An outbreak of HIV-1 subtype G among Italian injecting drug users.

We describe an outbreak of subtype G among injecting drug users (IDU) in northern Italy newly infected with HIV. We analysed pol gene sequences from samples of 139 individuals from different risk groups. Non-B subtypes were more frequently detected among IDU than in homosexual or heterosexual contacts. All G subtypes but one were found among IDU. The phylogenetic analysis indicated that the outbreak was of monophyletic origin and was caused by HIV-1 strains similar to those from western Africa.

Unexpected drug-drug interaction between tipranavir/ritonavir and enfuvirtide.

Fifty-five patients placed on tipranavir/ritonavir 500/200 mg twice a day (27 with enfuvirtide and 28 without) underwent tipranavir and ritonavir plasma concentration measurements by high-pressure liquid chromatography. Markedly higher tipranavir and ritonavir trough concentrations were observed in enfuvirtide recipients. The modelling of sparse plasma samples using a first order absorption and elimination monocompartmental model without time lag predicted higher tipranavir elimination half-life and volume of distribution in enfuvirtide takers. This unexpected drug-drug interaction warrants further investigation.

Resolution of HCV infection after highly active antiretroviral therapy in a HIV-HCV coinfected patient.

The effect of HAART on HCV infection and HCV-RNA plasma levels is controversial. We describe a patient with HIV-HCV coinfection with persistent disappearance of HCV-RNA after immunological and virological response to HAART, and we briefly discuss similar cases reported in the literature.

Vpr and HIV-1 disease progression: R77Q mutation is associated with long-term control of HIV-1 infection in different groups of patients.

BACKGROUND: Vpr (viral protein R) is a 96 amino acids soluble protein that is expressed late during viral replication. Recent studies have focused on the role of a mutation at position 77 that might be associated with the condition of long-term non-progression, but data are still controversial. PATIENTS AND METHODS: Fifteen long-term non-progressors (LTNP), 19 therapy-naive HIV-1-infected patients with progressive disease (Pr), 23 HIV-1-infected patients receiving sub-optimal therapy with dual nucleoside [nucleoside reverse transcriptase inhibitor (NRTI)] therapy but efficiently controlling viral replication (STP) and 19 antiretroviral therapy multi-experienced patients with actively replicating virus (MEP) were analysed. HIV-RNA was extracted from plasma samples, the Vpr region was amplified, cloned and sequenced. The Pol gene was amplified, directly sequenced and analysed using Sequence Navigator software. RESULTS: A significantly higher prevalence of the R77Q mutation was evidenced both in LTNP (86.7%) and STP (73.9%) in comparison with Pr (42.1%) and MEP (42.1%), (P = 0.007). Comparing groups of patients with progressive disease (Pr + MEP) and groups with non-progressive disease (LTNP + STP) the probability of harbouring the R77Q mutation was significantly higher in non-progressors (odds ratio, 5.16; P = 0.001). CONCLUSIONS: Our results support the hypothesis of the association of R77Q mutation in the Vpr gene with delayed progression of HIV-1 disease. R77Q does not seem to be linked to a particular viral strain but might be associated to immunologic selection. The R77Q mutation might reduce CD4+ T-cell depletion possibly affecting T-cell survival in vivo by altering the pro-apoptotic activity of Vpr.

The presence of anti-Tat antibodies is predictive of long-term nonprogression to AIDS or severe immunodeficiency: findings in a cohort of HIV-1 seroconverters.

The human immunodeficiency virus (HIV) type 1 Tat protein plays a key role in the life cycle of the virus and in pathogenesis and is highly conserved among HIV subtypes. On the basis of this and of safety, immunogenicity, and efficacy findings in monkeys, Tat is being tested as a vaccine in phase 1 trials. Here, we evaluated the incidence and risk of progression to advanced HIV disease by anti-Tat serostatus in a cohort of 252 HIV-1 seroconverters. The risk of progression was lower in the anti-Tat-positive subjects than in the anti-Tat-negative subjects. Progression was faster in the persistently anti-Tat-negative subjects than in the transiently anti-Tat-positive subjects, and no progression was observed in the persistently anti-Tat-positive subjects.

The effect of hepatitis C on progression to AIDS before and after highly active antiretroviral therapy.

OBJECTIVES: To assess the effect of infection with hepatitis C virus (HCV) on the progression of human immunodeficiency virus (HIV) disease, before and after the introduction of highly active antiretroviral therapy (HAART). METHODS: We used data from a multi-centre prospective study of HIV seroconverters. Survival analyses were performed to compare the progression to AIDS by HCV serostatus in the period before HAART (i.e. June 1991-May 1996) and in the HAART era (i.e. June 1996-June 2001), controlling for duration of HIV infection. RESULTS: Among the 1052 persons enrolled, 595 (56.6%) were co-infected; the median follow-up time was 9.7 years. Adjusting for demographic variables (age at HIV seroconversion and gender), HCV infection had no effect on the progression to AIDS in the pre-HAART era [relative hazard (RH) = 0.84; 95% confidence interval (CI), 0.63-1.11], whereas it increased the risk in the HAART era (RH = 1.77; 95% CI, 1.15-2.73). In the HAART era, the proportion of person-time spent on HAART out of the total time at risk was significantly lower among co-infected persons (30 versus 40% for non-co-infected persons; P-value = 0.001); no significant difference was found for dual-therapy (29 versus 25%, respectively; P-value = 0.205); a significant difference was found for mono-therapy (15 versus 8%, respectively; P-value < 0.001). CONCLUSIONS: HCV infection was not a determinant of HIV disease progression in the pre-HAART era, whereas since the introduction of HAART, co-infected individuals seem to have had a faster disease progression. This may in part be explained by differences in person-time spent on different antiretroviral regimens.

The relationship between nevirapine plasma concentrations and abnormal liver function tests.

Abnormal liver function tests are frequently observed in HIV-infected individuals receiving nevirapine (NVP). Here we investigate the relationship between total and unbound plasma concentrations of NVP and the liver enzymes alanine aminotransferase (ALT) and gamma-glutamyl transferase (gammaGT). HIV-infected individuals [n = 85, 22 female, 34 hepatitis C or B virus (HCV or HBV(+))] receiving NVP (200 mg bd; median duration 66 weeks, range 3-189) and two nucleoside reverse transcriptase inhibitors (NRTIs) were enrolled into this study. Blood samples were taken at C(trough) (12 hr postdose) for measurement of NVP and liver function tests (ALT and gammaGT). Plasma protein bound and unbound drug was separated using ultrafiltration and NVP concentrations quantified using HPLC-MS/MS. A linear relationship was observed between total and unbound NVP C(trough) (r(2) = 0.77, p < 0.0001). Patients with elevated ALT (>37 IU/liter; n = 31) had higher NVP unbound C(trough) than those with ALT within the normal range (median 2268 vs. 1694 ng/ml, p = 0.04) but there was no difference in total C(trough). Logistic regression revealed no association between higher NVP C(trough) and ALT elevations. Significantly higher NVP total and unbound C(trough) were observed in patients with increased gammaGT (>40 IU/liter; n = 63; total 6747 vs. 4530 ng/ml, p = 0.001; unbound 2113 vs. 1557 ng/ml, p = 0.03). Significantly higher unbound NVP C(trough) was observed in HCV/HBV(+) (median 2275 vs. 1726 ng/ml, p = 0.02) and on bivariate analysis, higher NVP C(trough) was associated with HCV/HBV coinfection (chi(2) = 4.228; p = 0.04). Overall we found no strong association between NVP concentrations and hepatotoxicity. Although in this study NVP was well tolerated in HCV/HBV coinfected patients, higher plasma concentrations were observed.

Lopinavir protein binding in vivo through the 12-hour dosing interval.

Most protease inhibitors available for the treatment of human immunodeficiency virus (HIV) infection are highly bound to plasma proteins, mainly alpha-1 acid glycoprotein. Therapeutic drug monitoring (TDM) of total protease inhibitor (PI) concentrations has been increasing in the past few years; however, the pharmacological activity of the PIs is dependent on unbound drug entering cells harboring HIV. There is little information available on unbound drug concentrations of these drugs in vivo. The aim of the study was to measure unbound plasma concentrations of lopinavir (LPV) and to relate them to the total plasma concentrations to establish the unbound percentage in vivo during a full dosage interval. A pharmacokinetic study was performed in HIV-infected subjects (n = 23; median CD4 cell count = 290 x 10(6) cells x L(-1); viral load < 50 copies x mL(-1)) treated with a LPV/ritonavir (RTV)-containing regimen. Ultrafiltration was used to separate unbound LPV in all plasma samples (n = 115). Equilibrium dialysis was also used to compare with ultrafiltration measurements in 10/23 patients at baseline and 2 hours after drug intake. Total and unbound LPV concentrations were measured by a fully validated method using high-performance liquid chromatography-mass spectometry (HPLC-MS/MS). Based on a comparison of AUC(unbound)AUC(total), the mean (+/- SD) unbound percentage of LPV from all the samples studied (n = 115) was 0.92% (+/- 0.22) when measured with ultrafiltration and 1.32% (+/- 0.44) when equilibrium dialysis was used (n = 20), showing a higher drug recovery (P = 0.048). The unbound percentage of LPV was found to be significantly higher after 2 h than at baseline (P < 0.05 with both methods), suggesting a concentration-dependent binding of LPV that has not been observed in vitro. However, the clinical significance of such phenomena is still unclear.

Clearance of 14-3-3 protein from cerebrospinal fluid heralds the resolution of bacterial meningitis.

The 14-3-3 protein, a cerebrospinal fluid (CSF) marker of neuronal damage that was recently adopted for the diagnosis of Creutzfeldt-Jakob disease, is also found in the CSF of patients with a variety of neurological disorders. We prospectively studied 12 consecutive patients with purulent bacterial meningitis and found that 14-3-3 protein was detected in all patients at admission to the hospital. All patients who recovered cleared 14-3-3 protein from the CSF before discharge from the hospital (this was the first CSF marker to clear), whereas those who died never cleared the protein.

Identifying recent HIV infections using the avidity index and an automated enzyme immunoassay.

We evaluated a procedure for identifying recent HIV infections, using sequential serum samples from 47 HIV-positive persons for whom the seroconversion date could be accurately estimated. Each serum sample was divided into two aliquots: one diluted with phosphate-buffered saline and the other diluted with 1 M guanidine. We assayed the aliquots with the automated AxSYM HIV1/2gO test (Abbott Diagnostics Division), without modifying the manufacturer's protocol. We then calculated the avidity index (AI): the ratio of the sample/cutoff value for the guanidine aliquot to that of the phosphate-buffered saline aliquot. We analyzed 216 serum samples: 34 samples were collected within 6 months of seroconversion (recent seroconversions), and 182 were collected after 6 months. The mean AIs, by time from seroconversion, were 0.68 +/- 0.16 (within 6 months) and 0.98 +/- 0.10 (after 6 months) (P < 0.0001). AI of <0.90 correctly identified 88.2% of recent infections but misclassified as recent infections 13.2% of serum samples collected afterward. The probability of an infection being classified as recent and having AI of > or = 0.90 would be 0.7% in a population with 5% recent infections. AI can identify with a certain degree of accuracy recent HIV infections, and being a quantitative index, it provides different levels of sensitivity and specificity, depending on the selected cutoff value. The standard assay procedure is not modified. This test is simple and inexpensive and could be used for surveillance, decision-making in treatment, and prevention.

Pharmacokinetics of saquinavir co-administered with cimetidine.

The present study evaluated the effect of cimetidine, a histamine H(2) receptor antagonist able to inhibit cytochrome P450 metabolism, on the steady-state pharmacokinetics of saquinavir soft gel. Twelve healthy volunteers (eight males and four females) participated in an open-label, double-phase pharmacokinetic study. Volunteers took saquinavir soft gel 1200 mg three times a day for 13 days and then saquinavir soft gel 1200 mg twice a day with cimetidine 400 mg twice a day from day 14 to 26. The pharmacokinetics of saquinavir on days 13 and 26 were compared. All 12 volunteers completed the study. The association of cimetidine with saquinavir soft gel 1200 mg twice a day resulted in a significant increase in saquinavir AUC(0-24) (120%; P = 0.023) and C(max) (179%; P = 0.019), whereas C(trough) did not differ significantly (32% increase; P = 0.272). Increased exposure to saquinavir was observed in healthy volunteers after co-administration with cimetidine. The most significant increase involved C(max). Further pharmacokinetic studies in HIV-infected subjects are warranted to confirm the boosting effect of cimetidine and to investigate any impact that the increase in saquinavir C(max) may have on intracellular accumulation of the drug.

Precision and accuracy of a procedure for detecting recent human immunodeficiency virus infections by calculating the antibody avidity index by an automated immunoassay-based method.

We evaluated the precision and accuracy of a procedure for detecting recent human immunodeficiency virus (HIV) infections, specifically, the avidity index (AI) calculated using a method based on an automated AxSYM HIV 1/2gO assay (Abbott). To evaluate precision, we performed multiple replicates on eight HIV-positive serum samples. To evaluate the accuracy in identifying recent infections (i.e., within 6 months of seroconversion), we used 216 serum samples from 47 persons whose dates of seroconversion were known. To evaluate the sensitivity and specificity of the procedure for different AI cutoff values, we performed receiver operating characteristic (ROC) analysis. To determine the effects of antiretroviral treatment, advanced stage of the disease (i.e., low CD4-cell count), and low HIV viral load on the AI, we analyzed 15 serum samples from 15 persons whose dates of seroconversion were unknown. The precision study showed that the procedure was robust (i.e., the total variance of the AI was lower than 10%). Regarding accuracy, the mean AI was significantly lower for samples collected within 6 months of seroconversion, compared to those collected afterwards (0.68 +/- 0.16 versus 0.99 +/- 0.10; P < 0.0001), with no overlap of the 95% confidence intervals. The ROC analysis revealed that an AI lower than 0.6 had a sensitivity of 33.3% and a specificity of 98.4%, compared to 87.9 and 86.3%, respectively, for an AI lower than 0.9. Antiretroviral treatment, low CD4-cell count, and low viral load had no apparent effect on the AI. In conclusion, this procedure is reproducible and accurate in identifying recent infections; it is automated, inexpensive, and easy to perform, and it provides a quantitative result with different levels of sensitivity and specificity depending on the selected cutoff.

Clinical use of lopinavir/ritonavir in a salvage therapy setting: pharmacokinetics and pharmacodynamics.

Lopinavir/ritonavir was administered to 35 HIV-infected patients after therapeutic failure with other protease inhibitors. The pharmacokinetics (trough concentrations) and baseline viral genotype were determined, together with the immunovirological outcome. The 22 responders had significantly higher mean lopinavir concentrations and lower baseline numbers of mutations. On multivariate analysis, a lopinavir concentration of 5.7 microg/ml or greater was an independent predictor of viral suppression over a 9-month follow-up period.

The unbound percentage of saquinavir and indinavir remains constant throughout the dosing interval in HIV positive subjects.

AIMS: To measure the unbound plasma concentrations of saquinavir (SQV) and indinavir (IDV) and to relate them to the total plasma concentrations in order to establish the unbound percentage of protease inhibitors in vivo during a full dosage interval profile. METHODS: HIV-infected subjects (n = 35; median CD4 cell count = 340 x 10(6) cells l-1, range: 120-825; viral load < 50 copies ml-1 in 22/35) treated with SQV or IDV containing regimens were studied. Plasma drug samples were collected at 0, 2, 4, 8 and 12 h postdose for the twice daily regimens and 0, 1, 2, 4 and 8 h for the three times daily regimens. Ultra-filtration was used to separate unbound IDV and SQV in plasma and their respective concentrations were measured by a fully validated method using high performance liquid chromatography-mass spectometry (h.p.l.c.-MS/MS). RESULTS: Based on the ratio AUCunbound/AUCtotal, the median unbound percentage (95% CI for differences) of SQV and IDV from all the samples studied was 1.19% (0.99, 1.58%) and 36.3% (35.1, 44.2%), respectively. No significant difference was seen in the percentage binding of SQV between patients receiving SQV alone (median = 1.49%) or with ritonavir (median = 1.09%; P = 0.141; 95% CI for difference between medians = -0.145, 0.937) over the pharmacokinetic profile. Similarly, no significant difference was seen in the percentage binding of IDV in patients receiving IDV alone (median 35.2%) or with ritonavir (median = 41.3%; P = 0.069; 95% CI for difference between medians = -0.09, 15.4). The unbound concentrations of SQV (P < 0.0001; 95% CI for r(2) = 0.634, 0.815) and IDV (P < 0.0001; 95% CI for r(2) = 0.830, 0.925) remained constant as a proportion of total concentration over the full dosing profile. CONCLUSIONS: These in vivo data confirm previously published in vitro measurements of SQV and IDV protein binding. The unbound percentage of both protease inhibitors remained constant over the dosing interval.