Emergence of Resistance in HIV-1 Integrase with Dolutegravir Treatment in a Pediatric Population from the IMPAACT P1093 Study.

P1093 is a multicenter, open-label, phase I/II study of pharmacokinetics, safety, and tolerability of dolutegravir plus an optimized background regimen in pediatric participants aged 4 weeks to <18 years with HIV-1. Most participants were highly treatment experienced. We report the mechanisms of emergent integrase strand transfer inhibitor (INSTI) resistance among adolescents and children receiving dolutegravir. Plasma was collected at screening and near protocol-defined virologic failure (PDVF) for population-level and, for some samples, clonal-level integrase genotyping, phenotyping, and replication capacity. HIV-1 RNA was assessed in all available plasma samples. Phylogenetic analysis of clonal integrase sequences and homology modeling of HIV-1 intasome complexes containing resistance-associated substitutions were performed. Treatment-emergent INSTI resistance was detected in 8 participants who met PDVF criteria. The rare INSTI resistance-associated substitution G118R or R263K developed in 6 participants. The on-study secondary integrase substitution E157Q or L74I was observed in 2 participants. G118R reduced dolutegravir susceptibility and integrase replication capacity more than R263K and demonstrated greater reduction in susceptibility and integrase replication capacity when present with specific secondary integrase substitutions, including L74M, T66I, and E138E/K. Continuing evolution after R263K acquisition led to reduced dolutegravir susceptibility and integrase replication capacity. Structural examination revealed potential mechanisms for G118R- and R263K-mediated INSTI resistance. G118R and R263K INSTI resistance substitutions, which are distinct to second-generation INSTIs, were detected in adolescents and children with prior virologic failure who received dolutegravir. This study provides additional molecular and structural characterization of integrase to aid in the understanding of INSTI resistance mechanisms in antiretroviral-experienced populations. (This study has been registered at ClinicalTrials.gov under identifier NCT01302847.).

Preclinical Characterization and In Vivo Efficacy of GSK8853, a Small-Molecule Inhibitor of the Hepatitis C Virus NS4B Protein.

The hepatitis C virus (HCV) NS4B protein is an antiviral therapeutic target for which small-molecule inhibitors have not been shown to exhibit in vivo efficacy. We describe here the in vitro and in vivo antiviral activity of GSK8853, an imidazo[1,2-a]pyrimidine inhibitor that binds NS4B protein. GSK8853 was active against multiple HCV genotypes and developed in vitro resistance mutations in both genotype 1a and genotype 1b replicons localized to the region of NS4B encoding amino acids 94 to 105. A 20-day in vitro treatment of replicons with GSK8853 resulted in a 2-log drop in replicon RNA levels, with no resistance mutation breakthrough. Chimeric replicons containing NS4B sequences matching known virus isolates showed similar responses to a compound with genotype 1a sequences but altered efficacy with genotype 1b sequences, likely corresponding to the presence of known resistance polymorphs in those isolates. In vivo efficacy was tested in a humanized-mouse model of HCV infection, and the results showed a 3-log drop in viral RNA loads over a 7-day period. Analysis of the virus remaining at the end of in vivo treatment revealed resistance mutations encoding amino acid changes that had not been identified by in vitro studies, including NS4B N56I and N99H. Our findings provide an in vivo proof of concept for HCV inhibitors targeting NS4B and demonstrate both the promise and potential pitfalls of developing NS4B inhibitors.

HIV-1 transmission patterns in antiretroviral therapy-naïve, HIV-infected North Americans based on phylogenetic analysis by population level and ultra-deep DNA sequencing.

Factors that contribute to the transmission of human immunodeficiency virus type 1 (HIV-1), especially drug-resistant HIV-1 variants remain a significant public health concern. In-depth phylogenetic analyses of viral sequences obtained in the screening phase from antiretroviral-naïve HIV-infected patients seeking enrollment in EPZ108859, a large open-label study in the USA, Canada and Puerto Rico (ClinicalTrials.gov NCT00440947) were examined for insights into the roles of drug resistance and epidemiological factors that could impact disease dissemination. Viral transmission clusters (VTCs) were initially predicted from a phylogenetic analysis of population level HIV-1 pol sequences obtained from 690 antiretroviral-naïve subjects in 2007. Subsequently, the predicted VTCs were tested for robustness by ultra deep sequencing (UDS) using pyrosequencing technology and further phylogenetic analyses. The demographic characteristics of clustered and non-clustered subjects were then compared. From 690 subjects, 69 were assigned to 1 of 30 VTCs, each containing 2 to 5 subjects. Race composition of VTCs were significantly more likely to be white (72% vs. 60%; p = 0.04). VTCs had fewer reverse transcriptase and major PI resistance mutations (9% vs. 24%; p = 0.002) than non-clustered sequences. Both men-who-have-sex-with-men (MSM) (68% vs. 48%; p = 0.001) and Canadians (29% vs. 14%; p = 0.03) were significantly more frequent in VTCs than non-clustered sequences. Of the 515 subjects who initiated antiretroviral therapy, 33 experienced confirmed virologic failure through 144 weeks while only 3/33 were from VTCs. Fewer VTCs subjects (as compared to those with non-clustering virus) had HIV-1 with resistance-associated mutations or experienced virologic failure during the course of the study. Our analysis shows specific geographical and drug resistance trends that correlate well with transmission clusters defined by HIV sequences of similarity. Furthermore, our study demonstrates the utility of molecular and epidemiological analysis of VTCs for identifying population-specific risks associated with HIV-1 transmission and developing effective local healthcare strategies.

In vitro characterization of GSK2485852, a novel hepatitis C virus polymerase inhibitor.

GSK2485852 (referred to here as GSK5852) is a hepatitis C virus (HCV) NS5B polymerase inhibitor with 50% effective concentrations (EC50s) in the low nanomolar range in the genotype 1 and 2 subgenomic replicon system as well as the infectious HCV cell culture system. We have characterized the antiviral activity of GSK5852 using chimeric replicon systems with NS5B genes from additional genotypes as well as NS5B sequences from clinical isolates of patients infected with HCV of genotypes 1a and 1b. The inhibitory activity of GSK5852 remained unchanged in these intergenotypic and intragenotypic replicon systems. GSK5852 furthermore displays an excellent resistance profile and shows a <5-fold potency loss across the clinically important NS5B resistance mutations P495L, M423T, C316Y, and Y448H. Testing of a diverse mutant panel also revealed a lack of cross-resistance against known resistance mutations in other viral proteins. Data from both the newer 454 sequencing method and traditional population sequencing showed a pattern of mutations arising in the NS5B RNA-dependent RNA polymerase in replicon cells exposed to GSK5852. GSK5852 was more potent than HCV-796, an earlier inhibitor in this class, and showed greater reductions in HCV RNA during long-term treatment of replicons. GSK5852 is similar to HCV-796 in its activity against multiple genotypes, but its superior resistance profile suggests that it could be an attractive component of an all-oral regimen for treating HCV.

Prevalent polymorphisms in wild-type HIV-1 integrase are unlikely to engender drug resistance to dolutegravir (S/GSK1349572).

The majority of HIV-1 integrase amino acid sites are highly conserved, suggesting that most are necessary to carry out the critical structural and functional roles of integrase. We analyzed the 34 most variable sites in integrase (>10% variability) and showed that prevalent polymorphic amino acids at these positions did not affect susceptibility to the integrase inhibitor dolutegravir (S/GSK1349572), as demonstrated both in vitro (in site-directed mutagenesis studies) and in vivo (in a phase IIa study of dolutegravir monotherapy in HIV-infected individuals). Ongoing clinical trials will provide additional data on the virologic activity of dolutegravir across subject viruses with and without prevalent polymorphic substitutions.

Virus susceptibility analyses from a phase IV clinical trial of inhaled zanamivir treatment in children infected with influenza.

A zanamivir postapproval efficacy study was conducted in children (n = 279) in Japan during three influenza seasons. Pharyngeal swab specimens (n = 714) were obtained for detailed resistance analysis. From 371 cultured viruses, 3 viruses (A/H1N1) from two subjects showed reduced susceptibility to zanamivir at day 1 (before treatment), 1 had an N74S amino acid substitution (fold shift, 46), and 2 (day 1 and day 2) had a Q136K amino acid substitution (fold shifts, 292 and 301). Q136K was detected only in cultured virus and not in the swab. From the remaining 118 cultured viruses obtained during or after treatment with zanamivir, no shifts in virus susceptibility were detected. Neuraminidase (NA) population sequencing showed that viruses from 12 subjects had emergent amino acid substitutions, but 3 with susceptibility data were not zanamivir resistant. The remainder may be natural variants. Further analysis is planned. Hemagglutinin (HA) sequencing showed that viruses from 20 subjects had 9 HA amino acid substitutions that were previously implicated in resistance to neuraminidase inhibitors in in vitro assays or that were close to the receptor binding site. Their role in in vivo resistance appears to be less important but is not well understood. NA clonal sequence analysis was undertaken to determine if minority species of resistant viruses were present. A total of 1,682 clones from 90 subjects were analyzed. Single clones from 12 subjects contained amino acid substitutions close to the NA active site. It is unclear whether these single amino acid substitutions could have been amplified after drug pressure or are just chance mutations introduced during PCR.

Computed tomography perfusion prognostic maps do not predict reversible and irreversible neurological dysfunction following reperfusion therapies.

BACKGROUND: We aimed to evaluate the ability of commercially available computed tomography perfusion (CTP) prognostic maps software to identify reversibly and irreversibly damaged brain functions in the best case scenario: patients who achieved early and complete tissue reperfusion. METHODS: Consecutive ischemic stroke patients who received reperfusion therapies, those with early (less than two-hours from treatment initiation) and complete Thrombolysis in Myocardial Infarction grade III or equivalent reperfusion were included in the analysis. Computed tomography perfusion prognostic maps were assigned as 'red,' or irreversible if cerebral blood volume declined below 2 ml/100 g and 'green,' or recoverable if the affected/unaffected mean transit time ratio was >1.45. Only patients with middle cerebral artery territory affected were included and subcomponents of the National Institutes of Health Stroke Scale scale pre- and posttreatment were analyzed based on anatomical correlation of the affected CTP areas and corresponding neurological functions. RESULTS: Among 109 consecutive patients who had intra-arterial reperfusion procedures, 16 (age 60 ± 17 years, 56% men, median National Institutes of Health Stroke Scale 13 . 5, interquartile range 7-18) had pretreatment CTP and had early complete reperfusion. We identified 44 affected areas on CTP (red 12 (27%), green 32 (73%)) with corresponding measurable neurological deficits including aphasia, arm, face weakness, and inattention. Red areas correctly identified 5/12 (42%) of functions that did not recover despite early reperfusion. Green areas correctly identified 18/32 (56%) of functions that recover after early reperfusion (OR 0.92, 95% CI 0.25-3.39, P = 1.0). CONCLUSIONS: In-patients achieving early and complete reperfusion, pretreatment CTP prognostic maps were not predictive for irreversibly or reversibly lost neurologic functions.

Tenofovir (TDF)-selected or abacavir (ABC)-selected low-frequency HIV type 1 subpopulations during failure with persistent viremia as detected by ultradeep pyrosequencing.

Detection of drug resistance is critical for determining antiretroviral treatment options. Ultradeep pyrosequencing (UDPS; 454 Life Sciences) is capable of detecting virus variant subpopulations with much greater sensitivity than population sequencing, which typically has a detection limit around 20%. UDPS of the HIV-1 reverse transcriptase (RT) (amino acids 56-120) was performed to detect the key mutations K65R and L74V associated with tenofovir and abacavir use. Plasma specimens from subjects with persistent rebound viremia following suppression on tenofovir (n = 8) or abacavir (n = 9)-based therapy were studied. Samples from a subject treated with zidovudine/lamivudine/efavirenz with a similar loss of virologic response served as a control. HIV-1 plasma RNA was ≥3.68 log(10) copies/ml at all time points sequenced. The median number of UDPS sequences analyzed/time point was 33,246. Among the eight tenofovir-treated subjects, three showed high-frequency (>20%) RT K65R at the time of failure, whereas one showed low-frequency (<20%) L74V; no low-frequency K65R was detected in these subjects. Among the nine abacavir-treated subjects, three showed low-frequency K65R; no L74V was detected in these patients. No K65R or L74V was detected in the samples from the control subject. At failure, other RT mutations were detected, including low-frequency NNRTI-resistant species detected at ≥1 time point in nine subjects; the key NNRTI mutation K103N, however, was always observed at >20% frequency. Although UDPS is useful in the detection of low-frequency subpopulations with transmitted resistance in antiviral-naive patients, it may have less utility in treatment-experienced patients with persistent viremia on therapy.

A near-linear time algorithm for haplotype determination on general pedigrees.

Abstract An O(nmα(m)) time algorithm is given for inferring haplotypes from genotypes of non-recombinant pedigree data, where n is the number of members, m is the number of sites, and α(m) is the inverse of the Ackermann function. The algorithm works on both tree and general pedigree structures with cycles. Constraints between pairs of heterozygous sites are used to resolve unresolved sites for the pedigree, enabling the algorithm to avoid problems previously experienced for non-tree pedigrees.

Impact of low abundance HIV variants on response to ritonavir-boosted atazanavir or fosamprenavir given once daily with tenofovir/emtricitabine in antiretroviral-naive HIV-infected patients.

Population genotyping (PG) can underestimate resistance if resistance-containing low abundance variants go undetected. PG and clonal analysis (CA) results were compared in virologic failures (VFs) from a 48-week clinical trial that evaluated once-daily fosamprenavir/ritonavir (FPV/r) 1400 mg/100 mg or atazanavir/ritonavir (ATV/r) 300 mg/100 mg, each combined with tenofovir/emtricitabine, in antiretroviral-naive patients. VF was defined as confirmed HIV-1 RNA > or =400 copies/ml at > or =24 weeks or viral rebound >400 copies/ml any time following viral suppression. All patients had baseline PG. One hundred and six patients enrolled (53/arm). Baseline resistance mutations were more prevalent in patients receiving FPV/r (10/53) than ATV/r (3/53). Seven patients (7%) were VFs-four on FPV/r and three on ATV/r. In the four FPV/r-treated VFs, baseline HIV TAMs combinations and/or PI mutations were detected in one by PG at VF (RT: L210W + T215C; PR: M46I + L76V) and three others by CA alone (RT: L210W + T215Y; RT: M41L; RT: K65R + K70R; PR: I47V); all four had study drug-associated mutations (CA detecting more HIV-1 resistance mutations than PG). In the three ATV/r VFs, no baseline drug-associated mutations were detected by PG; for one patient CA detected RT: K65R; PR: I84V. Phylogenetic analysis revealed tight clustering for FPV/r-treated VFs with highly related clones, whereas HIV-1 from ATV/r-treated VFs had no outgrowth from baseline of low abundance resistance-containing variants. In conclusion, low-abundance HIV resistance-containing variants were detected in baseline samples from patients with VF. The archived viruses that reemerged under selection pressure and acquired additional mutations were found primarily in patients in the FPV/r arm. Despite this and a baseline resistance imbalance between the two arms, FPV/r and ATV/r provided similar virologic suppression through 48 weeks; however, these findings highlight the necessity for the development of quick and inexpensive methods for detection of minority species to better guide therapy selection.

Diagnostic criteria and yield of real-time transcranial Doppler monitoring of intra-arterial reperfusion procedures.

BACKGROUND AND PURPOSE: Intra-arterial (IA) rescue procedures are increasingly used to treat acute ischemic stroke. We implemented continuous transcranial Doppler (TCD) monitoring during these procedures to detect any potentially harmful flow changes. Here, we report diagnostic criteria and yield of TCD monitoring. METHODS: We studied consecutive acute stroke patients who underwent IA reperfusion procedures. TCD flow signatures during these procedures were analyzed and any abnormal findings were documented. RESULTS: Patients were included only if there was successful insonation through the skull; of 56 eligible patients, 51 were included. IA procedures included IA tissue plasminogen activator, use of the Merci retriever, the Penumbra system, balloon angioplasty, and stenting. On TCD monitoring, contrast injections produced high-intensity signals and increased the mean flow velocity (MFV). Deployment of the Merci device appeared as high-intensity, short-duration signals with a transient MFV decrease of 11.5%. The Penumbra system produced lower-intensity signals with a greater transient decrease in MFV during aspiration. IA tissue plasminogen activator significantly increased MFV by 7.5% over Merci and Penumbra flow velocity changes. Power motion Doppler-TCD detected reocclusion in 13 patients, artery-to-artery embolization in 2 patients, air embolism in 2 patients, and hyperperfusion in 6 patients. Overall, the yield of TCD monitoring was positive in 23 (49%) patients who received IA reperfusion procedures. CONCLUSIONS: Our velocity, intensity, and flow signatures criteria for TCD monitoring of IA reperfusion procedures detect reocclusion, hyperperfusion, or thromboembolism and air embolism in nearly half of all procedures. This hemodynamic information can be particularly helpful when neurological assessment is limited or delayed.

Intravenous thrombolysis followed by intra-arterial thrombolysis and mechanical thrombectomy for the treatment of pediatric ischemic stroke.

Experience with systemic or selective local administration of thrombolytic agents in pediatric ischemic stroke is limited to sporadic case reports, since patients of age less than 18 years were systematically excluded from randomised controlled trials. We report a case of childhood IS attributable to the terminal internal carotid artery occlusion that was treated successfully with mechanical thrombectomy which followed unsuccessful attempts to recanalize the artery with intravenous and intra-arterial thrombolytics. Combined systemic and intra-arterial thrombolysis followed by mechanical thrombectomy can be feasible and may be considered as means of achieving reperfusion in pediatric ischemic stroke patients with persisting arterial occlusion.

MRA or CTA, that's the question.

A 26-year-old woman developed a painful left pupil-involving oculomotor palsy. Magnetic resonance imaging of the brain and orbits with and without fat suppression and gadolinium and magnetic resonance angiography of the head were normal. A computed tomographic angiogram showed a left 7-mm posterior communicating artery aneurysm. The different neuroimaging modalities used to diagnose intracranial aneurysms are discussed.

Improving treatment services for substance abusers with comorbid depression.

Early identification of patients with comorbid depression and their subsequent enrollment in an enhanced psychiatric case management (PCM) intervention were examined as an effective way to engage depressed substance abuse patients into psychiatric treatment. Depression was screened using the Global Appraisal of Individual Needs (GAIN) and a DSM-IV checklist. Patients positive on both evaluations were assigned to PCM (n = 10) or to no case management, or treatment as usual (TAU) (n = 10). An examination of outcomes at six weeks indicated that PCM services are feasible and appear to be effective in encouraging use of psychiatric referral by depressed substance abusers.