Population pharmacokinetic modelling and evaluation of different dosage regimens for darunavir and ritonavir in HIV-infected individuals.

OBJECTIVES: Darunavir is a protease inhibitor that is administered with low-dose ritonavir to enhance its bioavailability. It is prescribed at standard dosage regimens of 600/100 mg twice daily in treatment-experienced patients and 800/100 mg once daily in naive patients. A population pharmacokinetic approach was used to characterize the pharmacokinetics of both drugs and their interaction in a cohort of unselected patients and to compare darunavir exposure expected under alternative dosage regimens. METHODS: The study population included 105 HIV-infected individuals who provided darunavir and ritonavir plasma concentrations. Firstly, a population pharmacokinetic analysis for darunavir and ritonavir was conducted, with inclusion of patients' demographic, clinical and genetic characteristics as potential covariates (NONMEM(®)). Then, the interaction between darunavir and ritonavir was studied while incorporating levels of both drugs into different inhibitory models. Finally, model-based simulations were performed to compare trough concentrations (Cmin) between the recommended dosage regimen and alternative combinations of darunavir and ritonavir. RESULTS: A one-compartment model with first-order absorption adequately characterized darunavir and ritonavir pharmacokinetics. The between-subject variability in both compounds was important [coefficient of variation (CV%) 34% and 47% for darunavir and ritonavir clearance, respectively]. Lopinavir and ritonavir exposure (AUC) affected darunavir clearance, while body weight and darunavir AUC influenced ritonavir elimination. None of the tested genetic variants showed any influence on darunavir or ritonavir pharmacokinetics. The simulations predicted darunavir Cmin much higher than the IC50 thresholds for wild-type and protease inhibitor-resistant HIV-1 strains (55 and 550 ng/mL, respectively) under standard dosing in >98% of experienced and naive patients. Alternative regimens of darunavir/ritonavir 1200/100 or 1200/200 mg once daily also had predicted adequate Cmin (>550 ng/mL) in 84% and 93% of patients, respectively. Reduction of darunavir/ritonavir dosage to 600/50 mg twice daily led to a 23% reduction in average Cmin, still with only 3.8% of patients having concentrations below the IC50 for resistant strains. CONCLUSIONS: The important variability in darunavir and ritonavir pharmacokinetics is poorly explained by clinical covariates and genetic influences. In experienced patients, treatment simplification strategies guided by drug level measurements and adherence monitoring could be proposed.

[An update on hepatitis E]. / Mise à jour sur l'hépatite E.

The hepatitis E virus (HEV) is an RNA virus transmitted via the fecal-oral route or through uncooked animal meat products. Of the 4 known genotypes, genotype 3 is responsible for autochthonous infections in industrialized countries, with a seroprevalence in Switzerland estimated as high as 22%. The majority of infections is asymptomatic but a minority of patients, notably men over 50 or with underlying liver disease, can present with severe acute hepatitis. Chronic hepatitis E with HEV of genotype 3 has been observed in immunosuppressed patients, mostly transplant recipients. Serology is not sufficiently sensitive, especially in immunosuppressed patients, making PCR identification the preferred test for diagnosing active infection. Ribavirin or interferon-alpha can be used to treat chronic hepatitis E if reduction of immunosuppressive treatment does not result in viral elimination.

A LC-tandem MS assay for the simultaneous measurement of new antiretroviral agents: Raltegravir, maraviroc, darunavir, and etravirine.

Raltegravir (RAL), maraviroc (MVC), darunavir (DRV), and etravirine (ETV) are new antiretroviral agents with significant potential for drug interactions. This work describes a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of plasma drug levels. Single-step extraction of RAL, MVC, DRV, ETV and RTV from plasma (100 microl) is performed by protein precipitation using 600 microl of acetonitrile, after the addition of 100 microl darunavir-d(9) (DRV-d(9)) at 1000 ng/ml in MeOH/H(2)O 50/50 as internal standard (I.S.). The mixture is vortexed, sonicated for 10 min, vortex-mixed again and centrifuged. An aliquot of supernatant (150 microl) is diluted 1:1 with a mixture of 20 mM ammonium acetate/MeOH 40/60 and 10 microl is injected onto a 2.1 x 50 mm Waters Atlantis-dC18 3 microm analytical column. Chromatographic separations are performed using a gradient program with 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile with 0.1% formic acid. Analytes quantification is performed by electrospray ionisation-triple quadrupole mass spectrometry using the selected reaction monitoring detection in the positive mode. The method has been validated over the clinically relevant concentrations ranging from 12.5 to 5000 ng/ml, 2.5 to 1000 ng/ml, 25 to 10,000 ng/ml, 10 to 4000 ng/ml, and 5 to 2000 ng/ml for RAL, MRV, DRV, ETV and RTV, respectively. The extraction recovery for all antiretroviral drugs is always above 91%. The method is precise, with mean inter-day CV% within 5.1-9.8%, and accurate (range of inter-day deviation from nominal values -3.3 to +5.1%). In addition our method enables the simultaneous assessment of raltegravir-glucuronide. This is the first analytical method allowing the simultaneous assay of antiretroviral agents targeted to four different steps of HIV replication. The proposed method is suitable for the Therapeutic Drug Monitoring Service of these new regimen combinations administered as salvage therapy to patients having experienced treatment failure, and for whom exposure, tolerance and adherence assessments are critical.

[A primer on the management of antiviral resistance in chronic hepatitis B]. / Prévention et prise en charge de la résistance antivirale dans l'hépatite B chronique.

Treatment options for chronic hepatitis B have significantly expanded over the last decade. Six nucleoside or nucleotide analogs (NA) with activity against the hepatitis B virus are currently available. Prolonged NA treatment is required in many cases to maintain viral suppression, with an inherent risk of the development of antiviral resistance. The purpose of this concise review is to provide an introduction to the prevention, diagnosis and management of antiviral resistance in chronic hepatitis B.

Pharmacogenetics-based population pharmacokinetic analysis of efavirenz in HIV-1-infected individuals.

Besides CYP2B6, other polymorphic enzymes contribute to efavirenz (EFV) interindividual variability. This study was aimed at quantifying the impact of multiple alleles on EFV disposition. Plasma samples from 169 human immunodeficiency virus (HIV) patients characterized for CYP2B6, CYP2A6, and CYP3A4/5 allelic diversity were used to build up a population pharmacokinetic model using NONMEM (non-linear mixed effects modeling), the aim being to seek a general approach combining genetic and demographic covariates. Average clearance (CL) was 11.3 l/h with a 65% interindividual variability that was explained largely by CYP2B6 genetic variation (31%). CYP2A6 and CYP3A4 had a prominent influence on CL, mostly when CYP2B6 was impaired. Pharmacogenetics fully accounted for ethnicity, leaving body weight as the only significant demographic factor influencing CL. Square roots of the numbers of functional alleles best described the influence of each gene, without interaction. Functional genetic variations in both principal and accessory metabolic pathways demonstrate a joint impact on EFV disposition. Therefore, dosage adjustment in accordance with the type of polymorphism (CYP2B6, CYP2A6, or CYP3A4) is required in order to maintain EFV within the therapeutic target levels.

Liver enzyme elevation after lamivudine withdrawal in HIV-hepatitis B virus co-infected patients: the Swiss HIV Cohort Study.

Background The principal causes of liver enzyme elevation among HIV-hepatitis B virus (HBV) co-infected patients are the hepatotoxic effects of antiretroviral therapy (ART), alcohol abuse, ART-induced immune reconstitution and the exacerbation of chronic HBV infection. Objectives To investigate the incidence and severity of liver enzyme elevation, liver failure and death following lamivudine (3TC) withdrawal in HIV-HBV co-infected patients. Methods Retrospective analysis of the Swiss HIV Cohort Study database to assess the clinical and biological consequences of the discontinuation of 3TC. Variables considered for analysis included liver enzyme, HIV virological and immunological parameters, and medication prescribed during a 6-month period following 3TC withdrawal. Results 3TC was discontinued in 255 patients on 363 occasions. On 147 occasions (109 patients), a follow-up visit within 6 months following 3TC withdrawal was recorded. Among these patients, liver enzyme elevation occurred on 42 occasions (29%), three of them (2%) with severity grade III and five of them (3.4%) with severity grade IV elevations (as defined by the AIDS Clinical Trials Group). Three patients presented with fulminant hepatitis. One death (0.7%) was recorded. Conclusions HBV reactivation leading to liver dysfunction may be an under-reported consequence of 3TC withdrawal in HIV-HBV co-infected patients. Regular monitoring of HBV markers is warranted if active therapy against HBV is discontinued.

The evolutionary history of the CD209 (DC-SIGN) family in humans and non-human primates.

The CD209 gene family that encodes C-type lectins in primates includes CD209 (DC-SIGN), CD209L (L-SIGN) and CD209L2. Understanding the evolution of these genes can help understand the duplication events generating this family, the process leading to the repeated neck region and identify protein domains under selective pressure. We compiled sequences from 14 primates representing 40 million years of evolution and from three non-primate mammal species. Phylogenetic analyses used Bayesian inference, and nucleotide substitutional patterns were assessed by codon-based maximum likelihood. Analyses suggest that CD209 genes emerged from a first duplication event in the common ancestor of anthropoids, yielding CD209L2 and an ancestral CD209 gene, which, in turn, duplicated in the common Old World primate ancestor, giving rise to CD209L and CD209. K(A)/K(S) values averaged over the entire tree were 0.43 (CD209), 0.52 (CD209L) and 0.35 (CD209L2), consistent with overall signatures of purifying selection. We also assessed the Toll-like receptor (TLR) gene family, which shares with CD209 genes a common profile of evolutionary constraint. The general feature of purifying selection of CD209 genes, despite an apparent redundancy (gene absence and gene loss), may reflect the need to faithfully recognize a multiplicity of pathogen motifs, commensals and a number of self-antigens.

Pharmacogenetics of anti-HIV drugs.

Pharmacogenetics holds promise in HIV treatment because of the complexity and potential toxicity of multidrug therapies that are prescribed for long periods. Thus far, few candidate genes have been examined for a limited number of allelic variants, but a number of confirmed associations have already emerged. A change in paradigm emerges from the availability of the HapMap, the wealth of data on less-common genetic polymorphisms, and new genotyping technology. This review presents a comprehensive analysis of the existing literature on pharmacogenetic determinants of antiretroviral drug exposure, drug toxicity, as well as genetic markers associated with the rate of disease progression. It is expected that larger-scale comprehensive genome approaches will profoundly change the landscape of knowledge in the future.

Influence of inhibitory killer immunoglobulin-like receptors and their HLA-C ligands on resolving hepatitis C virus infection.

An estimated 2%-3% of the world's population is chronically infected with hepatitis C virus (HCV) and this is a major cause of liver disease worldwide. Following acute infection, outcome is variable with acute HCV successfully resolved in some individuals (20%-30%), but in the majority of cases the virus is able to persist. Co-infection with human immunodeficiency virus has been associated with a negative impact on the course of HCV infection. The host's immune response is an important correlate of HCV infection outcome and disease progression. Natural killer (NK) cells provide a major component of the antiviral immune response by recognising and killing virally infected cells. NK cells modulate their activity through a combination of inhibitory and activatory receptors such as the killer immunoglobulin-like receptors (KIRs) that bind to human leukocyte antigen (HLA) Class I molecules. In this workshop component, we addressed the influence of KIR genotypes and their HLA ligands on resolving HCV infection and we discuss the implications of the results of the study of Lopez-Vazquez et al. on KIR and HCV disease progression.

Predictive value of known and novel alleles of CYP2B6 for efavirenz plasma concentrations in HIV-infected individuals.

To assess the association of CYP2B6 allelic diversity with efavirenz (EFV) pharmacokinetics, we performed extensive genotyping of 15 relevant single nucleotide polymorphism in 169 study participants, and full resequencing of CYP2B6 in individuals with abnormal EFV plasma levels. Seventy-seven (45.5%) individuals carried a known (CYP2B6*6, *11, *15, or *18) or new loss/diminished-function alleles. Resequencing defined two new loss-of-function alleles: allele *27 (marked by 593T>C [M198T]), that results in 85% decrease in enzyme activity and allele *28 (marked by 1132C>T), that results in protein truncation at arginine 378. Median AUC levels were 188.5 microg h/ml for individuals homozygous for a loss/diminished-function allele, 58.6 microg h/ml for carriers, and 43.7 microg h/ml for noncarriers (P<0.0001). Individuals with a poor metabolizer genotype had a likelihood ratio of 35 (95% CI, 11-110) of presenting very high EFV plasma levels. CYP2B6 poor metabolizer genotypes explain to a large extent EFV pharmacokinetics and identify individuals at risk of extremely elevated EFV plasma levels.

Determination of the novel non-peptidic HIV-protease inhibitor tipranavir by HPLC-UV after solid-phase extraction.

An HPLC method previously described for the assay of amprenavir (APV), ritonavir (RTV), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV), lopinavir (LPV), atazanavir (ATV), nevirapine (NVP) and efavirenz (EFV) can be also conveniently applied, with minor gradient program adjustment, for the determination of the novel non-peptidic HIV protease inhibitor tipranavir (TPV) in human plasma, by off-line solid-phase extraction (SPE) followed by HPLC coupled with UV-diode array detection (DAD). After viral inactivation by heat, the plasma is diluted with phosphate buffer (pH 7), and subjected to a SPE on a C18 cartridge. Matrix components are eliminated with a solution of 0.1% H3PO4 solution neutralised to pH 7, and TPV is eluted with MeOH. The resulting eluate is evaporated and reconstituted in 100 microl MeOH/H2O 50/50. A 40 microl volume is injected onto a Nucleosil C18 AB column and TPV is analysed by UV detection at 201 nm using a gradient elution program constituted of MeCN and phosphate buffer adjusted to pH 5.12 and containing 0.02% sodium heptanesulfonate. The calibration curves are linear up to 75 microg/ml, with a lower limit of quantification of 0.125 microg/ml. The mean absolute recovery of TPV is 77.1+/-4.0%. The method is precise with mean inter-day coefficient of variations (CVs) within 2.2-3.4%, and accurate (range of inter-day deviations from 0.7 to 1.2%). The method has been validated and is currently applied to the monitoring of TPV plasma levels in HIV patients.

Intracellular measurements of anti-HIV drugs indinavir, amprenavir, saquinavir, ritonavir, nelfinavir, lopinavir, atazanavir, efavirenz and nevirapine in peripheral blood mononuclear cells by liquid chromatography coupled to tandem mass spectrometry.

A sensitive and accurate liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the intracellular determination of nine antiretroviral drugs in human peripheral blood mononuclear cells (PBMCs) is proposed. PBMCs are isolated by density gradient centrifugation using Vacutainer CPT tubes and cell count is performed with a Coulter instrument. Single-step extraction of drugs from PBMCs pellets is performed with MeOH 50% (with clozapine added as internal standard, I.S.) and the supernatant is injected onto a 2.1 mm x 30 mm SymmetryShield 3.5 microm-RP18 column equipped with a 2.1 x 10 mm guard column. Chromatographic separations are performed using a gradient program with a mixture of 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile with 0.1% formic acid. Analytes quantification is performed by electro-spray ionisation-triple quadrupole mass spectrometry using the selected reaction monitoring (SRM) detection mode. The positive mode is used for the HIV protease inhibitors (PIs) indinavir, amprenavir, saquinavir, ritonavir, nelfinavir, lopinavir, atazanavir and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine, and the negative mode is applied for efavirenz. The calibration curves are prepared using blank PBMCs spiked with antiretroviral drugs at concentrations ranging from 0.5 to 100 ng/ml of cell extracts and fitted to a quadratic regression model weighted by 1/(concentration)(2). The lower limit of quantification is less than 0.5 ng/ml. The mean extraction recovery for all PIs/NNRTIs is always above 88%. The method is precise, with mean inter-day CV% within 0.6-10.2%, and accurate (range of inter-day deviation from nominal values -7.2 to +8.3%). This analytical method can be conveniently used in clinical research for the assessment of intracellular levels of all PIs/NNRTIs commercially available at present using a simple one-step cell extraction of PBMCs followed by liquid chromatography coupled with tandem triple quadripole mass detection.

[The genetics HIV cohort of the Swiss HIV Cohort Study--facing the new challenges in medicine]. / Bereit für neue medizinische Herausforderungen--die "Genetics HIV Cohort" der Schweizerischen HIV Kohortenstudie.

Host genetic factors determine the individual natural course of HIV infection and influence the response to therapy and the occurrence of adverse events to treatment. Variants of multiple genes are associated with faster but also slower development of severe immunodeficiency. However, only very rarely the variant of one single gene explains a specific clinical phenotype. But multiple genetic marker form a complex trait, which is difficult to analyse biostatistically. Research in this rapidly evolving field asks for structures in which hypotheses can be generated and evaluated and which combine basic and diagnostic and therapeutic research. The large amount of prospective information on HIV disease natural history and treatment response of the Swiss HIV Cohort Study will make of the Genetics project an excellent test-setting for some of the immediate difficulties in this research field: validation of new markers and modelling of complex traits.

Determination of the new HIV-protease inhibitor atazanavir by liquid chromatography after solid-phase extraction.

An HPLC method previously described for the simultaneous assay of amprenavir, ritonavir, indinavir, saquinavir, nelfinavir and efavirenz is proposed here for the simultaneous analysis of the new HIV protease inhibitor atazanavir (ATV) in human plasma, by off-line solid-phase extraction (SPE) followed by HPLC coupled with UV-diode array detection. After viral inactivation by heat (60 degrees C for 60 min), plasma (600 microl) with clozapine (internal standard) is diluted 1 + 1 with phosphate buffer pH 7 and subjected to a SPE on a C18 cartridge. Matrix components are eliminated with 2 x 500 microl of a solution of 0.1% H(3)PO(4) neutralised with NaOH to pH 7. ATV is eluted with 3 x 500 microl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 100 microl MeOH/H(2)O 50/50. A 40 microl volume is injected onto a Nucleosil 100-5 microm C18 AB column. ATV is analysed by UV detection at 201 nm using a gradient elution program with solvents constituted of MeCN and phosphate buffer adjusted to pH 5.14. The mobile phase also contains 0.02% sodium heptanesulfonate, enabling an excellent separation of ATV from the other HIV protease inhibitors (PIs) amprenavir, indinavir, saquinavir, ritonavir, lopinavir, nelfinavir and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine. The calibration curves are linear up to 10 microg/ml, with a lower limit of quantification of 0.2 microg/ml. The mean absolute recovery of ATV is 96.4 +/- 3.2%. The method is precise with mean inter-day CVs within 1.1-6.1%, and accurate (range of inter-day deviations +0.3 to +2.3%). The method has been validated and is currently applied to the monitoring of ATV in HIV patients.

CCTTT-repeat polymorphism of the inducible nitric oxide synthase is not associated with HIV pathogenesis.

Nitric oxide (NO) produced by the inducible form of nitric oxide synthase (iNOS) has bactericidal and virocidal effects. Although NO synthesis and iNOS expression in macrophages affect several aspects of human immunodeficiency virus (HIV) type-1 pathogenesis, their role in HIV disease remains largely unknown. In humans, the expression of iNOS is influenced by a functional CCTTT-repeat polymorphism in the promoter region of the gene. We investigated the association of this polymorphism with HIV pathogenesis in naive HIV-infected patients before the initiation of antiretroviral therapy. The allele frequencies of the iNOS CCTTT-repeat polymorphism were assessed by PCR in 857 patients from the Swiss HIV Cohort Study, including rapid progressors and long-term nonprogressors, and in 240 healthy volunteers. In HIV-infected patients, the initial viral load and the decline in total CD4 cells was calculated to estimate disease progression. Allele frequencies of the iNOS CCTTT-repeat polymorphism were similar between the HIV-infected and noninfected blood donors. In treatment-naive HIV-positive patients, there was no association of the iNOS polymorphism with viral load or with the course of CD4 cells. Regulation of iNOS expression by the functional CCTTT-polymorphism does not modify HIV pathogenesis.

[Antiviral therapy and resistance formation exemplified by HIV infection]. / Antivirale Therapie und Resistenzentwicklung am Beispiel der HIV-Infektion.

In the last few years the antiretroviral combination therapy has drastically reduced the mortality and morbidity in HIV-infected patients. The aim of this treatment is the suppression of viral replication by a combination of at least three active drugs. However, about 20-40% of the patients experience a virological treatment failure, mostly due to development of drug resistant viruses on the background of insufficient drug levels. This resistances can be detected geno- and phenotypically by validated techniques. In the individual case of treatment failure resistance testing and therapeutic drug monitoring can help to find the most appropriate drug combination in the right dosage.

Ritonavir plus saquinavir versus single protease inhibitor therapy in protease inhibitor-naive HIV-infected patients: the Swiss HIV Cohort Study.

OBJECTIVES: To compare the response to ritonavir (RTV) plus saquinavir (SQV) with single protease inhibitor (PI) therapies among PI-naive HIV-1 infected individuals. METHODS: Response to treatment was analysed according to the intent-to-treat principle in a prospective observational cohort study of 177 patients who between May 1995 and March 2000 started a double PI therapy with RTV and SQV (nonboosting dosages) plus at least one nucleoside reverse transcriptase inhibitor (NRTI) and 2,214 patients with a single PI therapy plus two NRTIs. We used survival analysis and Cox's proportional hazard regression methods. The primary endpoint was the time to a plasma viral load of < 400 copies/mL. Secondary endpoints were taken as a gain in the CD4 count of >100 cells/microL, and change of initial PI for any reason. RESULTS: Baseline characteristics in both treatment groups were balanced. Median follow-up in both groups was 10.4 months. Time to an HIV-1 viral load of < 400 copies/mL and an increase in the CD4 count of >100 x 10(6) cells/L was shorter for RTV plus SQV compared with single PI regimens (log rank test for each endpoint P < 0.05). The adjusted hazard ratios of RTV plus SQV compared with single PI regimens were 1.21 (95% confidence interval 0.99-1.47) for achieving an HIV-1 viral load of < 400 copies/mL, 1.12 (0.88-1.42) for an increase in the CD4 count of > 100 cells/microL, and 0.90 (0.73-1.11) for change of first PI regimen. CONCLUSIONS: Treatment with RTV plus SQV compared with single PI regimens appeared to give similar results for virological or immunological response.

Determination of lopinavir and nevirapine by high-performance liquid chromatography after solid-phase extraction: application for the assessment of their transplacental passage at delivery.

An adaptation of the HPLC method previously described for the simultaneous assay of amprenavir, ritonavir, indinavir, saquinavir, nelfinavir and efavirenz after solid-phase extraction is proposed here for the separate analysis of the newer PI lopinavir (LPV) and the NNRTI nevirapine (NVP). After viral inactivation by heat (60 degrees C for 60 min), plasma (600 microl), with clozapine added as internal standard, is diluted 1+1 with phosphate buffer pH 7 and subjected to a solid-phase extraction on a C(18) cartridge. Matrix components are eliminated with 2 x 500 microl of a solution of 0.1% H(3)PO(4) neutralised with NaOH to pH 7. LPV and NVP are eluted with 3 x 500 microl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 100 microl MeOH 50%. A 40-microl volume is injected onto a Nucleosil 100, 5 microm C(18) AB column. LPV and NVP are analysed separately using a gradient elution program with solvents constituted of MeCN and phosphate buffer adjusted to pH 5.07 and containing 0.02% sodium heptanesulfonate. LPV and NVP are detected by UV at 201 and 282 nm, respectively. The calibration curves are linear up to 10 microg/ml. The mean absolute recovery of LPV and NVP is 91% and 88%, respectively. The method is precise with mean inter-day C.V.s within 2.1-6.6% and 0.9-1.7% for LPV and NVP, and accurate (range of inter-day deviations -1.1 to +2.4%, and -1.9 to +0.8%, for LPV and NVP, respectively). The method has been validated and is currently applied to the monitoring of LPV and NVP in HIV patients, and has been notably applied in a study aimed at assessing the extent of transplacental passage of nevirapine and PIs, notably lopinavir, at the time of delivery in pregnant HIV-infected women.

New developments in laboratory monitoring of HIV-1 infection.

The laboratory tests currently available to the clinician for day-to-day management of HIV infection are generally limited to the measurement of the viral load and of the CD4 cell count. More recently, analysis of drug resistance and of plasma drug levels have been added to the monitoring armamentarium. There are, however, numerous other techniques currently available to researchers that may in the future be incorporated into clinical routine. These include the analysis of human and viral genetic determinants of disease evolution, detailed analyses of immune recovery and reserve, pharmacogenetic determinants of treatment response, and toxicity. These approaches may in the future provide highly individualized disease management.