Egyptian children, who, if seropositive, would have acquired the virus through a nonsexual route. Methods: Sera from 196 children (

The usefulness of traditional methods of HIV plasma titration has been limited by poor detection capacity in the asymptomatic phase of HIV disease. We analyzed plasma samples from asymptomatic seropositive or early symptomatic patients, comparing the classic plasma culture method with the following techniques: phorbol 12-myristate 13-acetate treatment of target cells, centrifugal inoculation of the virus, heat treatment of cultures, addition of monocyte/macrophages to cultures, and polyethylene glycol (PEG) treatment of the plasma. Only PEG treatment significantly increased the percentage of HIV isolation. The increase of HIV isolation after PEG treatment is more evident in patients with higher CD4+ cell counts and those without detectable levels of p24 antigen. In the p24-negative samples, HIV was isolated in 17 of 24 (71%) with PEG treatment versus nine of 24 (37%) with the classic method (p0.01). A number of discordant samples were found using the classic and PEG methods. Combining the positive results obtained with either technique, we obtained an overall HIV detection rate of 76%. The increased sensitivity of the combination of PEG and classic methods may allow a wider use of plasma viremia as part of the virological evaluation of anti-HIV drug efficacy in asymptomatic patients.

Aim: To investigate the prevalence and genotypic profile of overt and occult hepatitis-B infection (OBI) among HIV-infected individuals in Cameroon. Methods: 212 HIV-infected Cameroonians, aged 37.6 [IQR: 32.6-46.6] followed-up at the University Health Centre in Yaounde, were tested for HBsAg, anti-HBs, anti-HBc IgG/IgM, HBV-DNA and anti-HCV IgG. HBV positive cases were tested for Hepatitis Delta virus (HDV) using anti-HDV IgG and HDV-RNA. Liver function was assessed by alanine and aspartate aminotransaminases. OBI was defined as negative-HBsAg and detectable HBV-DNA. In occult or overt HBVinfected participants, HBV reverse transcriptase (RT)/surface (S) sequences were analyzed for drug resistance, immuneescape mutants, and phylogeny. Results: Overall, 78.3% (166/212) participants had past/ongoing HBV-exposure, with 39.1% (83/212) carrying “HBcAbpositive alone”. Prevalence of overt HBV (positive-HBsAg) was 11.8% (25/212), prevalence of HBV and HDV was respectively 6.9% (12/175) and 12% (3/25). Phylogeny of HBV-RT/S revealed the co-circulation of genotypes A and E. All HBV-coinfected participants harbored HBV strains with at least one immune-escape mutation. Of note, one HBV variant carried the vaccine-escape mutation G145R that hinders HBsAg neutralization by antibodies. For the first time, a novel 9 aa-deletion (s115-s123), located in the HBsAg “a” determinant, was found concomitantly with OBI. A stop codon in the S region (associated with increased risk of hepatocellular carcinoma) was found in six cases. Conclusion: High prevalence of overt/occult HBV-infection and circulating atypical strains highlight the importance of HBV-surveillance among HIV-infected Cameroonians and strategies to detect OBI in highly endemic countries.

The aim of this study was to evaluate outcome after a genotype guided change of therapy in 18 patients failing HAART. Patients were divided into two groups according to the response to therapy: immune responders (12 patients with immune recovery defined as having more than 100 CD4 cells compared to baseline value), and 6 failing patients (without immune recovery). At month 12 after genotype change of therapy a significant difference in the decrease of HIV-RNA viral load between the two groups of patients was detected (mean -1.95 and +0.04 log HIV-RNA copies/ml, p=0.04). One year after the change of therapy, all but one patients experienced a decrease in the replication capacity of HIV strains. Particularly, the HIV replication capacity of HIV strains decreased from 52% (range 14-98%) to 15.2% (range 0.1-74.5%). The HIV strains of patients failing HAART showed a progressive impaired replication capacity. In patients failing HAART the impaired replication capacity of HIV strains could justify the persistence of an immune recovery.

In order to investigate the potential susceptibility of bone marrow stromal myoid cells to human immunodeficiency virus (HIV), a myoid cell population devoid of all other cellular components of marrow environment was isolated from 3 normal adult bone marrow samples. The bone marrow myoid cells were infected with 3 different strains of HIV-1, 2 strains isolated from patients with acquired immune deficiency syndrome (AIDS) and HTLV-IIIB strain. To demonstrate successful infection and HIV production, culture supernatants, harvested weekly until 2 months post-infection, were tested for the presence of p24 antigen and infectious virus. Myoid cell monolayers obtained from the 3 different bone marrow samples were shown to be susceptible to infection. In particular, infection led to the presence of p24 antigen and of infectious virus in culture supernatants up to day 49 post-infection. After day 49, it was not possible to demonstrate the presence of infectious virus in culture supernatants and HIV-DNA polymerase chain reaction (PCR) failed to show viral genome in any of the cultures assayed. Our results demonstrate the susceptibility of myoid stromal cells to HIV infection and may provide an in vitro model for studying the effects of HIV infection in disregulation of the haemopoietic function of bone marrow environment.

Quantitative culture of human immunodeficiency virus (HIV) was performed on 202 plasma samples obtained from asymptomatic and early symptomatic HIV-1 infected patients (mean CD4+ count: 186/mm3) before antiretroviral therapy was started. HIV could be isolated from 84% of the plasma samples (titers ranging from 10(0) to 10(2.75) TCID50/ml). Immune complex dissociated p24 antigen (ICD-p24) was detected in 66% of the samples. Only 23 samples (11%) were negative for both ICD-p24 as well as HIV culture. Discordant results were obtained in 55 samples, and 45 samples negative for ICD-p24 were positive for HIV culture. A significant proportion (42%) of patients that were negative for ICD-p24 belonged to a very advanced group with very low CD4+ cell count. However, almost 90% of these ICD-p24 negative samples were positive for HIV plasma viremia, stressing the value of this virological marker in patients with low CD4+ cell count and without any detectable ICD-p24 antigenemia. HIV-1 RNA was detected in all ICD-p24 negative plasma samples tested by the branched DNA (bDNA) assay. A very good correlation was found between high RNA copy number and HIV plasma isolation in samples obtained from patients with low CD4+ cell count, suggesting that HIV-1 RNA quantitation may also reflect viral infectivity of plasma.

Human herpesvirus 8 (HHV-8) has been identified in skin lesions of patients with Kaposi's sarcoma and is now considered a co-factor for the development of the neoplasm associated with this disease. Several laboratory tests are currently available for use in the detection of HHV-8 antibodies--immunofluorescence and enzyme-linked immunosorbant assays being the most frequently used tests. Epidemiological studies have demonstrated the existence of hyperendemic regions where HHV-8 infection is ubiquitous and often acquired early in childhood. The role of HHV-8 in the development of Kaposi's sarcoma has yet to be clarified completely and data collected from the epidemiological studies of HHV-8 prevalence may be useful in gaining a better understanding of the different factors involved in tumour development.