Protease Inhibitor Drugs Research Articles

Resistencia a drogas antirretrovirales en pacientes expuestos a terapia antirretroviral en Cali, Colombia, 2008-2010

Introduction: Little has been published in Colombia on HIV drug resistance in patients taking antiretroviral treatment (ART). Currently, the Colombian guidelines do not recommend the use of genotypic antiretroviral resistance tests (GART) for treatment-naive patients or for those experiencing a first therapeutic failure.Objective: To determine the frequency of relevant resistance mutations and the degree of susceptibility/resistance of HIV to antiretroviral drugs (ARVs) in ART-experienced patients.Materials and methods: A non-random sample of 170 ART-experienced HIV patients with virologic failure and who underwent GART was recruited. A study of HIV drug resistance was carried out in two groups of patients: one group that underwent early GART and the other group that received late GART testing.Results: The most frequent type of resistance affected the non-nucleoside class (76%). The late-GART group had higher risk of nucleoside analog and protease inhibitor drug resistance, a higher number of resistance mutations and more complex mutational profiles than the early-GART group. A high cross resistance level (30%) was found in the nucleoside analog class. The least affected medications were tenofovir and darunavir.Conclusions: Our results suggest that performing GART late is associated with levels of ARV resistance that could restrict the use of an important number of essential ARV in subsequent regimens. There is a need to revise the current recommendations to include GART prior to start of treatment and after the first virologic failure. doi: http://dx.doi.org/10.7705/biomedica.v33i4.1462

Implications of baseline polymorphisms for potential resistance to NS3 protease inhibitors in Hepatitis C virus genotypes 1a, 2b and 3a

The future interferon-free treatment of hepatitis C virus (HCV) infection could include NS3 protease inhibitors (PIs) for potent pan-genotypic effect. We studied the prevalence of pre-existing PI resistance associated amino acid variants (RAVs) in 126 treatment-naive patient samples of HCV genotypes 1a, 2b and 3a, the most common genotypes in Sweden. The NS3 genes were each amplified by nested PCR method with degenerated primers to enable a broad genotype analysis. Population sequencing method was used, and the sequences were aligned with the NS3 sequence from HCV genotype 1a H77 strain. Interpretation of fold-change resistance to NS3 candidate drugs were done from already published phenotypic resistance data. The prevalence of known PI RAVs at baseline in genotype 1a was 28% (15/53), either single (V36L or Q80K/R) or combinations (T54A/S and V55A/I) of mutation(s). In genotype 2b, specific mutations like V36L, Q80G and S122R of viral NS3 protease gene were found in 100% (11/11). These may be the natural polymorphisms unique to genotype 2b. Similarly, specific mutations like V36L and D168Q were found uniquely in all 3a samples (30/30). The natural PI RAVs found in genotype 1a, although with relatively weak resistance, could still render up to 10-fold-resistance to the approved (boceprevir and telaprevir) and the 2nd generation PIs (faldaprevir and simeprevir). Moreover, the natural polymorphisms in genotype 2b (i.e. S122R) and 3a (i.e. D168Q), with inherent PI drug resistance of up to 20 and 700 fold respectively, would explain why current PIs are primarily directed against genotype 1.

Prevalence of HIV-1 drug resistance among women screening for HIV prevention trials in KwaZulu-Natal, South Africa (MTN-009).

BackgroundA major concern with using antiretroviral (ARV)-based products for HIV prevention is the potential spread of drug resistance, particularly from individuals who are HIV-infected but unaware of their status. Limited data exist on the prevalence of HIV infection or drug resistance among potential users of ARV-based prevention products.MethodsA cross-sectional study of reproductive-aged women who presented to screen for an HIV prevention trial was conducted at 7 clinical sites in Durban, South Africa. CD4+T cell counts, HIV-1 RNA levels and population sequencing of the protease and reverse transcriptase genes were performed for all women with 2 positive HIV rapid tests. Resistance mutations were identified using the Stanford Calibrated Population Resistance Tool.ResultsOf the 1073 evaluable women, 400(37%) were confirmed as HIV-infected. Of those, plasma HIV-1 RNA was detectable in 365/400(91%) and undetectable(<40 copies/ml) in 35/400(9%) women. 156 women(39%) were eligible for antiretroviral therapy (CD4+T cell counts<350 cells/mm3) and 50(13%) met criteria for AIDS(CD4<200 cells/mm3). Of 352 plasma samples(>200 copies/ml) analyzed for drug resistance, 26(7.4%) had nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI) or protease inhibitor (PI) drug resistance mutations. Among those with resistance, 18/26 participants(62%) had single-class NNRTI resistance and 5/26(19%) had dual-class NRTI/NNRTI. Major mutations in reverse transcriptase included K65R(n = 1), L74I(n = 1), K103N(n = 19), V106M(n = 4), Y181C(n = 2), M184V(n = 4), and K219E/R(n = 2). Major PI-resistance mutations were rare: M46L(n = 1) and I85V(n = 1). All participants were infected with subtype C virus, except one infected with subtype A.ConclusionsIn women from Durban, South Africa screening for an HIV prevention trial, the HIV prevalence was high (37%) and HIV drug resistance prevalence was above 5%. This study highlights the potential challenges faced when implementing an ARV-based prevention product that overlaps with first-line antiretroviral therapy. Effective screening to exclude HIV infection among women interested in uptake of ARV-based HIV prevention will be essential in limiting the spread of ARV resistance.

CPC-062 Hepatitis C and Adherence

BackgroundGood adherence to hepatitis C treatment seems necessary to obtain a successful treatment, increasing sustained virological response (SVR) rates.Purpose To assess the adherence to chronic hepatitis C treatment.Materials and Methods...

High drug resistance prevalence among vertically HIV-infected patients transferred from pediatric care to adult units in Spain.

BackgroundAntiretroviral treatment (ART) has contributed to increased life expectancy of HIV-1 infected children. In developed countries, an increasing number of children reaching adulthood are transferred to adult units. The objectives were to describe the demographic and clinical features, ART history, antiviral drug resistance and drug susceptibility in HIV-1 perinatally infected adolescents transferred to adult care units in Spain from the Madrid Cohort of HIV-1 infected children.MethodsClinical, virological and immunological features of HIV-1 vertically infected patients in the Madrid Cohort of HIV-infected children were analyzed at the time of transfer. Pol sequences from each patient were recovered before transfer. Resistance mutations according to the InternationaI AIDS Society 2011 list were identified and interpreted using the Stanford algorithm. Results were compared to the non-transferred HIV-1 infected pediatric cohort from Madrid.ResultsOne hundred twelve infected patients were transferred to adult units between 1997 and 2011. They were mainly perinatally infected (93.7%), with a mean nadir CD4+-T-cells count of 10% and presented moderate or severe clinical symptoms (75%). By the time of transfer, the mean age was 18.9 years, the mean CD4+T-cells count was 627.5 cells/ml, 64.2% presented more than 350 CD4+T-cells/ml and 47.3% had ≤200 RNA-copies/ml. Most (97.3%) were ART experienced receiving Highly Active ART (HAART) (84.8%). Resistance prevalence among pretreated was 50.9%, 76.9% and 36.5% for Protease Inhibitors (PI), Nucleoside Reverse Transcriptase Inhibitors (NRTI) and Non-NRTI (NNRTI), respectively. Resistance mutations were significantly higher among transferred patients compared to non-transferred for the PI+NRTI combination (19% vs. 8.4%). Triple resistance was similar to non-transferred pediatric patients (17.3% vs. 17.6%).ConclusionDespite a good immunological and virological control before transfer, we found high levels of resistance to PI, NRTI and triple drug resistance in HIV-1 infected adolescents transferred to adult units.

HIV protease inhibitors in combination with boceprevir

HIV protease inhibitors in combination with boceprevir

Short Communication: Emerging Transmitted HIV Type 1 Drug Resistance Mutations Among Patients Prior to Start of First-Line Antiretroviral Therapy in Middle and Low Prevalence Sites in China

It is known that transmitted drug resistance (TDR) will most likely emerge in regions where antiretroviral therapy (ART) has been widely available for years. However, after a decade of rapid scale-up of ART in China, there are few data regarding TDR among HIV-infected patients prior to initiating first-line ART in China. A prospective, observational cohort study was performed at sentinel sites in five provinces or municipalities. Study participants were recruited at the county- or city-level centers for disease control (CDCs), during routine monitoring visits following referral from diagnosing parties (e.g., hospitals). Each province or municipality recruited 140 patients through sequential sampling throughout the 2011 calendar year. A total of 627 eligible subjects were included in the analysis. the median CD4(+) cell count was 206 cells/ml at the baseline survey. The majority of patients (93.5%) had plasma HIV viral load ≥1,000 copies/ml. Of the 627 patients, 17 (2.7%) had drug resistance mutations for any type of HIV drugs. The prevalence of drug resistance mutations to nonnucleoside reverse transcriptase inhibitor (NNRTI) drugs (8/627, 1.3%) was higher than to nucleoside reverse transcriptase inhibitor (NRTI) drugs (5/627, 0.8%) and protease inhibitor (PI) drugs (4/627, 0.6%). A logistic regression model showed that the only predictive factor was the route of infection through homosexual intercourse, i.e., men who have sex with men (MSM) status. As HIV prevalence is rising rapidly among Chinese MSM, it is essential to continue surveying this risk group and related high-risk populations with low awareness of HIV, and to develop new public health interventions that help to reduce the spread of drug-resistant HIV.

Characterization of Hepatitis C Virus (HCV) Quasispecies Dynamics upon Short-Term Dual Therapy with the HCV NS5B Nucleoside Polymerase Inhibitor Mericitabine and the NS3/4 Protease Inhibitor Danoprevir

In the INFORM-1 study, 73 patients with chronic hepatitis C virus infection received mericitabine plus danoprevir for up to 13 days. Seventy-two patients experienced a continuous decline in HCV RNA levels during treatment, and of these patients, 14 had viral loads that remained >1,000 IU/ml by day 13 and 1 met the definition for viral breakthrough. In-depth NS5B and NS3/4A population and clonal sequencing studies and mericitabine and danoprevir drug susceptibility testing were performed to assess the variability and quasispecies dynamics before and upon monotherapy or dual therapy. Sequence analysis of the viral quasispecies indicated that the mericitabine resistance mutation S282T was not present at baseline, nor was it selected (even at a low level) during treatment. Protease inhibitor resistance mutations, either as predominant or as minority species, were detected in 18 patients at baseline. No enrichment of minority protease inhibitor-resistant variants present at baseline was observed during treatment; viral population samples were fully susceptible to mericitabine and/or danoprevir, despite the presence within their quasispecies of minority variants confirmed to have reduced susceptibility to danoprevir or other protease inhibitors. It was also observed that certain NS3 amino acid substitutions affected protease inhibitor drug susceptibility in a compound-specific manner and varied with the genetic context. In summary, the slower kinetics of viral load decline observed in some patients was not due to the selection of danoprevir or mericitabine resistance during treatment. Over 2 weeks' therapy, mericitabine suppressed the selection of danoprevir resistance, results that could differ upon longer treatment periods.

Ginkgolic acid inhibits HIV protease activity and HIV infection in vitro

SummaryBackgroundSeveral HIV protease mutations, which are resistant to clinical HIV protease inhibitors (PIs), have been identified. There is a great need for second-generation PIs with different chemical structures and/or with an alternative mode of inhibition. Ginkgolic acid is a natural herbal substance and a major component of the lipid fraction in the nutshells of the Ginkgo biloba tree. The objective of this study was to determine whether ginkgolic acid could inhibit HIV protease activity in a cell free system and HIV infection in human cells.Material/MethodsPurified ginkgolic acid and recombinant HIV-1 HXB2 KIIA protease were used for the HIV protease activity assay. Human peripheral blood mononuclear cells (PBMCs) were used for HIV infection (HIV-1SF162 virus), determined by a p24gag ELISA. Cytotoxicity was also determined.ResultsGinkgolic acid (31.2 μg/ml) inhibited HIV protease activity by 60%, compared with the negative control, and the effect was concentration-dependent. In addition, ginkgolic acid treatment (50 and 100 μg/ml) effectively inhibited the HIV infection at day 7 in a concentration-dependent manner. Ginkgolic acid at a concentration of up to 150 μg/ml demonstrated very limited cytotoxicity.ConclusionsGinkgolic acid effectively inhibits HIV protease activity in a cell free system and HIV infection in PBMCs without significant cytotoxicity. Ginkgolic acid may inhibit HIV protease through different mechanisms than current FDA-approved HIV PI drugs. These properties of ginkgolic acid make it a promising therapy for HIV infection, especially as the clinical problem of viral resistance to HIV PIs continues to grow.

PIN43 The Total Cost of HIV Patients Treated with ARV Therapy: Real World Evidence from Three Italian Administrative Databases

To calculate the cost of Human Immunodeficiency Virus (HIV) patients treated with Anti-Retroviral therapy (ARV) including medications, hospitalizations, tests, and specialist visits over a 12 months follow-up period, 3 Italian Local Health Units databases were analysed. All records (patients ≥18 years) between January 1, 2008 and December 31, 2009 associated with nucleoside analogue reverse-transcriptase inhibitor (NRTI), non-nucleoside analogue reverse-transcriptase inhibitor (NNRTI), protease inhibitor (PI), or other drugs in ATC J05A group, were included. Data and costs were collected for medications, hospitalizations, diagnostic tests, and specialist visits for the 12 months after the first ARV prescription (follow-up). Records of treatment for the 24 month prior to the first ARV prescription were classified as naïve or experienced. Costs-per-unit for resource use were collected from DRGs, National Tariffs and Drugs Formulary. A total of 779 records were analyzed 515 male (46.4±9.7years) and 264 female (42.0±9.2years). Records were classified as naïve (12,7%) and experienced (87,3%). The most prescribed regimens were Efavirenz+Tenofovir/Emtricitabine (TDF/FTC) (22.1%), Atazanavir±ritonavir(r)+ TDF/FTC (17.2%), and Lopinavir/r+TDF/FTC (11.2%). No switching of therapy during the follow-up was found in 78.2% of the records. Amid non-switcher records, the annual average total cost (medications, hospitalizations, tests, and specialist visits) was €9,103.82±5,302.11, including €7,099.70 for ARV therapy (77%), €631.65 for HIV-related hospitalizations (7%), and €551.49 for HIV-related diagnostic tests/specialist visits (6%). Total costs for Efavirenz+TDF/FTC, Atazanavir±r+TDF/FTC and Lopinavir/r+TDF/FTC regimens amounted to 7,637.40€, 11,257.00€ and 9,426.94€ respectively, with higher total costs being associated with Atazanavir±r+TDF/FTC. In this administrative databases analysis, the annual total average cost of HIV patients was significantly influenced by specific ARV medications, suggesting that total cost of therapies could differ significantly from drug acquisition cost of a single drug. A payer's perspective should include all direct costs and not only drug acquisition costs.

A Randomized Clinical Trial Evaluating Therapeutic Drug Monitoring (TDM) for Protease Inhibitor–Based Regimens in Antiretroviral-Experienced HIV-Infected Individuals: Week 48 Results of the A5146 Study

Background: We devised an open-label, randomized trial to evaluate whether therapeutic drug monitoring (TDM) of protease inhibitors (PIs) and dose escalation based upon a normalized inhibitory quotient (NIQ), which integrates PI trough concentration and drug resistance, could improve virologic outcome in PI-experienced patients with treatment failure. Secondary analyses through 48 weeks are presented.Methods: Eligible HIV-infected subjects with a screening viral load of ≥1000 copies/mL initiated a new PI-based regimen at entry and had NIQ performed at week 2. Subjects with an NIQ ≤1 were randomized at week 4 to a standard-of-care (SOC) arm or TDM arm featuring PI dose escalation.Results: One hundred and eighty-three subjects were randomized. There was no significant treatment difference in change from randomization to week 48 in HIV-1 RNA [ P = .13, median (25th, 75th percentile log₁₀ copies/mL change): -0.03 (-0.74, 0.62) with TDM and 0.11 (-2.3, 0.82) with SOC]. In subgroup analysis, patients with ≥0.69 active PIs benefited from TDM compared to those with <0.69 active PIs ( P = .05).Conclusions: While the TDM strategy of PI dose escalation did not improve virologic response at week 48 overall, in subgroup analysis, TDM favorably impacted virologic outcome in subjects taking PI-based regimens with moderate antiviral activity.

Tetrahydrofuran, tetrahydropyran, triazoles and related heterocyclic derivatives as HIV protease inhibitors.

HIV/AIDS remains a formidable disease with millions of individuals inflicted worldwide. Although treatment regimens have improved considerably, drug resistance brought on by viral mutation continues to erode their effectiveness. Intense research efforts are currently underway in search of new and improved therapies. This review is concerned with the design of novel HIV-1 protease inhibitors that incorporate heterocyclic scaffolds and which have been reported within the recent literature (2005-2010). Various examples in this review showcase the essential role heterocycles play as scaffolds and bioisosteres in HIV-1 protease inhibitor drug development. This review will hopefully stimulate the widespread application of these heterocycles in the design of other therapeutic agents.

Surveillance of Transmitted HIV Type 1 Drug Resistance Among HIV Type 1-Positive Women Attending an Antenatal Clinic in Kakinada, India

The World Health Organizations HIV Drug Resistance (WHO HIVDR) Threshold survey method was used to assess transmitted HIVDR in newly diagnosed HIV-1-infected primigravida women attending the Prevention of Parent to Child Transmission (PPTCT) centers in Kakinada, in whom it is likely that the infection had recently occurred. Out of the 56 consecutively collected eligible specimens, 51 were tested using the ViroSeq RT-PCR method (Abbott Germany) to obtain 47 consecutive sequences for the HIV-1 protease (PR) and reverse transcriptase (RT) region. As per the 2009 WHO list of mutations for surveillance of transmitted HIVDR, only one nonnucleoside reverse transcriptase inhibitor (NNRTI) mutation was detected at K101E from all specimens tested, suggesting a low prevalence (<5%) of resistance to NNRTIs and no mutations were detected at other sites, suggesting a low prevalence (<5%) of resistance to nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitors (PI) drug classes as well. Phylogenetic analysis showed all sequences belonged to HIV-1 subtype C. In the wake of antiretroviral treatment (ART) scale-up, future evaluation of transmitted HIVDR is essential in Kakinada as well as in other regions of India.

Desarrollo y validación de un método para la determinación de darunavir en plasma mediante LC-MS/MS

Introduction Darunavir is a latest generation of protease inhibitors (PI) drugs used as a treatment of HIV infection owing to its improved efficacy and its better tolerance among other characteristics. Several studies have demonstrated a correlation between the dose and the effect of darunavir, although a therapeutic range for the drug concentrations has not yet been established. These factors, besides the high inter-individual variability and the adverse effects resulting from drug interactions, reinforce the need for having a sensitive method for monitoring its plasma concentrations in treated patients. Objective Technical validation of a new method for darunavir plasma concentration monitoring by LC/MS/MS. Materials and methods The method validation procedure was based on the recommendations published in the guidelines ICH Topic Q 2B Validation of Analytical Procedures: Methodology ( CPMP/ICH/281/95). Darunavir was extracted from plasma samples by a protein precipitation procedure. The chromatographic separation was achieved with a gradient program (acetonitrile/buffer) on an X-Bridge TM C18 3.5 μm 2.1×100 mm (Waters ®). Analytes quantification is performed by electrospray ionization in positive mode, a Quattro Micro triple quadrupole mass spectrometer. Results Linearity (0.1-10 μg/mL): y = 18.85×-150.4 r 2: 0.997. Precision: Intra-assay CV: 1.07-4.62%. Inter-assay CV: 2.72-4.70%. Accuracy: Intra-assay: 1-9%. Inter-assay: 2-7%. LOD: 0.05 μg/mL, LLOQ: 0.15 μg/mL. Conclusion The developed method based on LC/MS/MS has an adequate sensibility and reproducibility for the determination of plasma concentrations of darunavir in treated patients.

Three Residues in HIV-1 Matrix Contribute to Protease Inhibitor Susceptibility and Replication Capacity

Other than cleavage site mutations, there is little data on specific positions within Gag that impact on HIV protease inhibitor susceptibility. We have recently shown that non-cleavage site mutations in gag, particularly within matrix protein can restore replication capacity and further reduce protease inhibitor drug susceptibility when coexpressed with a drug-resistant (mutant) protease. The matrix protein of this patient-derived virus was studied in order to identify specific changes responsible for this phenotype. Three amino acid changes in matrix (R76K, Y79F, and T81A) had an impact on replication capacity as well as drug susceptibility. Introduction of these three changes into wild-type (WT) matrix resulted in an increase in the replication capacity of the protease mutant virus to a level similar to that achieved by all the changes within the mutant matrix and part of the capsid protein. Pairs of changes to wild-type matrix led to an increased replication capacity of the protease mutant (although less than with all three changes). Having only these three changes to matrix in a wild-type virus (with wild-type protease) resulted in a 5- to 7-fold change in protease inhibitor 50% effective concentration (EC₅₀). Individual changes did not have as great an effect on replication capacity or drug susceptibility, demonstrating an interaction between these positions, also confirmed by sequence covariation analysis. Molecular modeling predicts that each of the three mutations would result in a loss of hydrogen bonds within α-helix-4 of matrix, leading to the hypothesis that more flexibility within this region or altered matrix structure would account for our findings.

Virologic outcomes of first-line HAART and associated factors among Chinese patients with HIV in three sentinel antiretroviral treatment sites

To evaluate HIV drug resistance (HIVDR) among Chinese patients with HIV receiving first-line highly active antiretroviral therapy (HAART). Based on the WHO HIVDR surveys, a prospective cohort study with 12-month follow-up was conducted to estimate the prevalence of HIV RNA<1000 copies/ml and HIVDR. A total of 341 study subjects naïve to prior antiretroviral therapy (ART) were followed up for a median of 12.1 months. The overall mortality rate was 9.9 per 100 person-years. The median of CD4 counts increased from 182 cells/mm(3) at baseline to 268 cells/mm(3) at 12 months (P<0.0001). Of patients with plasma HIV-1 RNA concentrations ≥1000 copies/ml at 12 months, the proportions of resistance to non-nucleoside reverse transcriptase drugs, nucleoside/nucleotide reverse transcriptase inhibitors, and protease inhibitor drugs were 34.2%, 23.7% and 0%, respectively. The overall proportion of HIV RNA<1000 copies/ml was 85.7% at 12 months. Occupation of farmer (AOR=0.3, 95% CI: 0.08, 0.94; P=0.0393) and HAART counselling and instruction through telephone (AOR=2.8, 95% CI: 1.4, 5.6; P=0.0047) were significantly associated with HIV RNA<1000 copies/ml. Our study demonstrated that the community-based ART had significant effects on viral suppression and immune recovery. HIVDR should be monitored in the long term to guide informed decisions on preventing HIVDR and choices of first- and second-line regimens.

Recent Advances in Hepatitis C Virus Treatment: Review of HCV Protease Inhibitor Clinical Trials

Hepatitis C virus (HCV) infection affects millions of people world-wide, and chronic infection can result in end-stage liver disease and hepatocellular carcinoma. Conventional therapy to date has involved combination antiviral therapy including alpha-interferon and ribavirin; response rates with these drugs are variable based on both viral and host factors, such as HCV viral load, HCV genotype, HIV co-infection, host genetic polymorphisms (such as those in the IL28B region), and other factors. Recent advances in HCV treatment have included pegylated forms of alpha-interferon and, more recently, the development of specifically targeted antiviral therapy for HCV (STAT-C) with novel HCV protease inhibitors (PIs) for genotype 1 HCV. Although unlikely to be administered as monotherapy due to the potential for development of HCV PI drug resistance mutations, results of phase II trials of two PIs in development have recently been reported, demonstrating promising therapeutic efficacy of HCV PIs in combination with established conventional treatment. This review outlines the advances and the challenges in the development of these HCV PIs as effective HCV antiviral agents and their role in clinical practice.

Neither ritonavir nor darunavir affect cell surface expression of tetherin or Vpu-mediated tetherin down-modulation

Neither ritonavir nor darunavir affect cell surface expression of tetherin or Vpu-mediated tetherin down-modulation

Viremia and drug resistance among HIV-1 patients on antiretroviral treatment: a cross-sectional study in Soweto, South Africa.

We assessed risk factors for viremia and drug resistance among long-term recipients of antiretroviral therapy (ART) in South Africa. In 2008, we conducted a cross-sectional study among patients receiving ART for 12 months or more. Genotypic resistance testing was performed on individuals with a viral load higher than 400 RNA copies/ml. Multiple logistic regression analysis was used to assess associations. Of 998 participants, 75% were women with a median age of 41 years. Most (64%) had been on treatment for more than 3 years. The prevalence of viremia was 14% (n = 139): 12% (102/883) on first-line [i.e. nonnucleoside reverse-transcriptase inhibitor (NNRTI)-based regimen] and 33% (37/115) on second-line (i.e. protease inhibitor (PI)-based regimen) ART. Of viremic patients, 78% had drug resistance mutations. For NRTIs, NNRTIs and PIs, the prevalence of mutations was 64, 81 and 2%, respectively, among first-line failures and 29, 54 and 6%, respectively, among second-line failures. M184V/I, K103N and V106A/M were the most common mutations. Significant risk factors associated with viremia on first-line regimen included concurrent tuberculosis treatment [odds ratio (OR) 6.4, 95% confidence interval (CI) 2.2-18.8, P < 0.01] and a recent history of poor adherence (OR 2.7, 1.3-5.6, P = 0.01). Among second-line failures, attending a public clinic (OR 4.6, 95% CI 1.8-11.3, P < 0.01) and not having a refrigerator at home (OR 6.7, 95% CI 1.2-37.5, P = 0.03) were risk factors for virological failure. Risk factors for viral failure were line regimen dependent. Second-line ART recipients had a higher rate of viremia, albeit with infrequent PI drug resistance mutations. Measures to maintain effective virologic suppression should include increased adherence counseling, attention to concomitant tuberculosis treatment and heat-stable formulations of second-line ART regimens.

Ultrafast and high-throughput mass spectrometric assay for therapeutic drug monitoring of antiretroviral drugs in pediatric HIV-1 infection applying dried blood spots

Kaletra® (Abott Laboratories) is a co-formulated medication used in the treatment of HIV-1-infected children, and it contains the two antiretroviral protease inhibitor drugs lopinavir and ritonavir. We validated two new ultrafast and high-throughput mass spectrometric assays to be used for therapeutic drug monitoring of lopinavir and ritonavir concentrations in whole blood and in plasma from HIV-1-infected children. Whole blood was blotted onto dried blood spot (DBS) collecting cards, and plasma was collected simultaneously. DBS collecting cards were extracted by an acetonitrile/water mixture while plasma samples were deproteinized with acetone. Drug concentrations were determined by matrix-assisted laser desorption/ionization-triple quadrupole tandem mass spectrometry (MALDI-QqQ-MS/MS). The application of DBS made it possible to measure lopinavir and ritonavir in whole blood in therapeutically relevant concentrations. The MALDI-QqQ-MS/MS plasma assay was successfully cross-validated with a commonly used high-performance liquid chromatography (HPLC)–ultraviolet (UV) assay for the therapeutic drug monitoring (TDM) of HIV-1-infected patients, and it showed comparable performance characteristics. Observed DBS concentrations showed as well, a good correlation between plasma concentrations obtained by MALDI-QqQ-MS/MS and those obtained by the HPLC-UV assay. Application of DBS for TDM proved to be a good alternative to the normally used plasma screening. Moreover, collection of DBS requires small amounts of whole blood which can be easily performed especially in (very) young children where collection of large whole blood amounts is often not possible. DBS is perfectly suited for TDM of HIV-1-infected children; but nevertheless, DBS can also easily be applied for TDM of patients in areas with limited or no laboratory facilities.