Nelfinavir+M8 plasma levels determined with an ELISA test in HIV infected patients with or without HCV and/or HBV coinfection: the VIRAKINETICS II study.

Virakinetics II was designed as an observational, multicenter cohort study conducted in HIV-positive patients treated with NFV-based combinations. Trough (pre-dose) concentrations of NFV+M8 in plasma were determined using a novel ELISA test (NFV TDM-ELISA) and analyzed using clinical and laboratory parameters. Drug levels were sorted as below, within or above a given interval (<0.8 microg/mL, 0.8-3.5 microg/mL and >3.5 microg/mL, respectively). Longitudinal analysis was performed in a subset of patients who underwent two or more determinations. Ninety patients on NFV-containing HAART were enrolled and 43 were coinfected with HCV and/or HBV. Among coinfected patients, 10 subjects had a clinical or histological diagnosis of cirrhosis. Compared to the HIV-monoinfected, the coinfected patients were significantly older, more treatment-experienced, with higher frequency of lipodystrophy and altered liver function test values (all p values: <0.05). Coinfected patients were also more likely to be on a reduced dose of NFV than monoinfected (p=0.03). No significant difference was observed between the two groups with regard to NFV+M8 trough values and concentration range distribution. Median NFV+M8 C(trough) concentrations were higher in coinfected patients, but without reaching statistical significance (p=0.2). This new ELISA test proved to be a rapid, convenient and reliable tool for assessing NFV+M8 plasma levels in HIV-positive patients. It could be suitable for use within the framework of routine clinical practice even in peripheral centers without specialized laboratories.

Evaluation of Elisa test for therapeutic monitoring of Nelfinavir in HIV-positive patients.

Therapeutic drug monitoring (TDM) is an important tool in the management of antiretroviral (ARV) therapy. The gold standard for measuring drugs plasma levels is High-Performance Liquid Chromatographic Assay (HPLC) however it is technically-demanding and time-consuming. We evaluated a new immunoenzymatic test (TDM-ELISA, Biostrands, Trieste, Italy) for nelfinavir and its active metabolite M8 in comparison with HPLC. A statistically significant difference in Ctrough between the two different tests was demonstrated but this difference was no longer significant when a value of 29% due to M8 aliquot was deleted. This faster TDM-ELISA may have an important role for TDM in HIV patients taking ARVs.

Development and evaluation of an immunoassay for the monitoring of the anti-HIV drug amprenavir.

An assay for routine therapeutic drug monitoring of anti-HIV HAART drugs in clinical use is highly desirable, in order to rapidly measure the pharmacokinetic parameters on single patients. We have started a project to develop a panel of enzyme-linked immunosorbent assays (ELISA) for the whole set of HAART drugs, and the development, performance and evaluation of the assay for amprenavir is described here. A diazo conjugate of amprenavir has been used in order to raise polyclonal anti-amprenavir antibodies in rabbits. Antisera have been used to set up a quantitative and rapid competitive assay. Plasma samples are simply diluted in the assay buffer after thermal inactivation, before running the assay. The assay allows the detection of amprenavir in the quantification range 400-5000 ng/ml, in a diluted plasma sample. The assay has been compared with an HPLC reference technique, on 27 samples from treated patients. Within the quantification range, the ELISA data are well correlated with the HPLC results by a regression line close to the identity, and a Bland-Altman analysis shows the agreement between the two methods.