Oxamniquine resistance alleles are widespread in Old World Schistosoma mansoni and predate drug deployment

Do mutations required for adaptation occur de novo, or are they segregating within populations as standing genetic variation? This question is key to understanding adaptive change in nature, and has important practical consequences for the evolution of drug resistance. We provide evidence that alleles conferring resistance to oxamniquine (OXA), an antischistosomal drug, are widespread in natural parasite populations under minimal drug pressure and predate OXA deployment. OXA has been used since the 1970s to treat Schistosoma mansoni infections in the New World where S. mansoni established during the slave trade. Recessive loss-of-function mutations within a parasite sulfotransferase (SmSULT-OR) underlie resistance, and several verified resistance mutations, including a deletion (p.E142del), have been identified in the New World. Here we investigate sequence variation in SmSULT-OR in S. mansoni from the Old World, where OXA has seen minimal usage. We sequenced exomes of 204 S. mansoni parasites from West Africa, East Africa and the Middle East, and scored variants in SmSULT-OR and flanking regions. We identified 39 non-synonymous SNPs, 4 deletions, 1 duplication and 1 premature stop codon in the SmSULT-OR coding sequence, including one confirmed resistance deletion (p.E142del). We expressed recombinant proteins and used an in vitro OXA activation assay to functionally validate the OXA-resistance phenotype for four predicted OXA-resistance mutations. Three aspects of the data are of particular interest: (i) segregating OXA-resistance alleles are widespread in Old World populations (4.29–14.91% frequency), despite minimal OXA usage, (ii) two OXA-resistance mutations (p.W120R, p.N171IfsX28) are particularly common (>5%) in East African and Middle-Eastern populations, (iii) the p.E142del allele has identical flanking SNPs in both West Africa and Puerto Rico, suggesting that parasites bearing this allele colonized the New World during the slave trade and therefore predate OXA deployment. We conclude that standing variation for OXA resistance is widespread in S. mansoni

The quality of sulphadoxine-pyrimethamine and amodiaquine products in the Kenyan retail sector.

BACKGROUND AND OBJECTIVE: Malaria is a disease of major public health importance in Kenya killing 26,000 children under 5 years of age annually. This paper seeks to assess the quality of sulphadoxine-pyrimethamine (SP) and amodiaquine (AQ) products available over-the-counter to communities in Kenya as most malaria fevers are self-medicated using drugs from the informal retail sector. METHODS: A retail audit of 880 retail outlets was carried in 2002 in four districts in Kenya, in which antimalarial drug stocks and their primary wholesale sources were noted. In addition, the expiry dates on audited products and the basic storage conditions were recorded on a proforma. The most commonly stocked SP and AQ products were then sampled from the top 10 wholesalers in each district and samples subjected to standard United States Pharmacopoeia (USP) tests of content and dissolution. RESULTS AND DISCUSSION: SP and AQ were the most frequently stocked antimalarial drugs, accounting for approximately 75% of all the antimalarial drugs stocked in the four districts. Of 116 SP and AQ samples analysed, 47 (40.5%) did not meet the USP specifications for content and/or dissolution. Overall, approximately 45.3% of SP and 33.0% of AQ samples were found to be sub-standard. Of the sub-standard SP products, 55.2% were suspensions while 61.1% of the substandard AQ products were tablets. Most SP failures were because of the pyrimethamine component. CONCLUSION: There is a need to strengthen post-marketing surveillance systems to protect patients from being treated with sub-standard and counterfeit antimalarial drugs in Kenya

Migration and career success: testing a time-sequenced model

Purpose – The paper seeks to determine whether different aspects of migrant pre-migration characteristics (human capital and motivation to migrate) and post-migration behaviour (social integration and career self-management) predict migrants' post-migration career success. Design/methodology/approach – The research employed a survey questionnaire applied to a sample of 210 migrants who had migrated from Sri Lanka to New Zealand. Twenty-three independent and three dependent (career success – objective and subjective) variables were measured. Sequential multiple regression analysis was applied, mirroring the time-sequenced theory of career development. Findings – Overall, migrants' occupational status had declined markedly following migration. Variables representing human capital, social integration and career self-management perspectives all contributed substantially to explaining variances in career success, especially objective career success, but motivation to migrate did not. Human capital variables were especially influential in determining pre-migration success, acculturation in the host country and education in the host country in post-migration success. Effects of career self-management behaviours on success were relatively small. Research limitations/implications – A limitation is the cross-sectional design, and possible non-generalisability beyond a single migrant group and host country. Practical implications – The paper discusses implications for migrants, policy makers and future research. Originality/value – Migration, and interest in research on migrants' careers, is growing. This paper applies a wide range of predictor variables and a logical causal model to predicting migrant career success, indicates significant effects, and points to positive actions that may be taken by government, organisations and migrants

Determination of paraldehyde by gas chromatography in whole blood from children.

A rapid, sensitive and selective gas chromatographic method with flame ionization detection was developed for the determination of paraldehyde in small blood samples taken from children. Whole blood samples (300 microl) collected in a 3 ml Wheaton glass sample vial were spiked with acetone (internal standard: 15 ng) followed by addition of concentrated hydrochloric acid. The mixture was heated in the sealed airtight sample vial in a water bath (96 Celsius; 5 min) to depolymerize paraldehyde to acetaldehyde. A 2 ml aliquot of the headspace was analyzed by gas chromatography with flame ionization detector using a stainless steel column (3 m x 4 mm i.d.) packed with 10% Carbowax 20 M/ 2% KOH on 80/100 Chromosorb WAW. Calibration curves were linear from 1.0-20 microg (r2>0.99). The limit of detection was 1.5 microg/ml, while relative mean recoveries at 2 and 18 microg were 105.6 +/- 8.4 and 101.2 +/- 5.9%, respectively (n = 10 for each level). Intra- and inter-assay relative standard deviations at 2, 10 and 18 microg were <15%. There was no interference from other drugs concurrently used in children with severe malaria, such as anticonvulsants (diazepam, phenytoin, phenobarbitone), antipyretics/analgesics (paracetamol and salicylate), antibiotics (gentamicin, chloramphenicol, benzyl penicillin) and antimalarials (chloroquine, quinine, proguanil, cycloguanil, pyrimethamine and sulfadoxine). The method was successfully applied for pharmacokinetic studies of paraldehyde in children with convulsions associated with severe malaria

Determination of midazolam and its major metabolite 1'-hydroxymidazolam by high-performance liquid chromatography-electrospray mass spectrometry in plasma from children.

We have developed a sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) for the simultaneous quantification of midazolam (MDZ) and its major metabolite, 1'-hydroxymidazolam (1'-OHM) in a small volume (200 microl) of human plasma. Midazolam, 1'-OHM and 1'-chlordiazepoxide (internal standard) were extracted from alkalinised (pH 9.5) spiked and clinical plasma samples using a single step liquid-liquid extraction with 1-chlorobutane. The chromatographic separation was performed on a reversed-phase HyPURITY Elite C18 (5 microm particle size; 100 mm x 2.1mm i.d.) analytical column using an acidic (pH 2.8) mobile phase (water-acetonitrile; 75:25% (v/v) containing formic acid (0.1%, v/v)) delivered at a flow-rate of 200 microl/min. The mass spectrometer was operated in the positive ion mode at the protonated-molecular ions [M+l]+ of parent drug and metabolite. Calibration curves in spiked plasma were linear (r2 > or = 0.99) from 15 to 600 ng/ml (MDZ) and 5-200 ng/ml (1'-OHM). The limits of detection and quantification were 2 and 5 ng/ml, respectively, for both MDZ and 1'-OHM. The mean relative recoveries at 40 and 600 ng/ml (MDZ) were 79.4+/-3.1% (n = 6) and 84.2+/-4.7% (n = 8), respectively; for 1'-OHM at 30 and 200 ng/ml the values were 89.9+/-7.2% (n = 6) and 86.9+/-5.6% (n = 8), respectively. The intra-assay and inter-assay coefficients of variation (CVs) for MDZ were less than 8%, and for 1'-OHM were less than 13%. There was no interference from other commonly used antimalarials, antipyretic drugs and antibiotics. The method was successfully applied to a pharmacokinetic study of MDZ and 1'-OHM in children with severe malaria and convulsions following administration of MDZ either intravenously (i.v.) or intramuscularly (i.m.)

Determination of lorazepam in plasma from children by high-performance liquid chromatography with UV detection.

A simple, sensitive, selective, and reproducible reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection was developed for the determination of lorazepam (LZP) in human plasma, using oxazepam (OZP) as internal standard. LZP and OZP were extracted from alkalinized (pH 9.5) spiked and clinical plasma samples using a single step liquid-liquid extraction with a mixture of n-hexane-dichloromethane (70:30%; v/v). Chromatographic separation was performed on a reversed-phase Synergi Max RP analytical column (150 mmx4.6 mm i.d.; 4 microm particle size), using an aqueous mobile phase (10 mM KH2PO4 buffer (pH 2.4)-acetonitrile; 65:35%, v/v) delivered at a flow-rate of 2.5 ml/min. Retention times for OZP and LZP were 10.2 and 11.9 min, respectively. Calibration curves were linear from 10 to 300 ng with correlation coefficients (r2) better than 0.99. The limits of detection (LOD) and quantification (LOQ) were 2.5 and 10 ng/ml, respectively, using 0.5 ml samples. The mean relative recoveries at 20 and 300 ng/ml were 84.1+/-5.5% (n=6) and 72.4+/-5.9% (n=7), respectively; for OZP at 200 ng the value was 68.2+/-6.8% (n=14). The intra-assay relative standard deviations (R.S.D.) at 20, 150 and 270 ng/ml of LZP were 7.8%, 9.8% (n=7 in all cases) and 6.6% (n=8), respectively. The inter-assay R.S.D. at the above concentrations were 15.9%, 7.7% and 8.4% (n=7 in all cases), respectively. Intra- and inter-assay accuracy data were within the acceptance interval of +/-20% of the nominal values. There was no interference from other commonly co-administered anticonvulsant, antimicrobial, antipyretic, and antimalarial drugs. The method has been successfully applied to a pharmacokinetic study of LZP in children with severe malaria and convulsions following administration of a single intravenous dose (0.1 mg/kg body weight) of LZP

The difference between effectiveness and efficacy of antimalarial drugs in Kenya.

OBJECTIVE: To demonstrate the difference between effectiveness and efficacy of antimalarial (AM) drugs in Kenya. METHODS: We undertook a series of linked surveys in four districts of Kenya between 2001 and 2002 on (i) community usage of nationally recommended first- and second-line AM drugs; (ii) commonly stocked AM products in the retail and wholesale sectors; and (iii) quality of the most commonly available first- and second-line AM products. These were combined with estimates of adherence and clinical efficacy to derive overall drug effectiveness. RESULTS: The overall modelled effectiveness for sulphadoxine-pyrimethamine (SP) was estimated to be 62% compared with 85% for reported SP clinical efficacy. For amodiaquine the modelled effectiveness was 48% compared with 99% reported efficacy during the same time period. CONCLUSIONS: The quality of AM products and patient adherence to dosage regimens are important determinants of drug effectiveness, and should be measured alongside clinical efficacy. Post-registration measures to regulate drug quality and improve patient adherence would contribute significantly to AM drug performance