Insecticide treated mosquito nets for malaria control in India-experience from a tribal area on operational feasibility and uptake

The study assessed the operational feasibility and acceptability of insecticide-treated mosquito nets (ITNs) in one Primary Health Centre (PHC) in a falciparum malaria endemic district in the state of Orissa, India, where 74% of the people are tribes and DDT indoor residual spraying had been withdrawn and ITNs introduced by the National Vector Borne Disease Control Programme. To a population of 63,920, 24,442 ITNs were distributed free of charge through 101 treatment centers during July-August 2002. Interview of 1,130, 1,012 and 126 respondents showed that the net use rates were 80%, 74% and 55% in the cold, rainy and summer seasons, respectively. Since using ITNs, 74.5-76.6% of the respondents observed reduction of mosquito bites and 7.2-32.1% reduction of malaria incidence; 37% expressed willingness to buy ITNs if the cost was lower and they were affordable. Up to ten months post-treatment, almost 100% mortality of vector mosquitoes was recorded on unwashed and washed nets (once or twice). Health workers re-treated the nets at the treatment centers eight months after distribution on a cost-recovery basis. The coverage reported by the PHC was only 4.2%, mainly because of unwillingness of the people to pay for re-treatment and to go to the treatment centers from their villages. When the re-treatment was continued at the villages involving personnel from several departments, the coverage improved to about 90%.Interview of 126 respondents showed that among those who got their nets re-treated, 81.4% paid cash for the re-treatment and the remainder were reluctant to pay. Majority of those who paid said that they did so due to the fear that if they did not do so they would lose benefits from other government welfare schemes. The 2nd re-treatment was therefore carried out free of charge nine months after the 1st re-treatment and thus achieved coverage of 70.4%. The study showed community acceptance to use ITNs as they perceived the benefit. Distribution and re-treatment of nets was thus possible through the PHC system, if done free of charge and when personnel from different departments, especially those at village level, were involved.

A cell biological view of the siderophore pyochelin iron uptake pathway in Pseudomonas aeruginosa.

Pyochelin (PCH) is a siderophore produced and secreted by Pseudomonas aeruginosa for iron capture. Using (55) Fe uptake and binding assays, we showed that PCH-Fe uptake in P. aeruginosa involves, in addition to the highly studied outer membrane transporter FptA, the inner membrane permease FptX, which recognizes PCH-(55) Fe with an affinity of 0.6 ± 0.2 nM and transports the ferri-siderophore complex from the periplasm into the cytoplasm: fptX deletion inhibited (55) Fe accumulation in the bacterial cytoplasm. Chromosomal replacement was used to generate P. aeruginosa strains producing fluorescent fusions with FptX, PchR (an AraC regulator), PchA (the first enzyme involved in the PCH biosynthesis) and PchE (a non-ribosomic peptide-synthetase involved in a further step). Fluorescence imaging and cellular fractionation showed a uniform repartition of FptX in the inner membrane. PchA and PchE were found in the cytoplasm, associated to the inner membrane all over the bacteria and also concentrated at the bacterial poles. PchE clustering at the bacterial poles was dependent on PchA expression, but on the opposite PchA clustering and membrane association was PchE-independent. PchA and PchE cellular organization suggests the existence of a siderosome for PCH biosynthesis as previously proposed for pyoverdine biosynthesis (another siderophore produced by P. aeruginosa).

Species differences in the response of liver drug-metabolizing enzymes to (S)-4-O-tolylsulfanyl-2-(4-trifluormethyl-phenoxy)-butyric acid (EMD 392949) in vivo and in vitro.

Induction of drug-metabolizing enzymes (DMEs) is highly species-specific and can lead to drug-drug interaction and toxicities. In this series of studies we tested the species specificity of the antidiabetic drug development candidate and mixed peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist (S)-4-O-tolylsulfanyl-2-(4-trifluormethyl-phenoxy)-butyric acid (EMD 392949, EMD) with regard to the induction of gene expression and activities of DMEs, their regulators, and typical PPAR target genes. EMD clearly induced PPARalpha target genes in rats in vivo and in rat hepatocytes but lacked significant induction of DMEs, except for cytochrome P450 (P450) 4A. CYP2C and CYP3A were consistently induced in livers of EMD-treated monkeys. Interestingly, classic rodent peroxisomal proliferation markers were induced in monkeys after 17 weeks but not after a 4-week treatment, a fact also observed in human hepatocytes after 72 h but not 24 h of EMD treatment. In human hepatocyte cultures, EMD showed similar gene expression profiles and induction of P450 activities as in monkeys, indicating that the monkey is predictive for human P450 induction by EMD. In addition, EMD induced a similar gene expression pattern as the PPARalpha agonist fenofibrate in primary rat and human hepatocyte cultures. In conclusion, these data showed an excellent correlation of in vivo data on DME gene expression and activity levels with results generated in hepatocyte monolayer cultures, enabling a solid estimation of human P450 induction. This study also clearly highlighted major differences between primates and rodents in the regulation of major inducible P450s, with evidence of CYP3A and CYP2C inducibility by PPARalpha agonists in monkeys and humans.

Prevalence of antiretroviral drug resistance among treatment-naive and treated HIV-infected patients in Venezuela

An in-house, low-cost method was developed to determine the genotypic resistance of immunodeficiency virus type 1 (HIV-1) isolates. All 179 Venezuelan isolates analysed belonged to subtype B. Primary drug resistance mutations were found in 11% of 63 treatment-naïve patients. The prevalence of resistance in isolates from 116 HIV-positive patients under antiretroviral treatment was 47% to protease inhibitors, 65% to nucleoside inhibitors and 38% to non-nucleoside inhibitors, respectively. Around 50% of patients in the study harboured viruses with highly reduced susceptibility to the three classical types of drugs after only five years from their initial diagnoses.

The Chemotherapeutic Drug 5-Fluorouracil Promotes PKR-Mediated Apoptosis in a p53-Independent Manner in Colon and Breast Cancer Cells

The chemotherapeutic drug 5-FU is widely used in the treatment of a range of cancers, but resistance to the drug remains a major clinical problem. Since defects in the mediators of apoptosis may account for chemo-resistance, the identification of new targets involved in 5-FU-induced apoptosis is of main clinical interest. We have identified the ds-RNA-dependent protein kinase (PKR)as a key molecular target of 5-FU involved in apoptosis induction in human colon and breast cancer cell lines. PKR distribution and activation, apoptosis induction and cytotoxic effects were analyzed during 5-FU and 5-FU/IFNalpha treatment in several colon and breast cancer cell lines with different p53 status. PKR protein was activated by 5-FU treatment in a p53-independent manner,inducing phosphorylation of the protein synthesis translation initiation factor eIF-2alpha and cell death by apoptosis. Furthermore, PKR interference promoted a decreased response to 5-FU treatment and those cells were not affected by the synergistic antitumor activity of 5-FU/IFNalpha combination. These results, taken together, provide evidence that PKR is a key molecular target of 5-FU with potential relevance in the clinical use of this drug.

Prevalence of hepatitis C virus infection and associated factors among male illicit drug users in Cuiabá, Mato Grosso, Brazil

Intravenous drug injection has been reported as the main risk factor for hepatitis C virus (HCV) infection. The aim of the present study was to describe the prevalence and the epidemiological profile of HCV infection among abusers of illegal injected and non-injected drugs in Cuiabá, state of Mato Grosso, Central Brazil. A cross-sectional study including 314 male drug users from eight detoxification centres was performed. Out of 314 subjects studied, 48 (15.2%) were intravenous drug users. Participants were interviewed and had blood samples taken and tested for the presence of anti-HCV antibodies. Positive samples were tested for the presence of HCV RNA. Genotyping was performed on HCV RNA-positive samples. The overall prevalence of anti-HCV antibodies was 6.4% (n = 20). Out of 20 anti-HCV antibody-positive subjects, 16 (80%) were also HCV RNA-positive. Genotype 1 predominated (75%), followed by 3a (25%). Subtype 1a was more common than 1b. HCV infection was more prevalent among intravenous drug users (33%) than non-injecting users (1.5%). Logistic regression analyses showed independent associations between HCV infection and intravenous drug use, imprisonment and increasing age. In the present study, injecting drug use was the factor most strongly associated to HCV infection and inhaling or sniffing did not represent an increased susceptibility to infection.

The value of metabolic imaging to predict tumour response after chemoradiation in locally advanced rectal cancer

Background: We aim to investigate the possibility of using 18F-positron emission tomography/computer tomography (PET-CT) to predict the histopathologic response in locally advanced rectal cancer (LARC) treated with preoperative chemoradiation (CRT). Methods: The study included 50 patients with LARC treated with preoperative CRT. All patients were evaluated by PET-CT before and after CRT, and results were compared to histopathologic response quantified by tumour regression grade (patients with TRG 1-2 being defined as responders and patients with grade 3-5 as non-responders). Furthermore, the predictive value of metabolic imaging for pathologic complete response (ypCR) was investigated. Results: Responders and non-responders showed statistically significant differences according to Mandard's criteria for maximum standardized uptake value (SUVmax) before and after CRT with a specificity of 76,6% and a positive predictive value of 66,7%. Furthermore, SUVmax values after CRT were able to differentiate patients with ypCR with a sensitivity of 63% and a specificity of 74,4% (positive predictive value 41,2% and negative predictive value 87,9%); This rather low sensitivity and specificity determined that PET-CT was only able to distinguish 7 cases of ypCR from a total of 11 patients. Conclusions: We conclude that 18-F PET-CT performed five to seven weeks after the end of CRT can visualise functional tumour response in LARC. In contrast, metabolic imaging with 18-F PET-CT is not able to predict patients with ypCR accurately

Appropriate drug treatment for status epilepticus does not influence its prognosis

Objective: Status epilepticus (SE) prognosis, is mostly related to non-modifiable factors (especially age, etiology), but the specific role of treatment appropriateness (TA) has not been investigated. Methods: In a prospective cohort with incident SE (excluding postanoxic), TA was defined, after recent European recommendations, in terms of drug dosage (630% deviation) and sequence. Outcome at hospital discharge was categorized into mortality, new handicap, or return to baseline. Results: Among 225 adults, treatment was inappropriate in 37%. In univariate analyses, age, etiology, SE severity and comorbidity, but not TA, were significantly related to outcome. Etiology (95% CI 4.3-82.8) and SE severity (95% CI 1.2-2.4) were independent predictors of mortality, and of lack of return to baseline conditions (etiology: 95% CI 3.9-14.0; SE severity: 95% CI 1.4-2.2). Moreover, TA did not improve outcome prediction in the corresponding ROC curves. Conclusions: This large analysis suggests that TA plays a negligible prognostic role in SE, probably reflecting the outstanding importance of the biological background. Awaiting treatment trials in SE, it appears questionable to apply further resources in refining treatment protocols involving existing compounds; rather, new therapeutic approaches should be identified and tested.

A fatal overdose of cocaine associated with coingestion of marijuana, buprenorphine, and fluoxetine. Body fluid and tissue distribution of cocaine and its metabolites determined by hydrophilic interaction chromatography-mass spectrometry(HILIC-MS).

Chromatographic separation of highly polar basic drugs with ideal ionspray mass spectrometry volatile mobile phases is a difficult challenge. A new quantification procedure was developed using hydrophilic interaction chromatography-mass spectrometry with turbo-ionspray ionization in the positive mode. After addition of deuterated internal standards and simple clean-up liquid extraction, the dried extracts were reconstituted in 500 microL pure acetonitrile and 5 microL was directly injected onto a Waters Atlantis HILIC 150- x 2.1-mm, 3-microm column. Chromatographic separations of cocaine, seven metabolites, and anhydroecgonine were obtained by linear gradient-elution with decreasing high concentrations of acetonitrile (80-56% in 18 min). This high proportion of organic solvent makes it easier to be coupled with MS. The eluent was buffered with 2 mM ammonium acetate at pH 4.5. Except for m-hydroxy-benzoylecgonine, the within-day and between-day precisions at 20, 100, and 500 ng/mL were below 7 and 19.1%, respectively. Accuracy was also below +/- 13.5% at all tested concentrations. The limit of quantification was 5 ng/mL (%Diff < 16.1, %RSD < 4.3) and the limit of detection below 0.5 ng/mL. This method was successfully applied to a fatal overdose. In Switzerland, cocaine abuse has dramatically increased in the last few years. A 45-year-old man, a known HIV-positive drug user, was found dead at home. According to relatives, cocaine was self-injected about 10 times during the evening before death. A low amount of cocaine (0.45 mg) was detected in the bloody fluid taken from a syringe discovered near the corpse. Besides injection marks, no significant lesions were detected during the forensic autopsy. Toxicological investigations showed high cocaine concentrations in all body fluids and tissues. The peripheral blood concentrations of cocaine, benzoylecgonine, and methylecgonine were 5.0, 10.4, and 4.1 mg/L, respectively. The brain concentrations of cocaine, benzoylecgonine, and methylecgonine were 21.2, 3.8, and 3.3 mg/kg, respectively. The highest concentrations of norcocaine (about 1 mg/L) were measured in bile and urine. Very high levels of cocaine were determined in hair (160 ng/mg), indicating chronic cocaine use. A low concentration of anhydroecgonine methylester was also found in urine (0.65 mg/L) suggesting recent cocaine inhalation. Therapeutic blood concentrations of fluoxetine (0.15 mg/L) and buprenorphine (0.1 microg/L) were also discovered. A relatively high concentration of Delta(9)-THC was measured both in peripheral blood (8.2 microg/L) and brain cortex (13.5 microg/kg), suggesting that the victim was under the influence of cannabis at the time of death. In addition, fluoxetine might have enhanced the toxic effects of cocaine because of its weak pro-arrhythmogenic properties. Likewise, combination of cannabinoids and cocaine might have increase detrimental cardiovascular effects. Altogether, these results indicate a lethal cocaine overdose with a minor contribution of fluoxetine and cannabinoids.

A new approach for potential drug target discovery through in silico metabolic pathway analysis using Trypanosoma cruzi genome information

The current drug options for the treatment of chronic Chagas disease have not been sufficient and high hopes have been placed on the use of genomic data from the human parasite Trypanosoma cruzi to identify new drug targets and develop appropriate treatments for both acute and chronic Chagas disease. However, the lack of a complete assembly of the genomic sequence and the presence of many predicted proteins with unknown or unsure functions has hampered our complete view of the parasite's metabolic pathways. Moreover, pinpointing new drug targets has proven to be more complex than anticipated and has revealed large holes in our understanding of metabolic pathways and their integrated regulation, not only for this parasite, but for many other similar pathogens. Using an in silicocomparative study on pathway annotation and searching for analogous and specific enzymes, we have been able to predict a considerable number of additional enzymatic functions in T. cruzi. Here we focus on the energetic pathways, such as glycolysis, the pentose phosphate shunt, the Krebs cycle and lipid metabolism. We point out many enzymes that are analogous to those of the human host, which could be potential new therapeutic targets.

Usefulness of PCR-based assays to assess drug efficacy in Chagas disease chemotherapy: value and limitations

One major goal of research on Chagas disease is the development of effective chemotherapy to eliminate the infection from individuals who have not yet developed cardiac and/or digestive disease manifestations. Cure evaluation is the more complex aspect of its treatment, often leading to diverse and controversial results. The absence of reliable methods or a diagnostic gold standard to assess etiologic treatment efficacy still constitutes a major challenge. In an effort to develop more sensitive tools, polymerase chain reaction (PCR)-based assays were introduced to detect low amounts of Trypanosoma cruzi DNA in blood samples from chagasic patients, thus improving the diagnosis and follow-up evaluation after chemotherapy. In this article, I review the main problems concerning drug efficacy and criteria used for cure estimation in treated chagasic patients, and the work conducted by different groups on developing PCR methodologies to monitor treatment outcome of congenital infections as well as recent and late chronic T. cruzi infections.

Ergosterol biosynthesis and drug development for Chagas disease

This article presents an overview of the currently available drugs nifurtimox (NFX) and benznidazole (BZN) used against Trypanosoma cruzi, the aetiological agent of Chagas disease; herein we discuss their limitations along with potential alternatives with a focus on ergosterol biosynthesis inhibitors (EBI). These compounds are currently the most advanced candidates for new anti-T. cruzi agents given that they block de novo production of 24-alkyl-sterols, which are essential for parasite survival and cannot be replaced by a host's own cholesterol. Among these compounds, new triazole derivatives that inhibit the parasite's C14± sterol demethylase are the most promising, as they have been shown to have curative activity in murine models of acute and chronic Chagas disease and are active against NFX and BZN-resistant T. cruzi strains; among this class of compounds, posaconazole (Schering-Plough Research Institute) and ravuconazole (Eisai Company) are poised for clinical trials in Chagas disease patients in the short term. Other T. cruzi-specific EBI, with in vitro and in vivo potency, include squalene synthase, lanosterol synthase and squalene epoxidase-inhibitors as well as compounds with dual mechanisms of action (ergosterol biosynthesis inhibition and free radical generation), but they are less advanced in their development process. The main putative advantages of EBI over currently available therapies include their higher potency and selectivity in both acute and chronic infections, activity against NFX and BZN-resistant T. cruzi strains, and much better tolerability and safety profiles. Limitations may include complexity and cost of manufacture of the new compounds. As for any new drug, such compounds will require extensive clinical testing before being introduced for clinical use, and the complexity of such studies, particularly in chronic patients, will be compounded by the current limitations in the verification of true parasitological cures for T. cruzi infections.