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ObjectivesOur aim was to investigate the durability of different initial regimens in patients starting ART with CD4+ counts 5 log10 copies/mL.MethodsThis was a retrospective study of HIV-infected patients prospectively followed in the ICONA cohort. Those who started ART with boosted protease inhibitors (bPIs), NNRTIs or integrase strand transfer inhibitors (InSTIs), with CD4+ 5 log10 copies/mL, were included. The primary endpoint was treatment failure (TF), a composite endpoint defined as virological failure (VF, first of two consecutive HIV-RNA >50 copies/mL after 6 months of treatment), discontinuation of class of the anchor drug or death. Independent associations were investigated by Poisson regression analysis in a model including age, gender, mode of HIV transmission, CDC stage, HCV and HBV co-infection, pre-treatment HIV-RNA, CD4+ count and CD4+/CD8+ ratio, ongoing opportunistic disease, fibrosis FIB-4 index, estimated glomerular filtration rate, haemoglobin, platelets, neutrophils, calendar year of ART initiation, anchor drug class (treatment group) and nucleos(t)ide backbone.ResultsA total of 1195 patients fulfilled the inclusion criteria: 696 started ART with a bPI, 315 with an InSTI and 184 with an NNRTI. During 2759 person-years of follow up, 642 patients experienced TF. Starting ART with bPIs [adjusted incidence rate ratio (aIRR) (95% CI) 1.62 (1.29–2.03) versus starting with NNRTIs; P ConclusionsIn patients starting ART with 5 log10 HIV-RNA copies/mL, the durability of regimens based on InSTIs was longer than that of NNRTI- and bPI-based regimens.

Introduction Nowadays, the treatment of hypertension represents an important issue, particularly in developed countries. While in most cases the standard therapeutic approaches, consisting in the administration of 1 to 3 drugs, are adequate to reach adequate blood pressure levels, in some cases more drugs are needed: this condition is called “resistant hypertension”. In this context, the administration of a diuretic, such as spironolactone or canrenoate salts, represents a standard practice. Since a reliable discrimination of real cases of resistant hypertension from cases of poor therapeutic adherence is currently difficult to obtain, the adoption of therapeutic drug monitoring has been suggested as a useful tool for this purpose. In this work, the authors developed and validated a simple, cheap and fast dilute-and-shot method with UHPLC-PDA analysis for the quantification of spironolactone and its metabolite canrenone in human urine samples. Methods Standards and quality controls were prepared in urine. Only 100 μL of sample were added with 80 μL of internal standard (6,7-dimethyl-2,3-di(2-pyridyl)quinoxaline) working solution and 820 μL of phosphate buffer 10 mM pH 3.2 (phase A):acetonitrile (phase B) 90:10 v:v solution. Chromatographic separation was performed on an Acquity® UPLC HSS T3 1.8 μm 2.1 × 150 mm column, with a binary gradient for 11 min at 40 °C. Results Accuracy, intra-day and inter-day precision, selectivity and sensitivity fitted FDA guidelines for all analytes (LLOQ and LOD were 156.25 ng/mL and 78.12 ng/mL, respectively, for both analytes) and recovery resulted high and reproducible. Method performances were tested on urine samples from hypertensive patients with good results. Discussion This simple analytical method could represent a useful tool for the management of antihypertensive therapy.

Maraviroc is a CCR5 inhibitor approved in 2007 for treatment of therapy experienced adult patients infected with CCR5-tropic HIV-1 virus. International guidelines for HIV therapy indicate a plasma concentration cutoff of maraviroc for response. We developed and validated a new HPLC-MS method to quantify maraviroc concentrations in human plasma. 6,7-Dimethyl-2,3-di(2-pyridyl)quinoxaline was used as internal standard and added to 100μL of plasma. Samples were then treated with 500μL of acetonitrile for the protein precipitation procedure. An analytical T3 Atlantis column (150mm×4.6mm i.d.) with a particle size of 5μm was used to separate the compounds and ions were detected at m/z 257.5 and 313.3 for maraviroc and quinoxaline respectively. The calibration curve was linear up to 2500ng/mL. The mean recovery of maraviroc was 89.1%. All validation data results were in accordance to Food and Drug Administration and European Medicines Agency requirements. The HPLC-MS method reported here could be used routinely to monitor plasma concentrations of maraviroc in HIV-infected patients.

Objectives: Ritonavir, used at low doses as a boosting agent of other protease inhibitors (PIs), is known to be associated with metabolic complications and gastrointestinal disturbances. The rate of accumulation of ritonavir within cells is still debated due to scarce data and methodological limitations. Therefore, our aim was to evaluate intracellular ritonavir penetration when used with different boosted PIs in the clinical setting. Methods: Patients administered with atazanavir/ritonavir (300/100 mg, once daily), darunavir/ritonavir [600/ 100 mg, twice daily (darunavir-600) and 800/100 mg, once daily (darunavir-800)], lopinavir/ritonavir (400/ 100 mg, twice daily) and tipranavir/ritonavir (500/200 mg, twice daily) were considered. Blood sampling at the end of the dosing interval (Ctrough) was performed. Peripheral blood mononuclear cell (PBMC)-associated and plasma ritonavir and PI concentrations were measured by validated HPLC methods. PBMC count and individual mean cell volume (MCV) were measured using a Coulter Counter instrument. Results: One hundred patients were enrolled. Frequencies of ritonavir-boosted PIs were atazanavir, 37%; darunavir-600, 23%; lopinavir, 19%; tipranavir, 13%; and darunavir-800, 8%. The median intracellular and plasma concentrations of ritonavir were 1279 ng/mL (IQR 727– 2087) and 170 ng/mL (IQR 82– 384), respectively, accounting for a cellular accumulation ratio of 7.69 (5.7 –10.9). Significant differences in ritonavir intracellular concentrations emerged among different PIs (P,0.001): specifically between darunavir-600 and atazanavir (P,0.001), between darunavir-600 and tipranavir (P ¼ 0.009), between atazanavir and lopinavir (P,0.001) and between lopinavir and tipranavir (P ¼ 0.027). Conclusions: Our study showed a higher rate of ritonavir intracellular accumulation than previously reported, possibly due to the more accurate calculation of intracellular concentrations by MCV. The ratio varied according to concomitantly administered PIs, suggesting their influence on the rate of ritonavir intracellular penetration.

Ribavirin-induced hemolytic anemia is the main cause of discontinuation of the combination therapy with alpha-interferon-2b and ribavirin for the treatment of hepatitis C virus (HCV) infection. The determination of intracellular ribavirin levels in blood, including the levels of its phosphorylated metabolites, might be useful for predicting ribavirin-induced anemia, which could be caused by the accumulation of these molecules within the erythrocytes. In this work, we simplified and validated a previously developed assay method, to make it suitable for routine monitoring of cellular ribavirin. Whole blood diluted with a five-fold volume of ice-cold distilled underwent a process of acid phosphatase digestion to convert phosphorylated ribavirin metabolites to free ribavirin. The resulting mixture, spiked with an internal standard, was treated with a protein precipitation protocol in acetonitrile, followed by reverse-phase high-performance liquid chromatography analysis. The calibration curve for ribavirin levels in whole blood was linear at concentrations from 625 to 320,000 ng/mL (r(2)=0.998). Accuracy, intra-day and inter-day precision for ribavirin and phosphorylated-ribavirin quality controls were all below 9.0%. We tested this method by monitoring blood ribavirin concentrations in 13 HCV+ patients, receiving alpha interferon-plus ribavirin combination therapy.

BACKGROUND Functional variants of inosine triphosphatase (ITPA) were recently found to protect against ribavirin (RBV)-induced hemolytic anemia. However, no definitive data are yet available on the role of plasma RBV concentrations on hemoglobin (Hb) decrement. Moreover, no data have been published on the possible interplay between these 2 factors. METHODS A retrospective analysis included 167 patients. The ITPA variants rs7270101 and rs1127354 were genotyped and tested using the χ test for association with Hb reduction at week 4. We also investigated, using multivariate logistic regression, the impact of RBV plasma exposure on Hb concentrations. RESULTS Both single nucleotide polymorphisms were associated with Hb decrease. The carrier of at least 1 variant allele in the functional ITPA single nucleotide polymorphisms was associated with a lower decrement of Hb (-1.1 g/dL), as compared with patients without a variant allele (-2.75 g/dL; P = 4.09 × 10). RBV concentrations were not influenced by ITPA genotypes. A cut-off of 2.3 μg/mL of RBV was found to be associated with anemia (area-under-receiver operating characteristic = 0.630, sensitivity = 50.0%, and specificity = 69.5%, P = 0.008). In multivariate logistic regression analyses, the carrier of a variant allele (P = 0.005) and plasma RBV concentrations

A high-performance liquid chromatography method with UV detection was developed and validated for the simultaneous quantification of linezolid (LZD), rifampicin (RFP), levofloxacin (LEVO), and moxifloxacin (MOXI) in human plasma. The method is based on a simple organic protein precipitation that guarantees rapid sample preparation and a direct injection into the high-performance liquid chromatography system. The use of quinoxaline as internal standard improved accuracy (relative standard deviation, RSD%14.9%) and precision (RSD%14.3%). The recovery was 75.9% (RSD% = 5.8). The limits of quantification were 0.234 microg/mL for LEVO, 0.312 microg/mL for LZD, 0.156 microg/mL for MOXI, and 0.622 microg/mL for RFP. This method allows the simultaneous measurement of LEVO, LZD, MOXI, and RFP in human plasma and may be used for both routine clinical applications and pharmacokinetic studies.

An improved HPLC fluorimetric method for the quantification of enfuvirtide in plasma of HIV-infected subjects was described and validated. The use of an internal standard improved the reproducibility and precision of the analysis. Our method showed lower limits of detection and quantification (LOD = 32 ng/mL, LOQ = 78 ng/mL), lower intraday (RSD% 1.25-2.95) and interday (RSD% 1.75-4.69) coefficients of variation, greater recovery (>100%), lower duration (16 minutes) and lower cost than previously described fluorimetric methods. Therefore, this method can be used as a reliable tool for pharmacokinetic studies of enfuvirtide.

Tenofovir disoproxil fumarate is a prodrug of tenofovir used in the treatment of HIV and HBV infections: it is the most used antiretroviral worldwide. Tenofovir is nucleotidic HIV reverse trascriptase inhibitor that showed excellent long-term efficacy and tolerability. However renal and bone complications (proximal tubulopathy, hypophosphatemia, decreased bone mineral density, and reduced creatinine clearance) limit its use. Tenofovir renal toxicity has been suggested as the consequence of drug entrapment in proximal tubular cells: measuring tenofovir urinary concentrations may be a proxy of this event and it may be used as predictor of tenofovir side effects. No method is currently available for quantifying tenofovir in this matrix: then, the aim of this work was to validate a new LC-MS method for the quantification of urinary tenofovir. Chromatographic separation was achieved with a gradient (acetonitrile and water with formic acid 0.05%) on an Atlantis 5 μm T3, 4.6 mm × 150 mm, reversed phase analytical column. Detection of tenofovir and internal standard was achieved by electrospray ionization mass spectrometry in the positive ion mode. Calibration ranged from 391 to 100,000 ng/mL. The limit of quantification was 391 ng/mL and the limit of detection was 195 ng/mL. Mean recovery of tenofovir and internal standard were consistent and stable, while matrix effect resulted low and stable. The method was tested on 35 urine samples from HIV-positive patients treated with tenofovir-based HAARTs and did not show any significant interference with antiretrovirals or other concomitantly administered drugs. All the observed concentrations in real samples fitted the calibration range, confirming the capability of this method for the use in clinical routine. Whether confirmed in ad hoc studies this method may be used for quantifying tenofovir urinary concentrations and help managing HIV-positive patients treated with tenofovir.

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.

Objectives The rate of accumulation of atazanavir and ritonavir within cells is still debated due to methodological limitations. Our aim was to measure peripheral blood mononuclear cell (PBMC) concentrations of atazanavir and ritonavir and investigate whether single-nucleotide polymorphisms of OATP, ABCB1, CYP3A4 and PXR genes are involved in intracellular drug penetration. Methods HIV-positive patients administered 300 mg of atazanavir/100 mg of ritonavir were enrolled. Blood sampling was performed at the end of the dosing interval (Ctrough). PBMC-associated and plasma atazanavir and ritonavir concentrations were measured by validated HPLC coupled with a single mass detector (HPLC-MS) and HPLC-photodiode array (PDA) methods, respectively. Cell count and mean cellular volume were determined using a Coulter counter. Genotyping was conducted using real-time PCR. Results Thirty-five patients were enrolled. Median atazanavir and ritonavir intracellular concentrations were 1844 and 716 ng/mL, respectively. Median plasma concentrations were 645 ng/mL for atazanavir and 75 ng/mL for ritonavir, while median intracellular/plasma concentration ratios were 2.4 and 9.2, respectively. Median ritonavir intracellular concentrations were higher for OATP1B1 521 T→C TC or CC carriers and for PXR 44477 A→G AG or GG carriers. Atazanavir intracellular/plasma concentration ratios were higher in patients GG for the ABCB1 2677 G→T single-nucleotide polymorphism (SNP) compared with GT and TT groups. Conclusions Our study showed a higher intracellular ritonavir accumulation than previously reported. Ritonavir intracellular concentrations were associated with OATP1B1 521 and PXR 44477 SNPs while intracellular atazanavir exposure was associated with the ABCB1 2677 SNP. Further clinical studies are necessary in order to confirm these data.

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.

We studied a cohort of 299 HIV-positive individuals with known date of seroconversion to evaluate the role of Cytomegalovirus (CMV) in the natural history of HIV. The study population consisted of 236 initially CMV-positive patients, 55 CMV-negative subjects and 8 CMV seroconverters. The study endpoints were the decline to CD4+200 x 10(6) cells/l, AIDS, and death. The cumulative risk of CMV disease and the survival after CMV disease were also investigated. At intake, there was no inter-group difference in sex, age, risk behaviours, history of hairy leucoplakia or herpes zoster and antiretroviral treatment. During the follow-up, 108 patients fell below 200 CD4+ x 10(6) cells/l, 72 developed AIDS and 63 died. Twenty-one subjects had CMV disease. The cumulative incidence of CMV disease in the cohort was 18.9%, and 23.3% within 8 and 9 years for the initially CMV-positive patients and 33.3% and 66.7% for the CMV seroconverters (log-rank test: p = 0.101). The median survival after CMV disease was 153 days (range: 28-855, interquartile range: 261), with a cumulative survival of 45.1%, 16.9% and 4.3% within 6, 12 and 18 months, respectively. On Cox's regression, the acute HIV seroconversion was an independent predictor of each endpoint, history of hairy leucoplakia or herpes zoster being associated only with CD4+ cell decline. Baseline CMV seropositivity was related to short survival (p = 0.037) and 2 x 2 inter-group comparison showed that older individuals with sexually acquired HIV who seroconverted to CMV had higher rates of progression to the study endpoints. Our data suggest that CMV infection influences the natural history of HIV disease and that CMV disease strongly affects the survival of the HIV-positive patients.

Between March 1986 and March 1994, the seroconversion to HBV associated to the seroconversion to HIV was investigated in 120 HIV seroconverters drawn from 2368 i.v. drug misusers screened for HIV, HBV and STDs. Among the 185 individuals susceptible to HIV and HBV at intake (41/120 HIV seroconverters and 144/364 HIV-negative controls), HBV seroconversion was associated with the seroconversion to HIV (p = 0.006) and history of more than 3 sexual partners per year (p = 0.000). Only the history of more than 3 partners per year remained associated with the HBV seroconversion in the conditional regression. The associated seroconversion to HIV and HBV was linked to the short period of i.v. drug injections (p = 0.032), history of more than 3 partners per year (p = 0.000) and more than 3 i.v. drug injections per day (p = 0.016). Compared to the seroconverters to HBV alone, the seroconverters to HBV and HIV were likely to have higher frequency of i.v. drug injection per day on univariate (p = 0.031) and multivariate analysis (p = 0.024). The seroconverters to both the viruses differed from the seroconverters to HIV alone in the year of drug debut (p = 0.045), short period of i.v. drug use (p = 0.048) and high frequency of injection per day (p = 0.008). The multivariate analysis confirmed only the association with high frequency of injection per day (p = 0.033). Higher risk of HIV seroconversion from the debut of i.v. drug use was observed in the subjects with concurrent HBV seroconversion (Log-Rank test: p = 0.0008).

The response rate to treatment of chronic hepatitis C virus-genotype 1 and 4 infections was recently found to be strongly influenced by many polymorphisms. The aim of our study was to carry out an integrated analysis of the effects of polymorphisms and ribavirin (RBV) plasma exposure on outcome.The retrospective analysis included 174 patients. IL28B, CYP27B1, SLC29A1, SLC28A3, and SLC28A2 polymorphisms were genotyped and tested for association with sustained virological response. The impact of RBV plasma exposure during the first 3 months of therapy on outcome was also investigated.Considering patients infected by hepatitis C virus-1/4, 3 polymorphisms (IL28B rs8099917TT, CYP27B1 rs4646536TT, and CNT2 rs11854484TT) were associated with sustained virological response. The number of negative variant allele and low RBV exposure were correlated to percentage increasing to therapy failure, suggesting some degree of cumulative effect of the 4 factors. A cutoff of 2.5 μg/mL of RBV was found to be associated with outcome (area under ROC [AUROC] curve = 0.64, sensitivity = 55.0%, and specificity = 71.2%, P = 0.020). In multivariate logistic regression analyses, each variant allele and RBV plasma exposure cutoff were independently associated with outcome.In this study, we found that additional polymorphisms and RBV plasma exposure are also able to influence the achievement of response. Regardless of the magnitude of RBV pharmacokinetic exposure, the negative predictive value of the polymorphisms here investigated is much stronger than the positive one.

The mean corpuscular volume (MCV) of peripheral blood mononuclear cells (PBMCs) was determined by Coulter Counter, and data were used to calculate the intracellular drug concentrations. A total of 574 PBMC samples were collected from 190 patients. The MCV was 282.9 fl (minimum, 207.0; maximum, 354.6), with a standard deviation of 8.8%. Previous reports have often used a fixed value of 400 fl for the MCV, which may result in artificially low estimates of the intracellular concentrations of antivirals.

A sensitive and accurate high performance liquid chromatography–mass spectrometric (HPLC–MS) method for the intracellular determination of 14 antiretroviral drugs in peripheral blood mononuclear cells (PBMCs) for HIV+ patients was validated. PBMCs are isolated by Ficoll density gradient centrifugation and cells count and the relative mean volume is performed with a Coulter ® instrument. Extraction of drugs from PBMCs pellets was obtained with methanol:water (70:30, v/v), with quinoxaline added as internal standard, after a sonication step. Supernatant was dried and then dissolved in water/acetonitrile (60/40, v/v), before injection into a 2.1 mm × 150 mm Atlantis ® T3 3μ column. Chromatographic separations were performed using a gradient program with a mixture of water (0.05% formic acid), as mobile phase A and acetonitrile (0.05% formic acid), as mobile phase B. Analytes quantification was performed by electro-spray ionisation–single quadrupole mass spectrometry using the selected ion recording (SIR) detection mode. The positive ionization was used for the HIV protease inhibitors (PIs) indinavir, saquinavir, nelfinavir, nelfinavir M8 metabolite, amprenavir, darunavir, atazanavir, ritonavir, lopinavir, tipranavir, the integrase inhibitor (II) raltegravir and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and etravirine, while the negative ionization is applied for efavirenz. The calibration curves were built using blank PBMCs spiked with antiretroviral drugs at concentrations ranging from 0.1 to 32 ng/mL (1–320 ng/mL for tipranavir) and fitted to a quadratic regression model weighted by 1/ X . The mean extraction recovery for all PIs, II and NNRTIs was always above 82%. The method was precise, with a range of intra/inter-day percent standard deviation within 2.6–14.8%, and accurate with mean of percent coefficient of variation (CV%) from nominal values −7.85 to +9.7%. Each drug concentration evaluated was expressed in ng/mL and optimized using each patient medium corpuscolar volume and cell number. This analytical method is routinely used in our clinical research center for the assessment of intracellular levels of all PIs, raltegravir and NNRTIs commercially available at present.

To investigate the relationship between cytokine serum levels, peripheral blood lymphocyte subsets and clinical picture in acute primary HIV-1 infection.Absolute number/microliters total lymphocytes, CD4+, CD8+ and natural killer (NK) cells, as well as serum levels of soluble CD8 receptor, interleukin (IL)-1 beta, IL-2, IL-4, IL-6, tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, beta 2-microglobulin and 5'-neopterin were determined in 15 patients with acute primary HIV-1 infection, 16 asymptomatic HIV-1-seropositive individuals and 18 HIV-1-seronegative individuals at risk for HIV-1 infection.Acute primary HIV-1 infection was characterized by significant CD4+ lymphocytopenia with low IL-2 serum concentrations, and by high absolute number of circulating CD8+ and NK cells, with elevated serum levels of soluble CD8 receptor, IL-1 beta, IFN-gamma and 5'-neopterin. Follow-up of acute seroconverters showed a significant decrease in NK cell counts and IL-1 beta levels, with an increase of IL-6.In acute primary HIV-1 infection, significant alteration of cytokine release, possibly induced by viral antigens, could be responsible for both clinical picture and activation of cytotoxic cells through abnormal mechanisms.

We have developed and validated a high-performance liquid chromatography method coupled with a mass detector to quantify itraconazole, voriconazole, and posaconazole using quinoxaline as the internal standard. The method involves protein precipitation with acetonitrile. Mean accuracy (percent deviation from the true value) and precision (relative standard deviation percentage) were less than 15%. Mean recovery was more than 80% for all drugs quantified. The lower limit of quantification was 0.031 μg/ml for itraconazole and posaconazole and 0.039 μg/ml for voriconazole. The calibration range tested was from 0.031 to 8 μg/ml for itraconazole and posaconazole and from 0.039 to 10 μg/ml for voriconazole.

A simultaneous extraction method to measure daptomycin, amikacin, gentamicin, and rifampicin in human plasma, by high-performance liquid chromatography, was developed and validated. The method involved a rapid sample preparation by protein precipitation with acetonitrile followed by direct injection into a high-performance liquid chromatography system coupled with mass detection. Drug retention times were 10.00 +/- 0.25, 2.00 +/- 0.25, 3.50 +/- 0.25, 11.50 +/- 0.25, and 12.50 +/- 0.25 min for daptomycin, amikacin, gentamicin, rifampicin, and quinoxaline, respectively. Good linearity (mean r(2) = 0.998) was obtained for all drugs quantified over the range of clinically relevant concentrations in human plasma and the use of the internal standard quinoxaline improves accuracy (RSD%

A bioanalytical method for the determination of most commonly prescribed protease inhibitors (saquinavir, atazanavir, amprenavir, darunavir, lopinavir and ritonavir) and non-nucleoside reverse transcriptase inhibitors (etravirine, efavirenz and nevirapine) was developed, modifying our previous HPLC–MS chromatographic run, validated and a complete short and long term stability evaluation was carried out. One hundred microlitres of plasma were distributed on a collection glass paper filter (Glass-Microfibre from Sartorius), then the filter underwent thermal treatment, both for drying and for HIV inactivation, and stored at room temperature, 4 °C and −20 °C. The analytes were extracted from the filter disc using tert -butylmethylether with basic pH, after the addition of the internal standards quinoxaline. The extract was dried, reconstituted and the chromatographic separation was performed on a reversed-phase C-18 column (150 mm × 2.0 mm) and the analytes were quantified using a single quadrupole mass spectrometer. The method was validated considering the concentration ranges encountered in clinical trials and the routine clinical practice. The assay was linear over the concentration ranges tested. Accuracies ranged from 92.1% to 111.9% and intra-day and inter-day relative standard deviation for all quality control levels ranged from 0.2 to 12.9 and 3.1 to 14.4, respectively. Analytes in dried plasma spots were stable for longer time when dried/inactivation step was carried out before storage compared to samples not dried/inactivated before the analysis. The dried/inactivation step allows shipment of samples at room temperature without any risks, therefore the developed and validated method enables an easy and cheap sample shipment for therapeutic drug monitoring and pharmacokinetic studies.

Maraviroc is the first commercialized CCR5 inhibitor for HIV therapy. A new high-performance liquid chromatography-ultraviolet method to quantify maraviroc concentrations in human plasma was developed and validated. The method is based on a protein precipitation procedure, with an acidic solution of acetonitrile (trifluoroacetic acid 0.1%) and quinoxaline as internal standard. The analytes were eluted using a gradient run in 15 minutes on an analytical C18 Luna column (150 mm × 4.6 mm ID) with a particle size of 5 μm. Maraviroc and internal standard were detected by UV at 193 nm and 352 nm, respectively. The calibration curve was linear up to 2500 ng/mL. The mean recovery of maraviroc was 96%. All validation data were in accordance with U.S. Food and Drug Administration requirements. The new high-performance liquid chromatography-ultraviolet method reported here could be used routinely to monitor plasma concentrations of maraviroc in healthy volunteers and HIV-infected patients.

This study investigated pregnane X receptor polymorphisms in relation to unboosted atazanavir plasma concentrations in 2 cohorts of patients. The polymorphism 63396T--C predicted concentrations below the minimum effective concentration (150 ng/mL) with odds ratios of 18 (P = .008) and 5.13 (P = .02). Prospective studies determining potential clinical usefulness are now warranted.

A new solid-phase extraction (SPE) method has been developed and validated on a liquid chromatography (LC) coupled with a mass spectrometer for the determination of plasma concentrations of tenofovir (TNF) and emtricitabine (FTC) in HIV infected patients. Chromatographic separation was achieved with a gradient (acetonitrile and water with formic acid 0.05%) on an Atlantis 4.6 mm x 150 mm, reversed phase analytical column. Detection of TNF, FTC, and internal standard (IS) was achieved by electrospray ionization mass spectrometry (ESI-MS) in the positive ion mode. Calibration ranged from 15.6 to 4000 ng/mL for TNF and 11.7 to 3000 ng/mL for FTC. Plasma was analyzed, and the limit of quantitation was 15.6 ng/mL for TNF and 11.7 ng/mL for FTC; limit of detection was 2 ng/mL for TNF and 1.5 ng/mL for FTC. Mean recovery of TNF, FTC, and IS were 46.5% [relative standard deviation (RSD): 8.8%] and 88.8% (RSD: 1.0%), and 81.7% (RSD: 3.1%), respectively. The method did not show any significant interference with antiretrovirals or other concomitant drugs administered to patients, and no significant "matrix effects" were observed. The method was applied for the determination of antiretroviral plasma concentration of HIV-positive patients treated with FTC and/or TNF, in combination with various other antiretrovirals.

Early virological response (VR) to enfuvirtide-based salvage regimens at week 12 has been described as a predictor of long-term therapeutic success. The relationship between enfuvirtide plasma exposure and VR has not yet been investigated in the clinical setting. Our aim was to investigate the role of enfuvirtide plasma exposure as a determinant of early VR in the clinical setting.Forty-two multidrug-experienced patients starting a salvage enfuvirtide-based regimen were prospectively evaluated over a 12 week period. HIV-RNA levels and enfuvirtide C(trough) were regularly measured. VR was considered as achievement of viral load (VL) undetectability and/or a decrease of more than 1 log at week 12.Optimized background score (OBS) and enfuvirtide C(trough) concentrations were associated with VL decrease at week 12. An OBSor =2 and enfuvirtide C(trough)2100 ng/mL were associated with VR. The pharmacokinetic/pharmacodynamic (PK/PD) analysis confirmed this exposure-response relationship both in the total population and in different groups according to OBS2 oror =2. Higher estimates of IC(50) were calculated for the OBS2 group when compared with the OBSor =2 group (7551 versus 2330 ng/mL, respectively), without a marked difference in I(0) (0.31 versus 0.21 log) and I(max) (-2.64 versus -3.33 log).Enfuvirtide plasma exposure and OBS were found to significantly influence the magnitude and rate of early VR. The PK/PD modelling of enfuvirtide concentrations was different in our clinical setting, compared with previous data obtained under trial conditions. Therefore, optimization of enfuvirtide plasma exposure could deserve further evaluation as a determinant of therapeutic response in HIV-positive patients.

A high-performance liquid chromatography method for the determination of ertapenem in human plasma using ultraviolet detection (300 nm) was developed and validated. The method involved a simple organic protein precipitation that guarantees rapid sample preparation. By using meropenem as internal standard, it also guarantees improved precision (relative standard deviation

A new method using high-performance liquid chromatography coupled with photo diode array detection was developed and validated for the quantification of plasma concentrations of the human immunodeficiency syndrome integrase inhibitor raltegravir (RGV), the new nonnucleoside reverse transcriptase inhibitor etravirine (ETV), and 11 other antiretroviral agents: ritonavir, atazanavir, lopinavir, nevirapine, efavirenz, saquinavir, indinavir, nelfinavir, and its metabolite M-8, amprenavir, and darunavir. A simple solid phase extraction procedure was applied to 500 microL aliquots of plasma, and chromatographic separation of the drugs and internal standard (quinoxaline) was achieved with a gradient (acetonitrile and phosphate buffer) on an C-18 reverse-phase analytical column with a 28-minute analytical run time. Calibration curves were optimized according to expected ranges of drug concentrations in patients, and the coefficient of determination (r) was higher than 0.998 for all analytes. Mean intraday and interday precisions (percent relative SD) for all compounds were 3.67% and 6.39%, respectively, and mean accuracy (percent deviation from nominal concentration) was -1.17%. Extraction recovery ranged within 75% and 83% for all drugs analyzed. The solid phase extraction and high-performance liquid chromatography coupled with photo diode array method described allow a specific, sensitive, and reliable simultaneous determination of RGV, ETV, and 11 antiretroviral agents in plasma by a single assay. Good extraction efficiency and low limit of quantification make this a suitable method for use in clinical trials and for therapeutic drug monitoring of RGV, protease inhibitors, and nonnucleoside reverse transcriptase inhibitors, including ETV.

Tipranavir (TPV) is a nonpeptidic protease inhibitor with potent in vitro activity against most human immunodeficiency virus (HIV) type 1 (HIV-1) strains resistant to other protease inhibitors (PIs) (1, 14, 15). In vitro data have shown that resistance to TPV develops slowly (5). When TPV is coadministered with ritonavir (RTV) as a booster, TPV has been shown to have potent antiviral activity in multidrug-experienced patients (3, 6, 9, 12). In the RESIST-1 and the RESIST-2 studies, the efficacy and safety of TPV-RTV (500 mg/200 mg twice daily) in 1,509 highly treatment-experienced HIV-1-positive patients were assessed. Analysis at 48 weeks showed that the TPV-RTV-containing regimens significantly improved the immune and virological responses (VRs) compared to the responses to an RTV-boosted comparator PI plus an optimized background (OB) regimen (3, 6, 8). Different factors have been found to be associated with the virological and immunological responses: a lower viral load (VL) at the baseline, the use of enfuvirtide (T20) as a part of the OB regimen, the presence of two or more active drugs in the OB regimen (OB score [OBS], ≥2) (3, 6, 8), and the baseline numbers of specific TPV-associated resistance mutations (TPV RMs) (2). Moreover, the TPV trough concentration (Ctrough) and phenotypic inhibitory quotient (IQ) have also been shown to be associated with the VR at week 24 (12a, 16). The genotypic IQ (gIQ), which is the ratio between the PI Ctrough and the number of PI-associated mutations, is simpler to derive than the IQ in the clinical setting. The gIQ has previously been shown to be a predictor of the therapeutic response to PI-based salvage regimens, e.g., lopinavir or fosamprenavir (7, 10, 11, 13). Preliminary data showed that TPV-gIQ correlated with the early and the middle VRs (2a, 2b). However, no data are yet available on the predictive value of the TPV gIQ on the long-term efficacy of TPV-based regimens. Therefore, the aim of our study was to perform a pharmacokinetic/pharmacodynamic evaluation of the predictors of the VR at week 48 to salvage TPV-containing regimens in the clinical setting.

A new method using high performance liquid chromatography coupled with electrospray mass spectrometry (HPLC-MS) was developed and validated, for the quantification of plasma concentration of the new protease inhibitors darunavir (DRV) and other 11 antiretroviral agents (ritonavir, amprenavir, atazanavir, lopinavir, saquinavir, indinavir, nelfinavir and its metabolite M-8, nevirapine, efavirenz and tipranavir). A simple protein precipitation extraction procedure was applied on 50 microl of plasma aliquots and chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water with formic acid 0.05%) on an C-18 reverse phase analytical column with 25 min of analytical run. Calibration curves were optimised according to expected ranges of drug concentrations in patients, and correlation coefficient (r2) was higher than 0.998 for all analytes. Mean intra- and inter-day precision (relative standard deviation %) for all compounds were 8.4 and 8.3%, respectively, and mean accuracy (% of deviation from nominal level) was 3.9%. Extraction recovery ranged within 93 and 105% for all drugs analysed. This novel HPLC-MS methodology allows a specific, sensitive and reliable determination of DRV and 11 other antiretrovirals. In our hand, it was used to measure DRV and ritonavir plasma concentration in HIV-positive patients, and it is now successfully applied for routine therapeutic drug monitoring and pharmacokinetics studies.

A simple method for the quantification of tipranavir, the first non-peptidic HIV protease inhibitor, was developed and validated. Quinoxaline, as internal standard, was added to 50 microl of plasma before a liquid-liquid extraction by 600 microl of protein precipitation solution. The extracts were diluted before being injected in the chromatographic system. Chromatographic separation was made on a C18 column using potassium phosphate buffer (pH 3.2) and acetonitrile with gradient. Detection was performed by an UV detector at 260 nm. Relative error at three control quality concentrations ranged from -1.81 to 1.72%. Intra-day (CV%) and inter-day (CV%) precision ranged from 0.94 to 2.55% and from 3.07 to 4.24%, respectively. LOQ and LOD were 0.090 microg/ml and 0.035 microg/ml, respectively. Mean recovery was 87.1%+/-2.4%. Calibration curve was linear up to 180 microg/ml. Concentration range when optimized (0.703-180 microg/ml) proved to be adequate to measure tipranavir concentration in HIV-1-positive patients, therefore this method could be suitable for therapeutic drug monitoring of this drug.

Measurement of ribavirin plasma levels in HCV-positive patients have been shown to be useful in order to optimise individual ribavirin exposure. Efficacy and toxicity of this drug are shown to be concentration-dependant. A simple HPLC-UV method was developed and validated, which has an easy liquid/liquid extraction, sensitive limit of detection, without any interference peaks, reproducible and linear over the range of clinical relevant concentrations. The assay warrants further evaluation as a tool for ribavirin therapeutic drug monitoring in HCV-positive patients.

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 relationship between nevirapine plasma concentrations and the durability of both viral suppression (VS) and selection of nevirapine primary resistance mutations (PRMs) was evaluated. A nevirapine trough concentration ( C trough ) of >4,300 ng/ml was found to predict longer VS. Patients with nevirapine C trough s ranging from 3,100 to 4,300 ng/ml had higher probabilities of developing PRMs than those with nevirapine C trough s below and above this concentration interval.

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) (P0.0001). AI of0.90 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 ofor = 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.

The influence of hepatitis B virus (HBV) on the natural history of human immunodeficiency virus (HIV) infection was evaluated in a prospective study of 347 HIV-positive, AIDS-free individuals infected through injecting drug use and sex and with known seroconversion dates. End points were CD4+ cell count < 200 x 10(6) cell/L and AIDS diagnosis. At entry, 229 had seromarkers to HBV; during the study, 107 had a CD4+ cell count < 200 x 10(6) cells/L and 66 developed AIDS. HBsAg chronic carriers, HBV infection-free subjects and those with baseline evidence of prior HBV infection did not differ in rates of progression to end points. Sexual transmission of HIV was significant predictor of CD4+ cell decline to < 200 x 10(6) cells/l [Hazard ratio (HZ): 1.56, 95% confidence interval (CI): 1.06-2.29, p = 0.0232] and progression to AIDS (HZ: 1.91, CI: 1.17-3.11, p = 0.0091). 15 HIV-positive and HBV infection-free patients had HBV seroconversion. They did not differ from those who remained HBV infection-free in rates of progression to end points, but 40% of them became HBsAg chronic carriers. These results suggest that HBV has no influence on progression of HIV disease, but that patients who have HIV before their HBV infection are more likely to become HBsAg chronic carriers than those who are infected with HBV before HIV.

surveillance program, 1998–1999. Antimicrobial Agents and Che-motherapy 46, 879–81.7. Bell, J. M., Turnidge, J. D. & Jones, R. N. (2002). Antimicrobialresistance trends in community-acquired respiratory tract pathogens inthe Western Pacific Region and South Africa: report from the SENTRYantimicrobial surveillance program, (1998–1999) including an in vitroevaluation of BMS284756. International Journal of Antimicrobial Agents19, 125–32.8. National Committee for Clinical Laboratory Standards. (2003).Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria thatGrow Aerobically-Sixth Edition: Approved Standard M7-A6. NCCLS,Wayne, PA, USA.9. Hasegawa, K., Chiba, N., Kobayashi, R. et al. (2004). Rapidlyincreasing prevalence of b-lactamase-nonproducing, ampicillin-resist-ant Haemophilus influenzae type b in patients with meningitis.Antimicrobial Agents and Chemotherapy 48, 1509–14.10. Gross, M., Clements, J., Beckett, R. P. et al. (2004). Oral anti-pneumococcal activity and pharmacokinetic profiling of a novel peptidedeformylase inhibitor. Journal of Antimicrobial Chemotherapy 53,487–93.11. Lofland, D., Difuntorum, S., Waller, A. et al. (2004). In vitroantibacterial activity of the peptide deformylase inhibitor BB-83698.Journal of Antimicrobial Chemotherapy 53, 664–8.

Serum cytokine profiles, T-cell subsets, and general parameters of immune activation were evaluated in 15 patients with acute primary HIV-1 infection, and compared with those obtained from 18 patients with acute primary Epstein-Barr virus (EBV) infection and from 18 control subjects in order to elucidate possible defects of immune response to HIV in early phases of virus-host interaction. Mean CD4 + cell count, serum concentrations of interleukin (IL)-2, IL-4, soluble IL-2 receptor (sIL-2R), tumor necrosis factor (TNF)-α,5′-neopterin, and β 2 -microglobulin were significantly lower in acute HIV-1 infection than in EBV infection. Both acute HIV-1 and EBV infections were characterized by significantly higher mean CD8 + cell count and soluble CD8 antigen (sCD8) levels compared to control subjects, while acute HIV-1 infection was accompanied by the highest interferon (IFN)-γ serum concentrations. In primary HIV-1 infection, significant impairment of CD4 + -mediated T-helper function may lead to viral escape and persistence of infection despite an early and vigorous CD8 + T-lymphocyte activation.

Purpose of the study Atazanavir (ATV) is administered at the usual adult dose of 300 mg with 100 mg of ritonavir (RTV) once a day (boosted). However, 400 mg once a day (unboosted) is also used in some settings. ATV plasma concentrations are influenced by efflux transporters, influx transporters and metabolism enzymes. The expression of many of these proteins is regulated by nuclear receptors such as PXR. Recently polymorphisms in the regulatory region of the PXR gene have been reported to influence its expression and the activity of downstream genes, such as CYP3A4 and ABCB1. The aim of this study was to investigate whether polymorphisms in PXR influence trough concentrations (Ctrough) of boosted or unboosted ATV.

Objective To identify how reliably CD4+ (helper) lymphocyte count and p24 antigenaemia can predict mother-to-infant transmission of human immunodeficiency virus type 1 (HIV-1). Design Prospective study. Setting University of Turin Center for Intravenous Drug Users (IVDU) and/or HIV-1 seropositive pregnant women. Subjects Twenty-nine infants born to asymptomatic seropositive women from November 1985 to June 1991. Results Seven children (24%) developed symptomatic infection, while 22 healthy seronegative children at the age of 18 months were considered uninfected. A CD4+ lymphocyte count persistently