Mauro Sciandra

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

ABSTRACT 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.

An HPLC-PDA Method for the Simultaneous Quantification of the HIV Integrase Inhibitor Raltegravir, the New Nonnucleoside Reverse Transcriptase Inhibitor Etravirine, and 11 Other Antiretroviral Agents in the Plasma of HIV-Infected Patients

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 μL 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 (r2) 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.

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 manufacturers 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.

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.

Cytokine network and acute primary HIV-1 infection.

Objective:To investigate the relationship between cytokine serum levels, peripheral blood lymphocyte subsets and clinical picture in acute primary HIV-1 infection. Patients and methods:Absolute number/μl total lymphocytes, CD4+, CD8+ and natural killer (NK) cells, as well as serum levels of soluble CD8 receptor, interleukin (IL)-1β, IL-2, lL-4, IL-6, tumour necrosis factor (TNF)-α, interferon (IFN)-γ, β2-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. Results: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-1β, IFN-γ and 5′-neopterin. Follow-up of acute seroconverters showed a significant decrease in NK cell counts and 1L-1β levels, with an increase of IL-6. Conclusions: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.

Coinfection and Superinfection of Hepatitis B Virus in Patients Infected with Human Immunodeficiency Virus: No Evidence of Faster Progression to AIDS

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.

Association of a single-nucleotide polymorphism in the pregnane X receptor (PXR 63396C-->T) with reduced concentrations of unboosted atazanavir.

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.

HPLC–MS method for the quantification of nine anti-HIV drugs from dry plasma spot on glass filter and their long term stability in different conditions

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 degrees C and -20 degrees 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 x 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.

A HPLC–MS method for the simultaneous quantification of fourteen antiretroviral agents in peripheral blood mononuclear cell of HIV infected patients optimized using medium corpuscular volume evaluation

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.

Nevirapine Plasma Exposure Affects both Durability of Viral Suppression and Selection of Nevirapine Primary Resistance Mutations in a Clinical Setting

ABSTRACT 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 (Ctrough) of >4,300 ng/ml was found to predict longer VS. Patients with nevirapine Ctroughs ranging from 3,100 to 4,300 ng/ml had higher probabilities of developing PRMs than those with nevirapine Ctroughs below and above this concentration interval.