Artemisinin resistance in Plasmodium falciparum malaria.

BACKGROUND: Artemisinin-based combination therapies are the recommended first-line treatments of falciparum malaria in all countries with endemic disease. There are recent concerns that the efficacy of such therapies has declined on the Thai-Cambodian border, historically a site of emerging antimalarial-drug resistance. METHODS: In two open-label, randomized trials, we compared the efficacies of two treatments for uncomplicated falciparum malaria in Pailin, western Cambodia, and Wang Pha, northwestern Thailand: oral artesunate given at a dose of 2 mg per kilogram of body weight per day, for 7 days, and artesunate given at a dose of 4 mg per kilogram per day, for 3 days, followed by mefloquine at two doses totaling 25 mg per kilogram. We assessed in vitro and in vivo Plasmodium falciparum susceptibility, artesunate pharmacokinetics, and molecular markers of resistance. RESULTS: We studied 40 patients in each of the two locations. The overall median parasite clearance times were 84 hours (interquartile range, 60 to 96) in Pailin and 48 hours (interquartile range, 36 to 66) in Wang Pha (P<0.001). Recrudescence confirmed by means of polymerase-chain-reaction assay occurred in 6 of 20 patients (30%) receiving artesunate monotherapy and 1 of 20 (5%) receiving artesunate-mefloquine therapy in Pailin, as compared with 2 of 20 (10%) and 1 of 20 (5%), respectively, in Wang Pha (P=0.31). These markedly different parasitologic responses were not explained by differences in age, artesunate or dihydroartemisinin pharmacokinetics, results of isotopic in vitro sensitivity tests, or putative molecular correlates of P. falciparum drug resistance (mutations or amplifications of the gene encoding a multidrug resistance protein [PfMDR1] or mutations in the gene encoding sarco-endoplasmic reticulum calcium ATPase6 [PfSERCA]). Adverse events were mild and did not differ significantly between the two treatment groups. CONCLUSIONS: P. falciparum has reduced in vivo susceptibility to artesunate in western Cambodia as compared with northwestern Thailand. Resistance is characterized by slow parasite clearance in vivo without corresponding reductions on conventional in vitro susceptibility testing. Containment measures are urgently needed. (ClinicalTrials.gov number, NCT00493363, and Current Controlled Trials number, ISRCTN64835265.

Optimal dosing of dihydroartemisinin-piperaquine for seasonal malaria chemoprevention in young children.

Young children are the population most severely affected by Plasmodium falciparum malaria. Seasonal malaria chemoprevention (SMC) with amodiaquine and sulfadoxine-pyrimethamine provides substantial benefit to this vulnerable population, but resistance to the drugs will develop. Here, we evaluate the use of dihydroartemisinin-piperaquine as an alternative regimen in 179 children (aged 2.33-58.1 months). Allometrically scaled body weight on pharmacokinetic parameters of piperaquine result in lower drug exposures in small children after a standard mg per kg dosage. A covariate-free sigmoidal EMAX-model describes the interval to malaria re-infections satisfactorily. Population-based simulations suggest that small children would benefit from a higher dosage according to the WHO 2015 guideline. Increasing the dihydroartemisinin-piperaquine dosage and extending the dose schedule to four monthly doses result in a predicted relative reduction in malaria incidence of up to 58% during the high transmission season. The higher and extended dosing schedule to cover the high transmission period for SMC could improve the preventive efficacy substantially

The utility of an AMR dictionary as an educational tool to improve public understanding of antimicrobial resistance

Background: Communicating about antimicrobial resistance (AMR) to the public is challenging.   Methods: We developed a dictionary of terms commonly used to communicate about AMR. For each term, we developed learning points to explain AMR and related concepts in plain language.  We conducted a pilot evaluation in 374 high school students in Ubon Ratchathani, Thailand. In three 50-minute sessions, students were asked to answer five true/false questions using a paper-based questionnaire. The first session assessed their understanding of AMR at baseline, the second after searching the internet, and the third after the provision of the printed AMR dictionary and its web address.    Results: We developed the AMR dictionary as a web-based application (www.amrdictionary.net). The Thai version of the AMR dictionary included 35 terms and associated learning points, seven figures displaying posters promoting AMR awareness in Thailand, and 66 recommended online videos. In the pretest, the proportion of correct responses to each question ranged from 10% to 57%; 10% of the students correctly answered that antibiotics cannot kill viruses and 57% correctly answered that unnecessary use of antibiotics makes them ineffective. After the internet searches, the proportions of correct answers increased, ranging from 62% to 89% (all p&lt;0.001). After providing the AMR dictionary, the proportions of correct answers increased further, ranging from 79% to 89% for three questions (p&lt;0.001), and did not change for one question (p=0.15). Correct responses as to whether taking antibiotics often has side-effects such as diarrhoea reduced from 85% to 74% (p&lt;0.001). The dictionary was revised based on the findings and comments received. Conclusions: Understanding of AMR among Thai high school students is limited. The AMR dictionary can be a useful supportive tool to increase awareness and improve understanding of AMR. Our findings support the need to evaluate the effectiveness of communication tools in the real-world setting.</ns3:p

Simultaneous and enantiospecific quantification of primaquine and carboxyprimaquine in human plasma using liquid chromatography-tandem mass spectrometry

Background The enantiomers of the 8-aminoquinoline anti-malarial primaquine have different pharmacological properties. Development of an analytical method for simultaneous quantification of the enantiomers of primaquine and its metabolite, carboxyprimaquine, will support clinical pharmacometric assessments. Methods A simple and sensitive method consisting of liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) was developed for simultaneous and enantiospecific determination of primaquine and its metabolite, carboxyprimaquine, in human plasma. Stable isotopes were used as internal standards to compensate for potential interference and matrix effects. Plasma samples (100 µL) were precipitated with 1% formic acid in acetonitrile followed by phospholipid removal solid phase extraction. Primaquine and carboxyprimaquine enantiomers were separated on a Chiralcel OD-3R (150 mm × 4.6 mm; I.D. 3 μm) column using a LC gradient mode. For separation of racemic primaquine and carboxyprimaquine, the LC method was modified and validated using a reverse phase column (Hypersil Gold 100 mm × 4.6 mm; I.D. 3 µm) and a mobile phase composed of 10 mM ammonium acetate buffer, pH 3.5 and acetonitrile in the isocratic mode. Method validation was performed according to regulatory guidelines. Results The calibration range was set to 0.571–260 ng/mL and 2.44–2,500 ng/mL for primaquine and carboxyprimaquine enantiomers, respectively, resulting in a correlation coefficient (r2) ≥ 0.0998 for all calibration curves. The intra- and inter-day assay precisions were < 10% and the accuracy was between 94.7 to 103% for all enantiomers of primaquine and carboxyprimaquine. The enantiospecific method was also modified and validated to quantify racemic primaquine and carboxyprimaquine, reducing the total run time from 30 to 8 min. The inter-, intra-day assay precision of the racemic quantification method was < 15%. The absolute recoveries of primaquine and carboxyprimaquine were between 70 and 80%. Stability was demonstrated for up to 2 years in − 80 °C. Both the enantiomeric and racemic LC–MS/MS methods were successfully implemented in pharmacokinetic studies in healthy volunteers. Conclusions Simple, sensitive and accurate LC–MS/MS methods for the quantification of enantiomeric and racemic primaquine and carboxyprimaquine in human plasma were validated successfully and implemented in clinical routine drug analysis

Screening of phytochemicals and in vitro evaluation of antibacterial and antioxidant activities of leaves, pods and bark extracts of Acacia nilotica (L.) Del

The objective of this study was to determine the phytochemical content, antibacterial activity and antioxidant activity of leaves, bark and pods of Acacia nilotica. The different extracts of acacia were evaluated for total phenolic, flavonoid and protein contents, antibacterial (agar well diffusion and broth dilution methods) and antioxidant (DPPH; 1,1-diphenyl-2-picrylhydrazyl assay) activities. The characterization and identification of phenolic compounds was carried out by Liquid Chromatography-tandem Mass Spectrometry analysis. The MS2 fragmentation pattern showed the presence of galloylated catechins and gallocatechin derivatives in tested extracts. The results indicated that all parts of the plant, but especially leaves, were effective in inhibiting the growth of antibiotic resistant strains of Escherichia coli and Salmonella species obtained from clinical and food isolates. The leaves were found to be rich in total phenolic content, proteins and high antioxidant activity as compared to pods and bark. The presence of functional groups of active compounds was confirmed by Fourier transform infrared spectroscopy (FTIR) analysis of plant extracts. It was concluded that all tested parts of A. nilotica had antibacterial and antioxidant activities. These properties might be due to the presence of high total phenolic content, proteins and/or flavonoids. Hence the extracts of leaves, bark and pods of A. nilotica represent a potential source of antibacterial and antioxidant compounds that may be used in food, agriculture and/or pharmaceutical products

A liquid chromatographic-tandem mass spectrometric method for determination of artemether and its metabolite dihydroartemisinin in human plasma.

BACKGROUND: Artemether-lumefantrine is the most widely recommended artemisinin-based combination treatment for falciparum malaria. Quantification of artemether and its metabolite dihydroartemisinin in biological matrices has traditionally been difficult, with sensitivity being an issue. RESULTS: A high-throughput bioanalytical method for the analysis of artemether and its metabolite dihydroartemisinin in human plasma using solid-phase extraction in the 96-well plate format and liquid chromatography coupled to positive ion mode tandem mass spectroscopy has been developed and validated according to US FDA guidelines. The method uses 50 µl plasma and covers the calibration range 1.43-500 ng/ml with a limit of detection at 0.36 ng/ml. CONCLUSIONS: The developed liquid chromatography-tandem mass spectrometry assay is more sensitive than all previous methods despite using a lower plasma volume (50 µl) and is highly suitable for clinical studies where plasma volumes are limited, such as pediatric trials

Simultaneous and enantiospecific quantification of primaquine and carboxyprimaquine in human plasma using liquid chromatography-tandem mass spectrometry

Background The enantiomers of the 8-aminoquinoline anti-malarial primaquine have different pharmacological properties. Development of an analytical method for simultaneous quantification of the enantiomers of primaquine and its metabolite, carboxyprimaquine, will support clinical pharmacometric assessments. Methods A simple and sensitive method consisting of liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) was developed for simultaneous and enantiospecific determination of primaquine and its metabolite, carboxyprimaquine, in human plasma. Stable isotopes were used as internal standards to compensate for potential interference and matrix effects. Plasma samples (100 µL) were precipitated with 1% formic acid in acetonitrile followed by phospholipid removal solid phase extraction. Primaquine and carboxyprimaquine enantiomers were separated on a Chiralcel OD-3R (150 mm × 4.6 mm; I.D. 3 μm) column using a LC gradient mode. For separation of racemic primaquine and carboxyprimaquine, the LC method was modified and validated using a reverse phase column (Hypersil Gold 100 mm × 4.6 mm; I.D. 3 µm) and a mobile phase composed of 10 mM ammonium acetate buffer, pH 3.5 and acetonitrile in the isocratic mode. Method validation was performed according to regulatory guidelines. Results The calibration range was set to 0.571–260 ng/mL and 2.44–2,500 ng/mL for primaquine and carboxyprimaquine enantiomers, respectively, resulting in a correlation coefficient (r2) ≥ 0.0998 for all calibration curves. The intra- and inter-day assay precisions were

Development and validation of a liquid chromatographic-tandem mass spectrometric method for determination of oseltamivir and its metabolite oseltamivir carboxylate in plasma, saliva and urine.

A bioanalytical method for the analysis of oseltamivir (OP) and its metabolite oseltamivir carboxylate (OC) in human plasma, saliva and urine using off-line solid-phase extraction and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. OP and OC were analysed on a ZIC-HILIC column (50 mm x 2.1 mm) using a mobile phase gradient containing acetonitrile-ammonium acetate buffer (pH 3.5; 10mM) at a flow rate of 500 microL/min. The method was validated according to published FDA guidelines and showed excellent performance. The lower limit of quantification for OP was determined to be 1, 1 and 5 ng/mL for plasma, saliva and urine, respectively and for OC was 10, 10 and 30 ng/mL for plasma, saliva and urine, respectively. The upper limit of quantification for OP was determined to be 600, 300 and 1500 ng/mL for plasma, saliva and urine, respectively and for OC was 10,000, 10,000 and 30,000 ng/mL for plasma, saliva and urine, respectively. The within-day and between-day precisions expressed as R.S.D., were lower than 5% at all tested concentrations for all matrices and below 12% at the lower limit of quantification. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range. Matrix effects were thoroughly evaluated both graphically and quantitatively. No matrix effects were detected for OP or OC in plasma or saliva. Residues from the urine matrix (most likely salts) caused some ion suppression for both OP and its deuterated internal standard but had no effect on OC or its deuterated internal standard. The suppression did not affect the quantification of OP