Risk Factors for Sexual Transmission of Hepatitis C Virus Among Human Immunodeficiency Virus-Infected Men Who Have Sex With Men: A Case-Control Study.

Background. Since 2000, incidence of sexually acquired hepatitis C virus (HCV)-infection has increased among human immunodeficiency virus (HIV)-infected men who have sex with men (MSM). To date, few case-control and cohort studies evaluating HCV transmission risk factors were conducted in this population, and most of these studies were initially designed to study HIV-related risk behavior and characteristics. Methods. From 2009 onwards, HIV-infected MSM with acute HCV infection and controls (HIV-monoinfected MSM) were prospectively included in the MOSAIC (MSM Observational Study of Acute Infection with hepatitis C) study at 5 large HIV outpatient clinics in the Netherlands. Written questionnaires were administered, covering sociodemographics, bloodborne risk factors for HCV infection, sexual behavior, and drug use. Clinical data were acquired through linkage with databases from the Dutch HIV Monitoring Foundation. For this study, determinants of HCV acquisition collected at the inclusion visit were analyzed using logistic regression. Results. Two hundred thirteen HIV-infected MSM (82 MSM with acute HCV infection and 131 MSM without) were included with a median age of 45.7 years (interquartile range [IQR], 41.0-52.2). Receptive unprotected anal intercourse (adjusted odds ratio [aOR], 5.01; 95% confidence interval [CI], 1.63-15.4), sharing sex toys (aOR, 3.62; 95% CI, 1.04-12.5), unprotected fisting (aOR, 2.57; 95% CI, 1.02-6.44), injecting drugs (aOR, 15.62; 95% CI, 1.27-192.6), sharing straws when snorting drugs (aOR, 3.40; 95% CI, 1.39-8.32), lower CD4 cell count (aOR, 1.75 per cubic root; 95% CI, 1.19-2.58), and recent diagnosis of ulcerative sexually transmitted infection (aOR, 4.82; 95% CI, 1.60-14.53) had significant effects on HCV acquisition. Conclusions. In this study, both sexual behavior and biological factors appear to independently increase the risk of HCV acquisition among HIV-infected MSM.

Development and validation of an assay for the simultaneous determination of zidovudine, abacavir, emtricitabine, lamivudine, tenofovir and ribavirin in human plasma using liquid chromatography-tandem mass spectrometry.

This paper describes the development and validation of an assay for the simultaneous quantification of the antiviral and antiretroviral drugs zidovudine, abacavir, emtricitabine, lamivudine, tenofovir and ribavirin in human plasma using liquid chromatography coupled to tandem mass spectrometry. Sample pretreatment consisted of protein precipitation with 0.1% (v/v) formic acid in methanol, evaporation and reconstitution. Chromatographic separation was performed on a Synergy Polar reversed phase C18 column (150 mm × 2.0 mm ID, particle size 4 µm) using a stepwise gradient with 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in methanol at a flow rate of 300 µL/min. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for drug detection and quantification. Isotopically labeled zidovudine, lamivudine, tenofovir and ribavirin were used as internal standards. The method was validated over a clinical range of 20-2500 ng/mL for zidovudine, lamivudine and tenofovir, 4-500 ng/mL for abacavir and emtricitabine and 160-20,000 ng/mL for ribavirin. The inter and intra-assay accuracies and precisions were between -8.47% and 14.2% for zidovudine, emtricitabine and ribavirin. For abacavir, lamivudine and tenofovir, the inter and intra-assay accuracies and precisions at the lower limit of quantification were between -11.0% and 18.3%, whereas at all other levels these accuracies and precisions were between -11.7% and 12.0%. The described method is suitable for the determination of zidovudine, abacavir, emtricitabine, lamivudine, tenofovir and ribavirin in human plasma in clinical practice to monitor plasma concentrations in selected cases to optimize therapy.

Identification and profiling of circulating metabolites of atazanavir, a HIV protease inhibitor.

Atazanavir is a commonly prescribed protease inhibitor for treatment of HIV-1 infection. Thus far, only limited data are available on the in vivo metabolism of the drug. Three systemic circulating metabolites have been reported, but their chemical structures have not been released publicly. Atazanavir metabolites may contribute to its effectiveness but also to its toxicity and interactions. Thus, there is a need for extensive metabolic profiling of atazanavir. Our goals were to screen and identify previously unknown atazanavir metabolites and to develop a sensitive metabolite profiling method in plasma. Five atazanavir metabolites were detected and identified in patient samples using liquid chromatography coupled to linear ion trap mass spectrometry: one N-dealkylation product (M1), two metabolites resulting from carbamate hydrolysis (M2 and M3), a hydroxylated product (M4), and a keto-metabolite (M5). For sensitive semiquantitative analysis of the metabolites in plasma, the method was transferred to liquid chromatography coupled to triple quadrupole mass spectrometry. In 12 patient samples, all the metabolites could be detected, and possible other potential atazanavir keto-metabolites were found. Atazanavir metabolite levels were positively correlated with atazanavir levels, but interindividual variability was high. The developed atazanavir metabolic screening method can now be used for further clinical pharmacological research with this antiretroviral agent.

Effect of HIV viral load, CD4 cell count and antiretroviral therapy on human papillomavirus prevalence in urine samples of HIV-infected men.

HIV-infected patients are at increased risk for persistent human papillomavirus (HPV) infection, the major cause of anogenital cancer. The present study describes the HPV prevalence in urine samples of 243 HIV-infected men and a control group of 231 men. HPV DNA was amplified by the SPF10 polymerase chain reaction primer set. The overall HPV prevalence in HIV-infected men was 27.5% compared with 12.6% in controls (P < 0.01). Infections with high-risk and multiple HPV genotypes were present in both groups. Differences were not statistically significant. A multivariate logistic regression model showed a decreased HPV prevalence associated with use of a nucleoside and a non-nucleoside reverse transcriptase inhibitor combination (P = 0.03). A trend was observed towards a higher HPV prevalence and a lower CD4 cell count. Further prospective studies are needed to determine the role of HPV DNA testing in urine in future screening programmes for anal cancer in men.

Comprehensive longitudinal analysis of hepatitis C virus (HCV)-specific T cell responses during acute HCV infection in the presence of existing HIV-1 infection.

The aim of this study was to study the development of HCV-specific T cell immunity during acute HCV infection in the presence of an existing HIV-1 infection in four HIV-1 infected men having sex with men. A comprehensive analysis of HCV-specific T cell responses was performed at two time points during acute HCV infection using a T cell expansion assay with overlapping peptide pools spanning the entire HCV genome Three patients with (near) normal CD4+ T cell counts (range 400-970 x 10(6)/L) either resolved (n=1) or temporary suppressed HCV RNA. In contrast, one patient with low CD4+ T cell counts (330 x 10(6)/L), had sustained high HCV RNA levels. All four patients had low HCV-specific CD8+ T cell responses, and similar magnitudes of CD4+ T cell responses. Interestingly, individuals with resolved infection or temporary suppression of HCV-RNA had HCV-specific CD4+ T cell responses predominantly against nonstructural (NS) proteins. While the individual with high HCV RNA plasma concentrations had CD4+ T cell responses predominantly directed against Core. Our data show that an acute HCV infection in an HIV-1 infected person can be suppressed in the presence of HCV-specific CD4+ T cell response targeting non-structural proteins. However further research is needed in a larger group of patients to evaluate the role of HIV-1 on HCV-specific T cell responses in relation to outcome of acute HCV infection.

Quantification of the HIV-integrase inhibitor raltegravir and detection of its main metabolite in human plasma, dried blood spots and peripheral blood mononuclear cell lysate by means of high-performance liquid chromatography tandem mass spectrometry.

For the quantification of the HIV-integrase inhibitor raltegravir in human plasma, dried blood spots and peripheral blood mononuclear cell (PBMC) lysate, an assay was developed and validated, using liquid chromatography coupled with tandem mass spectrometry. The assay also allowed detection, but no quantification due to absence of reference substance, of the main metabolite, raltegravir-glucuronide. Raltegravir was extracted from plasma by means of protein precipitation with a mixture of methanol and acetonitrile using only 50microL plasma. Extraction from dried blood spots was performed with a simple one-step extraction with a mixture of methanol, acetonitrile and 0.2M zincsulphate in water (1:1:2, v/v/v) and extraction from cell lysate was performed in 50% methanol in water. Chromatographic separation was performed on a reversed phase C18 column (150mmx2.0mm, particle size 5microm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25mL/min. The analytical run time was 10min. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring was used for drug quantification. The method was validated over a range of 50-10,000ng/mL in plasma and dried blood spots and a range of 1-500ng/mL in PBMC lysate. Dibenzepine was used as the internal standard. The method was proven to be specific, accurate, precise and robust. Accuracies ranged from 104% to 105% in plasma, from 93% to 105% in dried blood spots and from 82% to 113% in PBMC lysate. Precision over the complete concentration range was less than 6%, 11% and 13% in plasma, dried blood spots and PBMC lysate, respectively. The method is now applied for therapeutic drug monitoring and pharmacological research in HIV-infected patients treated with raltegravir.

Quantification of HIV protease inhibitors and non-nucleoside reverse transcriptase inhibitors in peripheral blood mononuclear cell lysate using liquid chromatography coupled with tandem mass spectrometry.

For pharmacokinetic monitoring, measurement of antiretroviral agents in plasma is the gold standard. However, human immunodeficiency virus protease inhibitors (PIs) or non-nucleoside reverse transcriptase inhibitors (NNRTIs) exert their action within the infected cell. Cell-associated concentrations may therefore more adequately reflect therapy outcome. Therefore, for the quantification of nine PIs (amprenavir, atazanavir, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir and tipranavir), 1 active PI metabolite (nelfinavir M8) and 2 NNRTIs (efavirenz and nevirapine) in lysate of peripheral blood mononuclear cells (PBMCs) an assay was developed and validated, using liquid chromatography coupled with tandem mass spectrometry. Analytes were extracted from a PBMC pellet by means of a one-step extraction with 50% methanol containing the internal standards D6-indinavir, D5-saquinavir, 13C6-efavirenz and dibenzepine. Chromatographic separation was performed on a reversed phase C18 column (150mmx2.0mm, particle size 5microm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25mL/min. The analytical run time was 10min. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring was used for drug quantification. The method was validated over a range of 1-500ng/mL in PBMC lysate for all analytes. The method was proven to be specific, accurate, precise and robust. The mean precision and accuracy was less than +/-12% at all concentration levels. Using the developed assay and a previously developed assay for these analytes in plasma, the relationship between plasma and intracellular pharmacokinetics and their relationship with therapy outcome can now be determined.

Tissue expansion for correction of baldness in aplasia cutis congenita.

UNLABELLED: Aplasia cutis is a congenital absence of the skin, usually presenting on the scalp. In 20% of all cases, part of the skull is also absent. A residual area of baldness may still be present some years after surgical or conservative treatment. It is possible to excise the scarred hairless region and cover that area with expanded hair-bearing skin from the rest of the skull. We present three patients who underwent tissue expansion and discuss the indications and pitfalls of this procedure. CONCLUSION: Tissue expansion can be used to cover a residual alopecia defect in young children with aplasia cutis congenita and associated bone abnormalities. The quality of the bone appears to be normal in our three patients. We demonstrate that even in young children with aplasia cutis and an underlying bony defect, tissue expansion is a safe and effective modality as a second stage reconstruction procedure.

Quantification of etravirine (TMC125) in plasma, dried blood spots and peripheral blood mononuclear cell lysate by liquid chromatography tandem mass spectrometry.

For the quantification of the novel non-nucleoside reverse transcriptase inhibitor etravirine in human plasma, dried blood spots and peripheral blood mononuclear cell (PBMC) lysate, an assay was developed and validated, using liquid chromatography coupled with tandem mass spectrometry. Etravirine was extracted from plasma by means of protein precipitation with a mixture of methanol and acetonitrile using only 50 microL plasma. Extraction from dried blood spots was performed with a one-step extraction with a mixture of methanol, acetonitrile and 0.2M zinc sulphate in water (1:1:2, v/v/v) and extraction from cell lysate was performed in 50% methanol in water. Chromatographic separation was performed on a reversed phase C18 column (150mmx2.0mm, particle size 5microm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25mL/min. (13)C(6)-efavirenz was used as an internal standard. The analytical run time was only 10min. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring was used for drug quantification. The method was validated over a range of 25-5000ng/mL in plasma, 50-10,000ng/mL in dried blood spots and a range of 5-2500ng/mL in PBMC lysate. Accuracies ranged from 89% to 106% in plasma, from 94% to 109% in dried blood spots and from 91% to 105% in PBMC lysate. Precisions at the all concentration levels ranged from 1.9% to 14% in plasma, 4.7% to 20% in dried blood spots and from 3.1% to 11% in PBMC lysate. The bioanalytical assay was successfully incorporated with previously developed assays for the determination of all currently approved PIs and NNRTIs in plasma and dried blood spots and it is now applied for therapeutic drug monitoring and pharmacological research in HIV-infected patients treated with etravirine.

An aeration energy model for an immersed membrane bioreactor.

A simple model for evaluating energy demand arising from aeration of an MBR is presented based on a combination of empirical data for the membrane aeration and biokinetic modelling for the biological aeration. The model assumes that aeration of the membrane provides a proportion of the dissolved oxygen demanded for the biotreatment. The model also assumes, based on literature information sources, a linear relationship between membrane permeability and membrane aeration up to a threshold value, beyond which permeability is unchanged with membrane aeration. The model was benchmarked against two full-scale plant to obtain the most appropriate and conservative value of the slope of the flux:aeration curve and the blower efficiency. Benchmarking in this way produced a match to within 20% of all key process plant operational parameters. The model demonstrated that significant reductions in aeration energy could be obtained through operation at lower flux and reducing the membrane aeration requirement accordingly, so-called "proportional aeration" at lower flows. Similarly, increasing oxygen transfer from membrane aeration would also be expected to decrease energy demand. A sensitivity analysis of some of the key parameters revealed that, of the key operating parameters, loading, SOTE and MLSS concentration remain the most critical in determining energy demand. It is suggested that a key parameter representing membrane aeration in MBRs is the mean in-module air upflow velocity U, since this gives a reasonable representation of the shear applied through membrane aeration. U was found to vary between 0.04 and 0.1m/s across a number of modern large pilot and full-scale plant. An analysis reveals that significant reductions in energy demand are attained through operating at lower MLSS levels and membrane fluxes. Evidence provided from recent controlled pilot trials implies that halving the flux can reduce the aeration is suggested whereby the number of membrane tanks on line and/or the membrane aeration intensity is adjusted according to the flow, and thus flux, so as to reduce the overall aeration energy demand.

Quantification of protease inhibitors and non-nucleoside reverse transcriptase inhibitors in dried blood spots by liquid chromatography-triple quadrupole mass spectrometry.

A bioanalytical method for the determination of most commonly prescribed protease inhibitors (atazanavir, darunavir, lopinavir and ritonavir) and non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine) was developed and validated according to FDA guidelines. In brief, dried blood spots were punched out of a collection paper with a 0.25 in. diameter punch. The analytes were extracted from the punched-out disc using a mixture of acetonitrile, methanol and 0.2M zinc sulphate in water (1:1:2, v/v/v) containing the internal standards dibenzepine, 13C6-efavirenz and D5-saquinavir. 20 microL of the extract was injected onto the reversed-phase C18 column (150 mm x 2.0 mm) for separation from endogenous compounds and the analytes were quantified using a triple quadrupole mass spectrometer. The analytical run time was only 10 min. Validated concentration ranges covered the ranges encountered in routine clinical practice. The assay was linear over the concentration ranges tested (0.1-20 mg/L for atazanavir, lopinavir, nevirapine and efavirenz and 0.05-10 mg/L for darunavir and ritonavir). Accuracies and inter- and intra-run precisions at all levels ranged from 96.2 to 113.9% and 3.1 to 13.3%, respectively. Analytes in dried blood spots were stable for at least 7 days at 30 degrees C. The method enabled patient-friendly sample collection, easy and cheap sample shipment and non-hospital based sampling for therapeutic drug monitoring and pharmacokinetic studies.

Full-scale granular sludge Anammox process.

The start-up of the first full scale Anammox reactor is complete. The reactor shows stable operation, even at loading rates of 10 kg N/m3.d. This performance is the result of the formation of Anammox granules, which have a high density and settling velocities exceeding 100 m/h. With this performance, the Anammox granular sludge technology has been proven on full scale.

SHARON process evaluated for improved wastewater treatment plant nitrogen effluent quality.

New stricter nitrogen effluent standards and increasing influent loads require existing wastewater treatment plans (WWTPs) to extend or optimize. At WWTPs with limited aeration capacity, limited denitrification capacity or shortage of aerobic sludge age, implementation of SHARON to improve nitrogen effluent quality can be a solution. SHARON is a compact, sustainable and cost-effective biological process for treatment of nitrogen-rich rejection waters. At WWTP Rotterdam-Dokhaven and WWTP Utrecht a SHARON has been in operation for several years. For both WWTPs the effect of SHARON on the nitrogen effluent quality has been evaluated. WWTP Rotterdam-Dokhaven has limited aeration capacity. By implementation of SHARON, the ammonia load of the effluent was reduced by 50%. WWTP Utrecht had limited denitrification capacity. The implementation of SHARON improved the effluent nitrate load by 40%. The overall TN removal efficiency increased from 65% to over 75% and strict nitrogen effluents standards (TN = 10 mg N/l) could be reached. Through modelling and supported by full scale practice it has been shown that by implementation of SHARON in combination with enhanced influent pre-treatment, the aerobic sludge age can be extended to maintain total nitrogen removal at lower temperatures.

Population pharmacokinetics of lopinavir in combination with ritonavir in HIV-1-infected patients.

AIMS: To develop a population pharmacokinetic model for lopinavir in combination with ritonavir, in which the interaction between both drugs was characterized, and in which relationships between patient characteristics and pharmacokinetics were identified. METHODS: The pharmacokinetics of lopinavir in combination with ritonavir were described using NONMEM (version V, level 1.1). First, ritonavir data were fitted to a previously developed model to obtain individual Bayesian estimates of pharmacokinetic parameters. Hereafter, an integrated model for the description of the pharmacokinetics of lopinavir with ritonavir was designed. RESULTS: From 122 outpatients 748 lopinavir and 748 ritonavir plasma concentrations were available for analysis. The interaction between the drugs was described by a time-independent inverse relationship between the exposure to ritonavir over a dosing-interval and the apparent clearance (CL/F) of lopinavir. The model parameters volume of distribution and absorption rate constant were 61.6 l (95% prediction interval (PI) 22.4, 83.7) and 0.564 h(-1) (95% PI 0.208, 0.947), respectively. The model yielded a theoretical value for the CL/F of lopinavir without ritonavir of 14.8 l h(-1) (95%PI 12.1, 20.1), which translates to a value of 5.73 l h(-1) in the presence of ritonavir. The only factor with significant effect on the pharmacokinetics was concurrent use of non-nucleoside reverse transcriptase inhibitors (NNRTI), which increased the CL/F of lopinavir by 39% (P < 0.001). CONCLUSIONS: We have developed a model that has defined a time-independent inverse relationship between the exposure to ritonavir and the CL/F of lopinavir, and provided an adequate description of the pharmacokinetic parameters for the latter. Concomitant use of the NNRTIs efavirenz and nevirapine increased the CL/F of lopinavir.

Evaluation of clinical pharmacist interventions on drug interactions in outpatient pharmaceutical HIV-care.

OBJECTIVE: To evaluate the usefulness of intervention in drug interactions of antiretroviral drugs with coadministered agents by a clinical pharmacist in outpatient HIV-treatment. METHODS: The study design included two intervention arms (A and B), which were both preceded by a control observation period. In arm A, a complete list of the currently used drugs, extracted from pharmacy records was provided to the treating physician. In arm B the same list was provided but with a notification when a drug interaction was present and an advice how to handle this. The infectious disease specialist obtained the information before the patient's visit to the outpatient clinic (time point 0). Three months prior (time point -3) and 3 months after (time point +3) the intervention, pharmacy records were also screened for drug interactions. The number of drug interactions (total and per patient) was determined at the three different time points (-3, 0, +3). In addition, drug interactions encountered at time points -3 and 0 were checked for their presence at time points 0 and +3, respectively, for both intervention arms. RESULTS: Arms A and B included 115 and 105 patients, respectively. Patient characteristics of both intervention arms were similar at time point 0. The number of interactions and the number of patients with interactions were similar in both intervention arms at time point 0. There were 42 and 40 potential drug interactions in 30 and 24 patients in arms A and B, respectively. The reduction in the number of interactions per patient over time and after intervention was small but significant, and was equal in both intervention arms. The advice of the clinical pharmacist had thus no additional value. CONCLUSION: Both interventions were effective in reducing the number of drug interactions per patient. The advice of a clinical pharmacist was, however, redundant in the studied setting.

Differential CD4 T-cell response in HIV-1-infected patients using protease inhibitor-based or nevirapine-based highly active antiretroviral therapy.

OBJECTIVES: To study the dynamics of CD4 T-lymphocyte counts (CD4 counts) after the initiation of either protease inhibitor (PI)-based or nevirapine (NVP)-based first-line highly active antiretroviral therapy (HAART). DESIGN AND METHODS: A retrospective cohort study of 1029 HIV-infected antiretroviral therapy-naive patients initiating either PI-based or NVP-based HAART was carried out. Patients were censored as soon as they experienced virological failure, or changed their original antiretroviral regimen for any reason. RESULTS: In total, 920 and 109 patients initiated PI- and NVP-based HAART, respectively. The patients in the PI group more often had AIDS (15 vs. 6% in the NVP group), had a lower median baseline CD4 count (234 vs. 250 cells/microL in the NVP group) and had higher median baseline plasma HIV-1 RNA levels (pVL) (5.0 vs. 4.7 log10 HIV-1 RNA copies/mL in the NVP group). After 96 weeks of follow-up, the mean increase from baseline in CD4 count, adjusted for baseline CD4 count, age, gender and baseline pVL, was 310 cells/microL in the PI group and 212 cells/microL in the NVP group (P=0.003). This difference was mainly attributable to the patients in the NVP group initiating HAART with a baseline CD4 count below 200 cells/microL. There were no differences between the PI and NVP groups with respect to the change in the number of CD4 cells as a proportion of the total number of lymphocytes. CONCLUSION: Patients successfully treated with NVP-based HAART have a smaller increase in absolute CD4 cells compared with those treated with PI-based HAART.