Population pharmacokinetics of mefloquine, piperaquine and artemether-lumefantrine in Cambodian and Tanzanian malaria patients.

BACKGROUND: Inter-individual variability in plasma concentration-time profiles might contribute to differences in anti-malarial treatment response. This study investigated the pharmacokinetics of three different forms of artemisinin combination therapy (ACT) in Tanzania and Cambodia to quantify and identify potential sources of variability. METHODS: Drug concentrations were measured in 143 patients in Tanzania (artemether, dihydroartemisinin, lumefantrine and desbutyl-lumefantrine), and in 63 (artesunate, dihydroartemisinin and mefloquine) and 60 (dihydroartemisinin and piperaquine) patients in Cambodia. Inter- and intra-individual variabilities in the pharmacokinetic parameters were assessed and the contribution of demographic and other covariates was quantified using a nonlinear mixed-effects modelling approach (NONMEM®). RESULTS: A one-compartment model with first-order absorption from the gastrointestinal tract fitted the data for all drugs except piperaquine (two-compartment). Inter-individual variability in concentration exposure was about 40% and 12% for mefloquine. From all the covariates tested, only body weight (for all antimalarials) and concomitant treatment (for artemether only) showed a significant influence on these drugs' pharmacokinetic profiles. Artesunate and dihydroartemisinin could not be studied in the Cambodian patients due to insufficient data-points. Modeled lumefantrine kinetics showed that the target day 7 concentrations may not be achieved in a substantial proportion of patients. CONCLUSION: The marked variability in the disposition of different forms of ACT remained largely unexplained by the available covariates. Dosing on body weight appears justified. The concomitance of unregulated drug use (residual levels found on admission) and sub-optimal exposure (variability) could generate low plasma levels that contribute to selecting for drug-resistant parasites.

L'hémisphérotomie péri-insulaire: technique chirurgicale, monitoring EEG intraopératoire et résultats sur le contrôle de l'épilepsie [Periinsular hemispherotomy: surgical technique, intraoperative EEG monitoring and results on seizure outcome]

Peri-insular hemispherotomy is a surgical technique used in the treatment of drug-resistant epilepsy of hemispheric origin. It is based on the exposure of insula and semi-circular sulci, providing access to the lateral ventricle through a supra- and infra-insular window. From inside the ventricle, a parasagittal callosotomy is performed. The basal and medial portion of the frontal lobe is isolated. Projections to the anterior commissure are interrupted at the time of amygdala resection. The hippocampal tail and fimbria-fornix are disrupted posteriorly. We report our experience of 18 cases treated with this approach. More than half of them presented with congenital epilepsy. Neuronavigation was useful in precisely determining the center and extent of the craniotomy, as well as the direction of tractotomies and callosotomy, allowing minimal exposure and blood loss. Intra-operative monitoring by scalp EEG on the contralateral hemisphere was used to follow the progression of the number of interictal spikes during the disconnection procedure. Approximately 90% of patients were in Engel's Class I. We observed one case who presented with transient postoperative neurological deterioration probably due to CSF overdrainage and documented one case of incomplete disconnection in a patient presenting with hemimegalencephaly who needed a second operation. We observed a good correlation between a significant decrease in the number of spikes at the end of the procedure and seizure outcome. Peri-insular hemispherotomy provides a functional disconnection of the hemisphere with minimal resection of cerebral tissue. It is an efficient technique with a low complication rate. Intra-operative EEG monitoring might be used as a predictive factor of completeness of the disconnection and consequently, seizure outcome.

Added Value of Deep Sequencing Relative to Population Sequencing in Heavily Pre-Treated HIV-1-Infected Subjects.

OBJECTIVE: To explore the potential of deep HIV-1 sequencing for adding clinically relevant information relative to viral population sequencing in heavily pre-treated HIV-1-infected subjects. METHODS: In a proof-of-concept study, deep sequencing was compared to population sequencing in HIV-1-infected individuals with previous triple-class virological failure who also developed virologic failure to deep salvage therapy including, at least, darunavir, tipranavir, etravirine or raltegravir. Viral susceptibility was inferred before salvage therapy initiation and at virological failure using deep and population sequencing genotypes interpreted with the HIVdb, Rega and ANRS algorithms. The threshold level for mutant detection with deep sequencing was 1%. RESULTS: 7 subjects with previous exposure to a median of 15 antiretrovirals during a median of 13 years were included. Deep salvage therapy included darunavir, tipranavir, etravirine or raltegravir in 4, 2, 2 and 5 subjects, respectively. Self-reported treatment adherence was adequate in 4 and partial in 2; one individual underwent treatment interruption during follow-up. Deep sequencing detected all mutations found by population sequencing and identified additional resistance mutations in all but one individual, predominantly after virological failure to deep salvage therapy. Additional genotypic information led to consistent decreases in predicted susceptibility to etravirine, efavirenz, nucleoside reverse transcriptase inhibitors and indinavir in 2, 1, 2 and 1 subject, respectively. Deep sequencing data did not consistently modify the susceptibility predictions achieved with population sequencing for darunavir, tipranavir or raltegravir. CONCLUSIONS: In this subset of heavily pre-treated individuals, deep sequencing improved the assessment of genotypic resistance to etravirine, but did not consistently provide additional information on darunavir, tipranavir or raltegravir susceptibility. These data may inform the design of future studies addressing the clinical value of minority drug-resistant variants in treatment-experienced subjects.

Two approaches to discovering and developing new drugs for Chagas disease

This review will focus on two general approaches carried out at the Sandler Center, University of California, San Francisco, to address the challenge of developing new drugs for the treatment of Chagas disease. The first approach is target-based drug discovery, and two specific targets, cytochrome P450 CYP51 and cruzain (aka cruzipain), are discussed. A "proof of concept" molecule, the vinyl sulfone inhibitor K777, is now a clinical candidate. The preclinical assessment compliance for filing as an Investigational New Drug with the United States Food and Drug Administration (FDA) is presented, and an outline of potential clinical trials is given. The second approach to identifying new drug leads is parasite phenotypic screens in culture. The development of an assay allowing high throughput screening of Trypanosoma cruzi amastigotes in skeletal muscle cells is presented. This screen has the advantage of not requiring specific strains of parasites, so it could be used with field isolates, drug resistant strains or laboratory strains. It is optimized for robotic liquid handling and has been validated through a screen of a library of FDA-approved drugs identifying 65 hits.

Schistosoma mansoni: a method for inducing resistance to praziquantel using infected Biomphalaria glabrata snails

To elucidate the mechanisms of antischistosoma resistance, drug-resistant Schistosoma mansoni laboratory isolates are essential. We developed a new method for inducing resistance to praziquantel (PZQ) using successive drug treatments of Biomphalaria glabrata snails infected with S. mansoni. Infected B. glabrata were treated three times with 100 mg/kg PZQ for five consecutive days with a one-week interval between them. After the treatment, the cercariae (LE-PZQ) produced from these snails and the LE strains (susceptible) were used to infect mice. Forty-five days after infection, mice were treated with 200, 400 or 800 mg/kg PZQ. Thirty days post-treatment, we observed that the mean number of worms recovered by perfusion was significantly higher in the group of mice infected with the LE-PZQ isolate treated with 200 and 400 mg/kg in comparison to the LE strain with the same treatment. Moreover, there was a significant difference between the ED50 (effective dose required to kill 50% of the worms) of the LE-PZQ isolate (362 mg/kg) and the LE strain (68 mg/kg). In the in vitro assays, the worms of the LE-PZQ isolate were also less susceptible to PZQ. Thus, the use of infected snails as an experimental model for development of resistance to S. mansoni is effective, fast, simple and cheap.

Sentinel network for monitoring in vitro susceptibility of Plasmodium falciparum to antimalarial drugs in Colombia: a proof of concept

Drug resistance is one of the principal obstacles blocking worldwide malaria control. In Colombia, malaria remains a major public health concern and drug-resistant parasites have been reported. In vitro drug susceptibility assays are a useful tool for monitoring the emergence and spread of drug-resistant Plasmodium falciparum. The present study was conducted as a proof of concept for an antimalarial drug resistance surveillance network based on in vitro susceptibility testing in Colombia. Sentinel laboratories were set up in three malaria endemic areas. The enzyme linked immunosorbent assay-histidine rich protein 2 and schizont maturation methods were used to assess the susceptibility of fresh P. falciparum isolates to six antimalarial drugs. This study demonstrates that an antimalarial drug resistance surveillance network based on in vitro methods is feasible in the field with the participation of a research institute, local health institutions and universities. It could also serve as a model for a regional surveillance network. Preliminary susceptibility results showed widespread chloroquine resistance, which was consistent with previous reports for the Pacific region. However, high susceptibility to dihydroartemisinin and lumefantrine compounds, currently used for treatment in the country, was also reported. The implementation process identified critical points and opportunities for the improvement of network sustainability strategies.

In vitro susceptibility of Plasmodium falciparum Welch field isolates to infusions prepared from Artemisia annua L. cultivated in the Brazilian Amazon

Artemisinin is the active antimalarial compound obtained from the leaves of Artemisia annua L. Artemisinin, and its semi-synthetic derivatives, are the main drugs used to treat multi-drug-resistant Plasmodium falciparum (one of the human malaria parasite species). The in vitro susceptibility of P. falciparum K1 and 3d7 strains and field isolates from the state of Amazonas, Brazil, to A. annua infusions (5 g dry leaves in 1 L of boiling water) and the drug standards chloroquine, quinine and artemisinin were evaluated. The A. annua used was cultivated in three Amazon ecosystems (várzea, terra preta de índio and terra firme) and in the city of Paulínia, state of São Paulo, Brazil. Artemisinin levels in the A. annua leaves used were 0.90-1.13% (m/m). The concentration of artemisinin in the infusions was 40-46 mg/L. Field P. falciparum isolates were resistant to chloroquine and sensitive to quinine and artemisinin. The average 50% inhibition concentration values for A. annua infusions against field isolates were 0.11-0.14 μL/mL (these infusions exhibited artemisinin concentrations of 4.7-5.6 ng/mL) and were active in vitro against P. falciparum due to their artemisinin concentration. No synergistic effect was observed for artemisinin in the infusions.

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

Megazol and its bioisostere 4H-1,2,4-triazole: comparing the trypanocidal, cytotoxic and genotoxic activities and their in vitro and in silico interactions with the Trypanosoma brucei nitroreductase enzyme

Megazol (7) is a 5-nitroimidazole that is highly active against Trypanosoma cruzi and Trypanosoma brucei, as well as drug-resistant forms of trypanosomiasis. Compound 7 is not used clinically due to its mutagenic and genotoxic properties, but has been largely used as a lead compound. Here, we compared the activity of 7 with its 4H-1,2,4-triazole bioisostere (8) in bloodstream forms of T. brucei and T. cruzi and evaluated their activation by T. brucei type I nitroreductase (TbNTR) enzyme. We also analysed the cytotoxic and genotoxic effects of these compounds in whole human blood using Comet and fluorescein diacetate/ethidium bromide assays. Although the only difference between 7 and 8 is the substitution of sulphur (in the thiadiazole in 7) for nitrogen (in the triazole in 8), the results indicated that 8 had poorer antiparasitic activity than 7 and was not genotoxic, whereas 7 presented this effect. The determination of Vmax indicated that although 8 was metabolised more rapidly than 7, it bounds to the TbNTR with better affinity, resulting in equivalent kcat/KM values. Docking assays of 7 and 8 performed within the active site of a homology model of the TbNTR indicating that 8 had greater affinity than 7.

Characteristics and determinants of outcome of hospital-acquired bloodstream infections in intensive care units: the EUROBACT International Cohort Study.

PURPOSE: The recent increase in drug-resistant micro-organisms complicates the management of hospital-acquired bloodstream infections (HA-BSIs). We investigated the epidemiology of HA-BSI and evaluated the impact of drug resistance on outcomes of critically ill patients, controlling for patient characteristics and infection management. METHODS: A prospective, multicentre non-representative cohort study was conducted in 162 intensive care units (ICUs) in 24 countries. RESULTS: We included 1,156 patients [mean ± standard deviation (SD) age, 59.5 ± 17.7 years; 65 % males; mean ± SD Simplified Acute Physiology Score (SAPS) II score, 50 ± 17] with HA-BSIs, of which 76 % were ICU-acquired. Median time to diagnosis was 14 [interquartile range (IQR), 7-26] days after hospital admission. Polymicrobial infections accounted for 12 % of cases. Among monomicrobial infections, 58.3 % were gram-negative, 32.8 % gram-positive, 7.8 % fungal and 1.2 % due to strict anaerobes. Overall, 629 (47.8 %) isolates were multidrug-resistant (MDR), including 270 (20.5 %) extensively resistant (XDR), and 5 (0.4 %) pan-drug-resistant (PDR). Micro-organism distribution and MDR occurrence varied significantly (p < 0.001) by country. The 28-day all-cause fatality rate was 36 %. In the multivariable model including micro-organism, patient and centre variables, independent predictors of 28-day mortality included MDR isolate [odds ratio (OR), 1.49; 95 % confidence interval (95 %CI), 1.07-2.06], uncontrolled infection source (OR, 5.86; 95 %CI, 2.5-13.9) and timing to adequate treatment (before day 6 since blood culture collection versus never, OR, 0.38; 95 %CI, 0.23-0.63; since day 6 versus never, OR, 0.20; 95 %CI, 0.08-0.47). CONCLUSIONS: MDR and XDR bacteria (especially gram-negative) are common in HA-BSIs in critically ill patients and are associated with increased 28-day mortality. Intensified efforts to prevent HA-BSIs and to optimize their management through adequate source control and antibiotic therapy are needed to improve outcomes.

Successful surgical resection in non-lesional operculo-insular epilepsy without intracranial monitoring.

Pre-operative assessment and surgical management of patients with non-lesional extratemporal epilepsy remain challenging due to a lack of precise localisation of the epileptic zone. In most cases, invasive recording with depth or subdural electrodes is required. Here, we describe the case of 6.5-year-old girl who underwent comprehensive non-invasive phase I video-EEG investigation for drug-resistant epilepsy, including electric source and nuclear imaging. Left operculo-insular epilepsy was diagnosed. Post-operatively, she developed aphasia which resolved within one year, corroborating the notion of enhanced language plasticity in children. The patient remained seizure-free for more than three years.

Reduction of plasma alanine aminotransferase during vigabatrin treatment.

In 9 drug-resistant patients with partial seizures treated with vigabatrin, gamma-vinyl GABA (VGB), alanine aminotransaminase (ALAT) activity in plasma was significantly reduced. Comparison of in vitro with in vivo measurements led us to conclude that this reduction is mainly an in vivo phenomenon, perhaps due to cross-enzyme inhibition. The assessment of two biological variables linked with ALAT, glucose and alanine levels under fasting conditions, failed to show any significant metabolic alterations. VGB is an effective drug for partial epilepsy. Our observations do not suggest that reduced ALAT activity is of clinical concern.

Maladies infectieuses en pratique ambulatoire (sauf sida) [Infectious diseases (beside AIDS)]

A simplified version of the US guidelines for prophylaxis of infectious endocarditis was published in 2007. Changes are expected in Switzerland as well. Posaconsole is a new antifungal agent available mostly for prophylaxis in immunocompromised patients. Epidemiology of sexually transmitted infections warrants screening in young adults and after one episode. A meta-analysis clarified the impact of antibiotic therapy in patients with Campylobacter spp. infection. In the field of emerging diseases, we discuss Norovirus epidemics, community-acquired bacteria producing extended-spectrum betalactamases, extensively resistant tuberculosis, and new respiratory viruses. Finally, we address a basic research topic that may change practice in the future: the relationship between individual susceptibility to infection and innate immunity.

Chronic deep brain stimulation in mesial temporal lobe epilepsy.

The objective of this study was to evaluate the efficiency and the effects of changes in parameters of chronic amygdala-hippocampal deep brain stimulation (AH-DBS) in mesial temporal lobe epilepsy (TLE). Eight pharmacoresistant patients, not candidates for ablative surgery, received chronic AH-DBS (130 Hz, follow-up 12-24 months): two patients with hippocampal sclerosis (HS) and six patients with non-lesional mesial TLE (NLES). The effects of stepwise increases in intensity (0-Off to 2 V) and stimulation configuration (quadripolar and bipolar), on seizure frequency and neuropsychological performance were studied. The two HS patients obtained a significant decrease (65-75%) in seizure frequency with high voltage bipolar DBS (≥1 V) or with quadripolar stimulation. Two out of six NLES patients became seizure-free, one of them without stimulation, suggesting a microlesional effect. Two NLES patients experienced reductions of seizure frequency (65-70%), whereas the remaining two showed no significant seizure reduction. Neuropsychological evaluations showed reversible memory impairments in two patients under strong stimulation only. AH-DBS showed long-term efficiency in most of the TLE patients. It is a valuable treatment option for patients who suffer from drug resistant epilepsy and who are not candidates for resective surgery. The effects of changes in the stimulation parameters suggest that a large zone of stimulation would be required in HS patients, while a limited zone of stimulation or even a microlesional effect could be sufficient in NLES patients, for whom the importance of the proximity of the electrode to the epileptogenic zone remains to be studied. Further studies are required to ascertain these latter observations.

Limited clinical benefit of minority K103N and Y181C-variant detection in addition to routine genotypic resistance testing in antiretroviral therapy-naive patients.

OBJECTIVE: The presence of minority nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 variants prior to antiretroviral therapy (ART) has been linked to virologic failure in treatment-naive patients. DESIGN: We performed a large retrospective study to determine the number of treatment failures that could have been prevented by implementing minority drug-resistant HIV-1 variant analyses in ART-naïve patients in whom no NNRTI resistance mutations were detected by routine resistance testing. METHODS: Of 1608 patients in the Swiss HIV Cohort Study, who have initiated first-line ART with two nucleoside reverse transcriptase inhibitors (NRTIs) and one NNRTI before July 2008, 519 patients were eligible by means of HIV-1 subtype, viral load and sample availability. Key NNRTI drug resistance mutations K103N and Y181C were measured by allele-specific PCR in 208 of 519 randomly chosen patients. RESULTS: Minority K103N and Y181C drug resistance mutations were detected in five out of 190 (2.6%) and 10 out of 201 (5%) patients, respectively. Focusing on 183 patients for whom virologic success or failure could be examined, virologic failure occurred in seven out of 183 (3.8%) patients; minority K103N and/or Y181C variants were present prior to ART initiation in only two of those patients. The NNRTI-containing, first-line ART was effective in 10 patients with preexisting minority NNRTI-resistant HIV-1 variant. CONCLUSION: As revealed in settings of case-control studies, minority NNRTI-resistant HIV-1 variants can have an impact on ART. However, the implementation of minority NNRTI-resistant HIV-1 variant analysis in addition to genotypic resistance testing (GRT) cannot be recommended in routine clinical settings. Additional associated risk factors need to be discovered.