Low-density lipoprotein and ritonavir: an interaction between antiretrovirals and lipids mediated by P-glycoprotein.

BACKGROUND: Antiretroviral therapy has considerably reduced HIV disease progression, but complete eradication of HIV cannot actually be achieved. Moreover, prolonged use of protease inhibitors (PIs) causes profound changes in lipid metabolism with an increased risk of cardiovascular diseases. P-glycoprotein (P-gp) is expressed on many cell types, playing an important role in the efflux of drugs including PIs, limiting their intracellular concentration. Furthermore, several studies showed that P-gp is involved in lipid homeostasis and its activity is regulated by cholesterol. METHODS: THP-1 monocytes were used to study: (i) the influence of low-density lipoprotein (LDL) on P-gp expression and function, assessed by flow cytometry and quantitative real-time PCR analysis and measuring ritonavir and rhodamine-123 dye efflux, respectively; and (ii) the influence of ritonavir on cholesterol mobilization. The intracellular levels of ritonavir or cholesterol were measured by HPLC-UV and filipin staining, respectively. RESULTS: In THP-1 cells, LDL was able to yield an increase in both P-gp expression and activity. THP-1 cells treated with LDL showed a decrease in the intracellular ritonavir concentration in a dose-dependent manner. Notably, ritonavir induced reduced cholesterol mobilization in THP-1 cells, probably due to its inhibitory action on P-gp activity. CONCLUSIONS: Our data indicate a potential interplay between LDL and ritonavir mediated by P-gp. This interaction could influence both therapy effectiveness and cellular lipid metabolism, with important implications in the management of HIV patients treated with boosted PIs.

Determination of telaprevir in plasma of HCV-infected patients by HPLC-UV.

Telaprevir is a direct acting antiviral agent, used with pegylated interferon and ribavirin for the management of chronic hepatitis C virus (HCV) genotype 1 infection, in patients not responding to therapy with pegylated interferon and ribavirin only. Although 75% of patients achieve a sustained virological response after treatment with telaprevir, adverse drug-drug interactions and undesirable events often occur. Therefore, telaprevir monitoring is pivotal to improve the management of HCV infection. Here, the first High-Performance Liquid Chromatography-Ultraviolet (HPLC-UV) method to quantify telaprevir in human plasma of HCV-genotype 1-infected patients is reported. The volume of the plasma sample was 700 µL. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (divinylbenzene and N-vinylpyrrolidone). The extracted samples were reconstituted with 150 µL of 60/40 water/acetonitrile. Thirty microliters of these samples was injected into a HPLC-UV system, and the analytes were eluted on a X Terra(®) RP18 column (250 mm × 4.6 mm i.d.) with a particle size of 5 µm. The mobile phase (ammonium acetate buffer, 150 mM, pH 8.0, and methanol:acetonitrile 50:50) was delivered at 1.0 mL/min with linear gradient elution. The total run time for a single analysis was 16 min; telaprevir was detected by UV at 276 and 286 nm. The calibration curve was linear from 312.5 to 20,000 ng/mL (r(2) > 0.996). The absolute recovery of telaprevir ranged between 89 and 93% at concentrations of quality control samples of 800, 4,000, 8,000, and 16,000 ng/mL. Both precision and accuracy were always <15%. The HPLC-UV method reported here: (i) has been validated, (ii) is currently applied to monitor telaprevir in plasma of HCV-infected patients, and (iii) appears useful in a routine laboratory. ,

Varicella zoster virus infection presenting as isolated diplopia: a case report.

BACKGROUND: Involvement of trochlear nerve during Varicella Zoster Virus (VZV) Infection has been rarely described, and always in association with skin rash. CASE PRESENTATION: We describe the case of a patient with VZV infection presenting as isolated diplopia due to fourth cranial nerve palsy. The diagnosis has been obtained through the application of a standardized molecular diagnostic panel, and diplopia resolved after specific antiviral and corticosteroid therapy. CONCLUSION: This case evidences that clinicians should be aware of atypical VZV infection, even in the absence of the typical skin rash.

Study of genotypic and phenotypic HIV-1 dynamics of integrase mutations during raltegravir treatment: a refined analysis by ultra-deep 454 pyrosequencing.

BACKGROUND: The dynamics of raltegravir-resistant variants and their impact on virologic response in 23 HIV-1-infected patients, who started a salvage raltegravir-containing regimen, were investigated. METHODS: Integrase population sequencing and Ultra-Deep-454 Pyrosequencing (UDPS) were performed on plasma samples at baseline and at raltegravir failure. All integrase mutations detected at a frequency ≥1% were considered to be reliable for the UDPS analyses. Phylogenetic and phenotypic resistance analyses were also performed. RESULTS: At baseline, primary resistance mutations were not detected by both population and UDPS genotypic assays; few secondary mutations (T97A-V151I-G163R) were rarely detected and did not show any statistically association either with virologic response at 24-weeks or with the development of resistant variants at failure. At UDPS, not all resistant variants appearing early during treatment evolved as major populations during failure; only specific resistance pathways (Y143R-Q148H/R-N155H) associated with an increased rate of fitness and phenotypic resistance were selected. CONCLUSIONS: Resistance to raltegravir in integrase strand transfer inhibitor-naive patients remains today a rare event, which might be changed by future extensive use of such drugs. In our study, pathways of resistance at failure were not predicted by baseline mutations, suggesting that evolution plus stochastic selection plays a major role in the appearance of integrase-resistance mutations, whereas fitness and resistance are dominant factors acting for the late selection of resistant quasispecies.

Viral tropism by geno2pheno as a tool for predicting CD4 decrease in HIV-1-infected naive patients with high CD4 counts.

OBJECTIVES: To investigate the value of tropism (determined by genotypic testing) to predict CD4 depletion in HIV-infected antiretroviral-naive patients with high CD4 counts. METHODS: Viral tropism was determined by geno2pheno (false positive rate = 10%) in 223 HIV-infected subjects naive to antiretrovirals with CD4 count ≥350 cells/µL and HIV-RNA >500 copies/mL enrolled in the ICONA Foundation Study for whom a stored plasma sample (baseline) was retrospectively tested. We monitored CD4 cell count and identified predictors of decline before antiretroviral therapy initiation, applying a mixed linear model with covariates (age, gender, tropism, HIV risk factor, calendar year of HIV infection, months from HIV diagnosis to baseline, hepatitis C virus status, CD4 and HIV-RNA at sample collection and duration of follow-up). RESULTS: Two hundred and twenty-three subjects met the eligibility criteria; 137 (61%) were male and the median age was 35 (31-40) years. Median follow-up was 16.4 (3.2-37.2) months. Median CD4 decrease during follow-up was -157 (-278 to -13) cells/µL. At baseline, 192 (86%) subjects were defined as harbouring R5 virus and 31 (14%) non-R5. Median CD4 count was 571 (458-729) cells/µL and median HIV-RNA was 4.08 (3.57-4.55) log(10) copies/mL. At multivariable analysis, a greater mean CD4 decrease was associated with non-R5 viral tropism (-159.9 ± 12.22, P = 0.0002) at baseline. Other significant covariates were female gender, older age, intravenous drug use, longer duration of follow-up, and higher CD4 cell count and higher HIV-RNA at sample collection. CONCLUSIONS: In patients with CD4 counts ≥350 cells/µL, non-R5 viral tropism by geno2pheno is predictive of CD4 decrease independent of their viral set point and CD4 counts.

Simultaneous determination of lamivudine, lopinavir, ritonavir, and zidovudine concentration in plasma of HIV-infected patients by HPLC-MS/MS.

The nucleoside reverse transcriptase inhibitors lamivudine and zidovudine and the protease inhibitors lopinavir and ritonavir are currently used in anti-human immunodeficiency virus (HIV) therapy. Here, a high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) method, using a hybrid quadrupole time-of-flight mass analyzer, is reported for the simultaneous quantification of lamivudine, lopinavir, ritonavir, and zidovudine in plasma of HIV-infected patients. The volume of plasma sample was 600 µL. Plasma samples were extracted by solid-phase using 1 cc Oasis HLB Cartridge (divinylbenzene and N-vinylpyrrolidone) and evaporated in a water bath under nitrogen stream. The extracted samples were reconstituted with 100-µL methanol. Five microliters of the reconstituted samples were injected into a HPLC-MS/MS apparatus, and the analytes were eluted on a Vydac column (250 × 1.0 mm i.d.) filled with 3-µm C(18) particles. The mobile phase was delivered at 70 µL/min with a linear gradient elution, both acetonitrile and ultrapure water solvents contained 0.2% formic acid. The calibration curves were linear from 0.47 to 20 ng/mL. The absolute recovery ranged between 91 and 107%. The minimal concentration of lamivudine, lopinavir, ritonavir, and zidovudine detectable by HPLC-MS/MS is 0.47, 0.28, 0.30, and 0.66 ng/mL, respectively. The great advantage of the new HPLC-MS/MS method here reported is the possibility to achieve a very high specificity toward the selected anti-HIV drugs, despite the simple and rapid sample preparation. Moreover, this method is easily extendible to the analysis of co-administrated drugs.

Ultra-deep sequencing reveals hidden HIV-1 minority lineages and shifts of viral population between the main cellular reservoirs of the infection after therapy interruption.

Viral quasispecies population dynamics between monocytes and T-lymphocytes were analyzed in patients after highly active antiretroviral therapy (HAART) interruption, during a follow-up of 3-6 months. V3 env region underwent ultra-deep pyrosequencing. Co-receptor usage prediction was performed by Position Specific Score Matrix Analysis. Phylogenetic trees were constructed to evaluate the relationships between the variants. Gene flow was also investigated. Even though at the moment of therapy interruption monocyte-derived HIV-1 genomes presented higher genetic heterogeneity than that of T-lymphocytes, at subsequent times, this difference in genetic heterogeneity disappeared, due to different waves of expansion and reduction of quasispecies variability associated with monocytes and T-lymphocytes. Phylogenetic analysis and gene flow evaluation supported the hypothesis of extensive interchange of variants between cellular compartments of the infection. A spread of proviral X4 lineages hidden in monocytes to T cells was observed, but this was not associated with an overall shift towards CXCR4 using variants during the observation period.

Increase in standard cholesterol and large HDL particle subclasses in antiretroviral-naïve patients prescribed efavirenz compared to atazanavir/ritonavir.

BACKGROUND: Cardiovascular risk in HIV-infected patients is related, at least in part, to serum lipid alterations before and after HAART. Lipoprotein-particle subclasses may also have an effect, but comparative data after standard HAART regimens are limited. METHODS: This was a substudy of a trial in 91 antiretroviral-naïve patients randomized to tenofovir + emtricitabine + atazanavir/ritonavir (ATV/r) or efavirenz (EFV). Over-time trends from baseline to week 48 in total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), HDL particles (HDLp), and TC:HDL-C and TG:HDL-C ratios were analyzed by analysis of covariance (ANCOVA). Furthermore, confidence intervals for differences between the 2 groups at week 48 were calculated. Indications for lipid-lowering interventions and low HDL-C were also studied. RESULTS: ANCOVA showed that, with respect to patients receiving ATV/r, those prescribed efavirenz (EFV) had greater increases reported as mean differences in lipid values at week 48: 14 mg/dL (95% CI, 0.2 to 27) for TC, 14 mg/dL (95% CI, 4 to 25) for LDL-C, 5 mg/dL (95% CI, 2 to 9) for HDL-C, and 2.2 mg/dL (95% CI, 0.4 to 4) for large HDLp. Proportions of subjects with indications for lipid-lowering interventions and with HDL-C <40 mg/dL did not differ significantly. CONCLUSIONS: Patients prescribed EFV had greater increases in TC, LDL-C, and HDL-C. Although no significant differences were detected between the 2 groups for the TC:HDL ratio and for indications to start lipid-lowering interventions, large HDLp increased more in the EFV group compared to the ATV/r group, suggesting a protective effect associated with EFV use.

Haemolytic anaemia in an HIV-infected patient with severe falciparum malaria after treatment with oral artemether-lumefantrine.

Intravenous (i.v.) artesunate is now the recommended first-line treatment of severe falciparum malaria in adults and children by WHO guidelines. Nevertheless, several cases of haemolytic anaemia due to i.v. artesunate treatment have been reported. This paper describes the case of an HIV-infected patient with severe falciparum malaria who was diagnosed with haemolytic anaemia after treatment with oral artemether-lumefantrine.The patient presented with fever, headache, and arthromyalgia after returning from Central African Republic where he had been working. The blood examination revealed acute renal failure, thrombocytopaenia and hypoxia. Blood for malaria parasites indicated hyperparasitaemia (6%) and Plasmodium falciparum infection was confirmed by nested-PCR. Severe malaria according to the laboratory WHO criteria was diagnosed. A treatment with quinine and doxycycline for the first 12 hours was initially administered, followed by arthemeter/lumefantrine (Riamet(®)) for a further three days. At day 10, a diagnosis of severe haemolytic anaemia was made (Hb 6.9 g/dl, LDH 2071 U/l). Hereditary and autoimmune disorders and other infections were excluded through bone marrow aspiration, total body TC scan and a wide panel of molecular and serologic assays. The patient was treated by transfusion of six units of packed blood red cell. He was discharged after complete remission at day 25. At present, the patient is in a good clinical condition and there is no evidence of haemolytic anaemia recurrence.This is the first report of haemolytic anaemia probably associated with oral artemether/lumefantrine. Further research is warranted to better define the adverse events occurring during combination therapy with artemisinin derivatives.

Prospective evaluation of bone markers, parathormone and 1,25-(OH)2 vitamin D in HIV-positive patients after the initiation of tenofovir/emtricitabine with atazanavir/ritonavir or efavirenz.

BACKGROUND: Increased risk of fractures and osteoporosis have been associated with the use of antiretroviral drugs. There is a paucity of prospective evaluations of bone markers after the initiation of drugs currently recommended to treat HIV infection and results on the evolution of these markers are conflicting. Lastly, the effect of tenofovir on 1,25-(OH)2 vitamin D is uncertain. METHODS: We performed a prospective study on the evolution of bone markers, parathormone and 1,25-(OH)2 vitamin D before and after standard antiretroviral regimens. This was a sub-study of a trial conducted in antiretroviral-naïve patients randomized to tenofovir + emtricitabine in combination with either atazanavir/ritonavir (ATV/r) or efavirenz (EFV). Follow-up lasted 48 weeks. The following bone markers were analyzed: C-terminal cross-laps (CTx), osteocalcin (OC), osteoprotegerin (OPG), and receptor activator of nuclear factor κB ligand (RANKL). Mixed-factorial analysis of variance with random-coefficient general linear model was used to compare their trends over time and linear multivariable regression was performed with a backward selection method to assess predictors of their variations from baseline to week 48. Trends of parathormone and 1,25-(OH)2 vitamin D were also evaluated. RESULTS: Seventy-five patients were studied: 33 received EFV and 42 ATV/r. Significant increases were found for all markers except for RANKL. There was a significant direct association between CTx and OC increases. Multivariable analysis showed that higher glomerular filtration rate (estimated through cystatin C clearance) predicted greater OPG increase, while older age, higher HIV RNA at baseline and use of ATV/r predicted greater CTx increase. A significant increase of parathormone accompanied the evolution of the study markers. 1,25-(OH)2 vitamin D remained stable, though a seasonality variation was demonstrated. CONCLUSIONS: These data demonstrate CTx increase (bone resorption marker) corresponding to OC increase (bone formation marker) early upon HAART initiation. Moreover, predictors of bone marker increases have been suggested, possibly indicating that a stricter monitoring of bone health and pro-active interventions are needed in older patients, those with higher HIV RNA, prescribed ATV/r rather than EFV, and with decreased renal function at baseline. Further studies are needed to clarify the mechanisms responsible for up-regulation of bone turnover markers, as well as to understand if and what markers are best correlated or predictive of pathological fractures.

'Sentinel' mutations in standard population sequencing can predict the presence of HIV-1 reverse transcriptase major mutations detectable only by ultra-deep pyrosequencing.

OBJECTIVES: This proof-of-concept study aimed to identify whether mutations considered not yet relevant for drug resistance (but located at key drug-resistance positions) can act as 'sentinels' of minority resistant variants in HIV-1 drug-naive patients. METHODS: We focused our attention on three reverse transcriptase (RT) mutations (T69S, L210M and K103R) easily detected by standard population sequencing [i.e. the genotypic resistance test (GRT)]. Ultra-deep pyrosequencing (UDPS) of HIV-1 RT was performed using GS-FLX Roche, on plasma RNA from 40 drug-naive patients infected with HIV-1 subtype B without primary resistance detected by GRT. Only RT drug resistance mutations detected at >0.1% in both forward and reverse directions were considered. Associations between GRT sentinel mutations and UDPS drug resistance were assessed using Fisher's exact test. RESULTS: UDPS detected drug resistance mutations in 18/40 drug-naive patients. Patients carrying HIV-1 strains with T69S and L210M by GRT showed a trend to greater infection by minority drug-resistant variants than control patients infected by HIV-1 without these mutations (5/10 and 7/10 versus 3/10; P = not significant). No association was found for K103R by GRT. Notably, T69S and L210M (but not K103R or control viruses) were associated with GRT minority drug-resistant variants with a prevalence >1% (3/10 and 4/10 versus 0/20 in K103R and controls; P = 0.03 and P = 0.008, respectively). Moreover, the presence of L210M or T69S viruses by GRT significantly correlated with that of minority thymidine analogue mutations by UDPS (6/20 patients carrying HIV-1 strains with T69S/L210M versus 0/20 patients carrying HIV-1 having K103R or none of these mutations; P = 0.03). CONCLUSIONS: This proof-of-concept study suggests the existence of genetic markers, detectable by routine testing, potentially acting as sentinel mutations of minority drug resistance. Their identification may help in the selection of patients at high risk of resistance in reservoirs without the necessity of using UDPS.

Marked increase in etravirine and saquinavir plasma concentrations during atovaquone/proguanil prophylaxis.

The case of a 32-year-old Caucasian female with multi-drug resistant HIV-1 subtype B infection treated with a salvage regimen including maraviroc, raltegravir, etravirine and unboosted saquinavir who started atovaquone/proguanil prophylaxis, is reported. The potential interactions between atovaquone/proguanil and these anti-retroviral drugs are investigated. Pharmacokinetic analyses documented a marked increase in etravirine and saquinavir plasma concentrations (+55% and +274%, respectively), but not in raltegravir and maraviroc plasma concentrations. The evidence that atovaquone/proguanil significantly interacts with etravirine and saquinavir, but not with raltegravir and maraviroc, suggests that the mechanism of interaction is related to cytochrome P450.

A case of pulmonary tuberculosis presenting as diffuse alveolar haemorrhage: is there a role for anticardiolipin antibodies?

BACKGROUND: Diffuse alveolar haemorrhage (DAH) has been rarely reported in association with pulmonary infections. CASE PRESENTATION: We report the case of a 43 year old immunocompetent man presenting with dyspnoea, fever and haemoptysis. Chest imaging showed bilateral ground glass opacities. Microbiological and molecular tests were positive for Mycobacterium tuberculosis and treatment with isoniazid, rifampicin, ethambutol and pyrazinamide was successful. In this case the diagnosis of DAH relies on clinical, radiological and endoscopic findings. Routine blood tests documented the presence of anticardiolipin antibodies. In the reported case the diagnostic criteria of antiphospholipid syndrome were not fulfilled. CONCLUSIONS: The transient presence of anticardiolipin antibodies in association with an unusual clinical presentation of pulmonary tuberculosis is intriguing although a causal relationship cannot be established.

Use of novel antiretroviral agents in rescue regimens: a case of early virological failure to raltegravir.

In patients with virological failure during highly active antiretroviral therapy (HAART) and drug resistance, guidelines recommend the achievement of maximal virological suppression by the use of a new regimen with at least 2 active drugs. We describe the clinical outcome of a heavily antiretroviral-experienced patient who experienced early failure to raltegravir.

Massively parallel pyrosequencing highlights minority variants in the HIV-1 env quasispecies deriving from lymphomonocyte sub-populations.

BACKGROUND: Virus-associated cell membrane proteins acquired by HIV-1 during budding may give information on the cellular source of circulating virions. In the present study, by applying immunosorting of the virus and of the cells with antibodies targeting monocyte (CD36) and lymphocyte (CD26) markers, it was possible to directly compare HIV-1 quasispecies archived in circulating monocytes and T lymphocytes with that present in plasma virions originated from the same cell types. Five chronically HIV-1 infected patients who underwent therapy interruption after prolonged HAART were enrolled in the study. The analysis was performed by the powerful technology of ultra-deep pyrosequencing after PCR amplification of part of the env gene, coding for the viral glycoprotein (gp) 120, encompassing the tropism-related V3 loop region. V3 amino acid sequences were used to establish heterogeneity parameters, to build phylogenetic trees and to predict co-receptor usage. RESULTS: The heterogeneity of proviral and viral genomes derived from monocytes was higher than that of T-lymphocyte origin. Both monocytes and T lymphocytes might contribute to virus rebounding in the circulation after therapy interruptions, but other virus sources might also be involved. In addition, both proviral and circulating viral sequences from monocytes and T lymphocytes were predictive of a predominant R5 coreceptor usage. However, minor variants, segregating from the most frequent quasispecies variants, were present. In particular, in proviral genomes harboured by monocytes, minority variant clusters with a predicted X4 phenotype were found. CONCLUSION: This study provided the first direct comparison between the HIV-1 quasispecies archived as provirus in circulating monocytes and T lymphocytes with that of plasma virions replicating in the same cell types. Ultra-deep pyrosequencing generated data with some order of magnitude higher than any previously obtained with conventional approaches. Next generation sequencing allowed the analysis of previously inaccessible aspects of HIV-1 quasispecies, such as co-receptor usage of minority variants present in archived proviral sequences and in actually replicating virions, which may have clinical and therapeutic relevance.

Treatment with the fusion inhibitor enfuvirtide influences the appearance of mutations in the human immunodeficiency virus type 1 regulatory protein rev.

The gp41-encoding sequence of the env gene contains in two separate regions the Rev-responsive elements (RRE) and the alternative open reading frame of the second exon of the regulatory protein Rev. The binding of Rev to the RRE allows the transport of unspliced/singly spliced viral mRNAs out of the nucleus, an essential step in the life cycle of human immunodeficiency virus type 1 (HIV-1). In this study, we have investigated whether the fusion-inhibitor enfuvirtide (ENF) can induce mutations in Rev and if these mutations correlate with the classical ENF resistance gp41 mutations and with viremia and CD4 cell count. Specific Rev mutations were positively associated with ENF treatment and significantly correlated with classical ENF resistance gp41 mutations. In particular, a cluster was observed for the Rev mutations E57A (E57A(rev)) and N86S(rev) with the ENF resistance gp41 mutations Q40H (Q40H(gp41)) and L45M(gp41). In addition, the presence at week 48 of the E57A(rev) correlates with a significant viremia increase from baseline to week 48 and with a CD4 cell count loss from baseline to week 48. By modeling the RRE structure, we found that the Q40(gp41) and L45(gp41) codons form complementary base pairs in a region of the RRE involved in Rev binding. The conformation of this Rev-binding site is disrupted when Q40H(gp41) and L45M(gp41) occur alone while it is restored when both mutations are present. In conclusion, our study shows that ENF pressure may also affect both Rev and RRE structures and can provide an excellent example of compensatory evolution. This highlights the multiple roles of ENF (and perhaps other entry inhibitors) in modulating the correct interplay between the different HIV-1 genes and proteins during the HIV-1 life cycle.

Historical resistance profile helps to predict salvage failure.

BACKGROUND: This study compared the predictive value for treatment failure of extended resistance detected in the current genotype resistance test (GRT) versus those from GRT history in patients with multiple combination antiretroviral therapy (cART) failures. METHODS: Patients who underwent three GRT between 1999 and 2007 were included. Extended resistance at genotypic sensitivity score (GSS) using the Rega 7.1 interpretation system compared with a non-standard definition (defined as class-wide resistance [CWR] on the basis of International AIDS Society-USA mutations) was assessed both for current and historical GRTs (a combination of mutations was detected in all three tests). The predictive role of extended resistance for treatment failure was evaluated with an adjusted Cox proportional hazard model. RESULTS: Overall, 177 patients were included. The historical GRT increased the number of patients with extended resistance to all three major drug classes by 25% in comparison with the current GRT. Using the GSS method, the absence of detection of any active drug in any drug class was predictive of failure with both the current and historical GRTs. Similarly, the number of active drugs in the cART regimen after the third resistance test, used as continuous variable, was also predictive of failure. Using both GSS approaches, current genotype had a higher effect than historical genotype on risk of treatment failure. Using the non-standard definition (CWR), historical resistance predicted failure better than current resistance. CONCLUSIONS: Our results provide an epidemiological demonstration that analysis of a combined latest and historical GRT, which also considers archived mutations, might better identify of the more virologically impaired patients in order to assess the best salvage treatment.

Simultaneous determination of maraviroc and raltegravir in human plasma by HPLC-UV.

Therapeutic drug monitoring is pivotal to improve the management of HIV infection. Here, a new HPLC-UV method to quantify simultaneously maraviroc and raltegravir levels in human plasma is reported. Remarkably, this is the first method for maraviroc determination in human plasma. The volume of the plasma sample was 600 microL. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (30 mg divinylbenzene and N-vinylpyrrolidone) and evaporation in a water bath under nitrogen stream. The extracted samples were reconstituted with 200 microL 50/50 of mobile-phase solution (0.01 M KH(2)PO(4) and acetonitrile). Twenty microliters of these samples were injected into a HPLC-UV system, the analytes were eluted on an analytical dC18 Atlantis column (150 mm x 4.6 mm I.D.) with a particle size of 5 microm. The mobile phase (0.01 M KH(2)PO(4) and acetonitrile) was delivered at 1.0 mL/min with isocratic elution. The total run time for a single analysis was 10 min; maraviroc and raltegravir were detected by UV at 197 and 300 nm. The calibration curves were linear up to 2,500 ng/mL. The absolute recovery ranged between 93 and 100%. The HPLC-UV method reported here has been validated and is currently applied to monitor plasma levels of maraviroc and raltegravir in HIV-infected patients.

Sonographic assessment of facial HIV-related lypoatrophy.

OBJECTIVE: To investigate the utility of ultrasonography (US) for assessing and grading facial lypoatrophy (FLA) in patients with HIV. DESIGN: The social effect of FLA is huge and may reduce antiretroviral therapy adherence. Strategies for the early detection of FLA are crucial, because complete correction of FLA in late stages is unlikely. METHODS: Fifty-two HIV-positive patients undergoing highly active antiretroviral therapy underwent US with nasogenian transversal scan using a high-frequency broadband transducer (5-17 MHz) to detect FLA. Intra- and interobserver variability were calculated to assess US reproducibility. Concerning FLA grading, patients were categorized in five clinical classes and four US classes. RESULTS: Our results regarding inter- and intraobserver coefficients of variation permit the validation of US as a reproducible technique (p<.001), and a high correlation between US and clinical classification was obtained, with complete concordance for more advanced FLA classes. CONCLUSIONS: The lack of a reference objective method to quantify subcutaneous fat is a major difficulty in measuring HIV-related FLA. Our results, in accordance with data from the literature, suggest that US is an ideal tool for assessing and grading FLA. Furthermore, US may be suitable for routine evaluation in HIV-infected patients for early detection of FLA and to select its optimal management.

Randomized clinical trial on efficacy of fixed-dose efavirenz/tenofovir/emtricitabine on alternate days versus continuous treatment.

OBJECTIVE: Antiretrovirals with long half-lives, such as tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) and efavirenz (EFV), are suitable for reduced frequency dosing, with potential for improved adherence and reduced toxicity and costs. The objective of this study was to investigate the noninferiority of the TDF/FTC/EFV fixed-dose combination on alternate-days versus standard regimen in virologically suppressed patients. DESIGN: A randomized-controlled open-label noninferiority trial enrolling HIV-1-infected patients treated for at least 6 months with TDF/FTC/EFV fixed-dose combination, virologically suppressed (<40 HIV-RNA copies/ml) with EFV plasma concentrations greater than 1000 ng/ml, were randomized to maintain TDF/FTC/EFV standard-of-care regimen (SOC, Arm A) or to switch to TDF/FTC/EFV on AlTernAte Days (ATAD, Arm B). METHODS: Primary end-point was the proportion of patients with less than 40 HIV-RNA copies/ml at week 48. RESULTS: One hundred and ninety-seven patients were randomized (98 in the SOC and 99 in the ATAD arm). One hundred and seventy-nine (90.3%) were men, median age 43.2 years, 133 (67.5%) MSM. CD4+ T-cell count at baseline was 706 cells/µl in SOC and 632 cells/µl in ATAD arm. At week 48, 95 (96.9%) patients in SOC and 93 (93.9%) in ATAD had a virological response (-3.0% overall risk difference, 95% CI: -8.86%/2.86%). Median change from baseline at week 48 in CD4+ T-cell count was 29.4 cells/µl (95% CI: 2.5/56.4) in SOC (P = 0.008) and 61.0 cells/µl (95% CI: 32.1/89.9) in ATAD (P < 0.001). Median change of EFV concentration at week 48 from baseline was -6.5 ng/ml (95% CI: -103/55) in SOC (P = 0.877) and -1124 ng/ml (95% CI: -1375/-928) in ATAD arm (P < 0.001). CONCLUSION: Despite a significant decrease of EFV exposure, TDF/FTC/EFV on ATAD was noninferior to SOC regimen through 48 weeks.