Publications

Parasite clearance rates in Upper Myanmar indicate a distinctive artemisinin resistance phenotype: a therapeutic efficacy study

Artemisinin resistance in Plasmodium falciparum extends across Southeast Asia where it is associated with worsening partner drug resistance and a decline in the efficacy of frontline artemisinin-based combination therapy. Dihydroartemisinin-piperaquine (DP) is an essential component of preventive and curative treatment in the region, but its therapeutic efficacy has fallen in Cambodia. A prospective clinical and parasitological evaluation of DP was conducted at two sites in Upper Myanmar between August 2013 and December 2014, enrolling 116 patients with acute uncomplicated falciparum malaria. Patients received DP orally for 3 days together with primaquine 0.25 mg/kg on admission. Parasite clearance half-lives based on 6 hourly blood smears, and day 42 therapeutic responses were assessed as well as parasite K13 genotypes. Median parasite clearance half-life was prolonged, and clearance half-life was greater than 5 h in 21 % of patients. Delayed parasite clearance was significantly associated with mutations in the propeller region of the parasite k13 gene. The k13 F446I mutation was found in 25.4 % of infections and was associated with a median clearance half-life of 4.7 h compared with 2.7 h for infections without k13 mutations (p < 0.001). There were no failures after 42 days of follow-up, although 18 % of patients had persistent parasitaemia on day 3. The dominant k13 mutation observed in Upper Myanmar, F446I, appears to be associated with an intermediate rate of parasite clearance compared to other common mutations described elsewhere in the Greater Mekong Subregion. Discerning this phenotype requires relatively detailed clearance measurements, highlighting the importance of methodology in assessing artemisinin resistance.

Pharmacokinetics and Pharmacodynamics of Lumefantrine (Benflumetol) in Acute Falciparum Malaria

The objective of this study was to conduct a prospective population pharmacokinetic and pharmacodynamic evaluation of lumefantrine during blinded comparisons of artemether-lumefantrine treatment regimens in uncomplicated multidrug-resistant falciparum malaria. Three combination regimens containing an average adult lumefantrine dose of 1,920 mg over 3 days (four doses) (regimen A) or 2,780 mg over 3 or 5 days (six doses) (regimen B or C, respectively) were given to 266 Thai patients. Detailed observations were obtained for 51 hospitalized adults, and sparse data were collected for 215 patients of all ages in a community setting. The population absorption half-life of lumefantrine was 4.5 h. The model-based median (5th and 95th percentiles) peak plasma lumefantrine concentrations were 6.2 (0.25 and 14.8) μg/ml after regimen A, 9.0 (1.1 and 19.8) μg/ml after regimen B, and 8 (1.4 and 17.4) μg/ml after regimen C. During acute malaria, there was marked variability in the fraction of drug absorbed by patients (coefficient of variation, 150%). The fraction increased considerably and variability fell with clinical recovery, largely because food intake was resumed; taking a normal meal close to drug administration increased oral bioavailability by 108% (90% confidence interval, 64 to 164) ( P , 0.0001). The higher-dose regimens (B and C) gave 60 and 100% higher areas under the concentration-time curves (AUC), respectively, and thus longer durations for which plasma lumefantrine concentrations exceeded the putative in vivo MIC of 280 μg/ml (median for regimen B, 252 h; that for regimen C, 298 h; that for regimen A, 204 h [ P , 0.0001]) and higher cure rates. Lumefantrine oral bioavailability is very dependent on food and is consequently poor in acute malaria but improves markedly with recovery. The high cure rates with the two six-dose regimens resulted from increased AUC and increased time at which lumefantrine concentrations were above the in vivo MIC.

Single dose primaquine for clearance of Plasmodium falciparum gametocytes in children with uncomplicated malaria in Uganda: a randomised, controlled, double-blind, dose-ranging trial

Background Primaquine is the only available drug that clears mature Plasmodium falciparum gametocytes in infected human hosts, thereby preventing transmission of malaria to mosquitoes. However, concerns about dose-dependent haemolysis in people with glucose-6-phosphate dehydrogenase (G6PD) deficiencies have limited its use. We assessed the dose-response association of single-dose primaquine for gametocyte clearance and for safety in P falciparum malaria. Methods We undertook this randomised, double-blind, placebo-controlled trial with four parallel groups in Jinja district, eastern Uganda. We randomly allocated Ugandan children aged 1–10 years with uncomplicated falciparum malaria and normal G6PD enzyme function to receive artemether–lumefantrine, combined with either placebo or with 0·1 mg/kg, 0·4 mg/kg, or 0·75 mg/kg (WHO reference dose) primaquine base. Randomisation was done with computer-generated four-digit treatment assignment codes allocated to random dose groups in block sizes of 16. Study staff who provided care or assessed outcomes and the participants remained masked to the intervention group after assignment. The primary efficacy endpoint was the non-inferiority of the mean duration of gametocyte carriage in the test doses compared with the reference group of 0·75 mg primaquine per kg, with a non-inferiority margin of 2·5 days. The primary safety endpoint was the superiority of the arithmetic mean maximum decrease in haemoglobin concentration from enrolment to day 28 of follow-up in the primaquine treatment groups compared with placebo, with use of significance testing of pairwise comparisons with a cutoff of p=0·05. The trial is registered with ClinicalTrials.gov, number NCT01365598. Findings We randomly allocated 468 participants to receive artemether–lumefantrine combined with placebo (119 children) or with 0·1 mg/kg (116), 0·4 mg/kg (116), or 0·75 mg/kg (117) primaquine base. The mean duration of gametocyte carriage was 6·6 days (95% CI 5·3–7·8) in the 0·75 mg/kg reference group, 6·3 days (5·1–7·5) in the 0·4 mg/kg primaquine group (p=0·74), 8·0 days (6·6–9·4) in the 0·1 mg/kg primaquine group (p=0·14), and 12·4 days (9·9–15·0) in the placebo group (p<0·0001). No children showed evidence of treatment-related haemolysis, and the mean maximum decrease in haemoglobin concentration was not associated with the dose of primaquine received—it did not differ significantly compared with placebo (10·7 g/L, SD 11·1) in the 0·1 mg/kg (11·4 g/L, 9·4; p=0·61), 0·4 mg/kg (11·3 g/L, 10·0; p=0·67), or 0·75 mg/kg (12·7 g/L, 8·2; p=0·11) primaquine groups. Interpretation We conclude that 0·4 mg/kg primaquine has similar gametocytocidal efficacy to the reference 0·75 mg/kg primaquine dose, but a dose of 0·1 mg/kg was inconclusive for non-inferiority. Our findings call for the prioritisation of further trials into the efficacy and safety of doses of primaquine between 0·1 mg/kg and 0·4 mg/kg (including the dose of 0·25 mg/kg recently recommended by WHO), in view of the potential for widespread use of the drug to block malaria transmission. Funding Wellcome Trust and the Bill & Melinda Gates Foundation.

Polymorphic markers for identification of parasite population in Plasmodium malariae

Abstract Background Molecular genotyping in Plasmodium serves many aims including providing tools for studying parasite population genetics and distinguishing recrudescence from reinfection. Microsatellite typing, insertion-deletion (INDEL) and single nucleotide polymorphisms is used for genotyping, but only limited information is available for Plasmodium malariae, an important human malaria species. This study aimed to provide a set of genetic markers to facilitate the study of P. malariae population genetics. Methods Markers for microsatellite genotyping and pmmsp1 gene polymorphisms were developed and validated in symptomatic P. malariae field isolates from Myanmar (N=37). Fragment analysis was used to determine allele sizes at each locus to calculate multiplicity of infections (MOI), linkage disequilibrium, genetic richness index, heterozygosity and construct dendrograms. Nucleotide diversity (π), number of haplotypes, and genetic diversity (Hd) were assessed and a phylogenetic tree was constructed. Genome-wide microsatellite maps with annotated regions of newly identified markers were constructed. Results Six microsatellite markers were developed and tested in 37 P. malariae isolates which showed sufficient heterozygosity (0.530-0.922), genetic richness index (0.050-0.250) and absence of linkage disequilibrium (IAS = 0.03, p-value &gt; 0.05)(N=37). In addition, a tandem repeat (VNTR)-based pmmsp1 INDEL polymorphisms marker was developed and assessed in 27 P. malariae isolates showing a nucleotide diversity of 0.0976, haplotype gene diversity of 0.698 and identified 14 unique variants. The size of VNTR consensus repeat unit adopted as allele was 27 base pairs. The markers Pm12_426 andpmmsp1 showed greatest diversity with heterozygosity scores of 0.920 and 0.835, respectively. Using six microsatellites markers, the likelihood that any two parasite strains would have the same microsatellite genotypes was 8.46 × 10-4 and was further reduced to 1.66 × 10-4 when pmmsp1 polymorphisms were included. Conclusions Six novel microsatellites genotyping markers and a set of pmmsp1 VNTR-based INDEL polymorphisms markers for P. malariae were developed and validated. Each marker could be independently or in combination employed to access genotyping of the parasite. The newly developed markers may serve as a useful tool for investigating parasite diversity, population genetics, molecular epidemiology and for distinguishing recrudescence from reinfection in drug efficacy studies.

Oseltamivir Is Adequately Absorbed Following Nasogastric Administration to Adult Patients with Severe H5N1 Influenza

In the absence of a parenteral drug, oral oseltamivir is currently recommended by the WHO for treating H5N1 influenza. Whether oseltamivir absorption is adequate in severe influenza is unknown. We measured the steady state, plasma concentrations of nasogastrically administered oseltamivir 150 mg bid and its active metabolite, oseltamivir carboxylate (OC), in three, mechanically ventilated patients with severe H5N1 (male, 30 yrs; pregnant female, 22 yrs) and severe H3N2 (female, 76 yrs). Treatments were started 6, 7 and 8 days after illness onset, respectively. Both females were sampled while on continuous venovenous haemofiltration. Admission and follow up specimens (trachea, nose, throat, rectum, blood) were tested for RNA viral load by reverse transcriptase PCR. In vitro virus susceptibility to OC was measured by a neuraminidase inhibition assay. Admission creatinine clearances were 66 (male, H5N1), 82 (female, H5N1) and 6 (H3N2) ml/min. Corresponding AUC0–12 values (5932, 10,951 and 34,670 ng.h/ml) and trough OC concentrations (376, 575 and 2730 ng/ml) were higher than previously reported in healthy volunteers; the latter exceeded 545 to 3956 fold the H5N1 IC50 (0.69 ng/ml) isolated from the H5N1 infected female. Two patients with follow-up respiratory specimens cleared their viruses after 5 (H5N1 male) and 5 (H3N2 female) days of oseltamivir. Both female patients died of respiratory failure; the male survived. 150 mg bid of oseltamivir was well absorbed and converted extensively to OC. Virus was cleared in two patients but two patients died, suggesting viral efficacy but poor clinical efficacy.

Introduction

Comparison of plasma, venous and capillary blood levels of piperaquine in patients with uncomplicated falciparum malaria

Dihydroartemisinin-piperaquine (DP) is a fixed-dose artemisinin-based combination treatment. Field pharmacokinetic studies would be simplified and facilitated by being able to use small volume capillary assays rather than venous blood. The aim of this study was to describe the relationship between piperaquine concentrations measured in capillary blood, venous blood and venous plasma. Samples of plasma, whole blood obtained by venesection and capillary blood were taken simultaneously from patients with uncomplicated Plasmodium falciparum malaria treated with DP between 0 and 9 weeks after treatment. Piperaquine concentrations in venous and capillary samples were measured using solid phase extraction and analysis by liquid chromatography with ultraviolet detection. A total of 161 sets of the three measures were obtained from 54 patients. Piperaquine concentrations in the venous blood samples were approximately twofold higher and those in the capillary blood samples were threefold higher than the corresponding venous plasma concentrations. Capillary blood piperaquine concentrations were approximately 1.7-fold higher than venous blood concentrations, and this difference also increased with time. Differences in whole blood and plasma levels of piperaquine suggest compartmentalisation of the drug within blood cells, as also occurs with the structurally related quinoline chloroquine. The relationship between piperaquine concentrations in the venous plasma, venous blood and capillary blood is variable and unpredictable at low concentrations. However, within the range of concentrations usually present in patients between 3 and 21 days after treatment with currently recommended doses, the relationship between capillary and venous whole blood is predictable; consequently, capillary blood sampling can be used in field assessments.

A molecular barcode and web-based data analysis tool to identify imported Plasmodium vivax malaria

Traditionally, patient travel history has been used to distinguish imported from autochthonous malaria cases, but the dormant liver stages of Plasmodium vivax confound this approach. Molecular tools offer an alternative method to identify, and map imported cases. Using machine learning approaches incorporating hierarchical fixation index and decision tree analyses applied to 799 P. vivax genomes from 21 countries, we identified 33-SNP, 50-SNP and 55-SNP barcodes (GEO33, GEO50 and GEO55), with high capacity to predict the infection’s country of origin. The Matthews correlation coefficient (MCC) for an existing, commonly applied 38-SNP barcode (BR38) exceeded 0.80 in 62% countries. The GEO panels outperformed BR38, with median MCCs &gt; 0.80 in 90% countries at GEO33, and 95% at GEO50 and GEO55. An online, open-access, likelihood-based classifier framework was established to support data analysis (vivaxGEN-geo). The SNP selection and classifier methods can be readily amended for other use cases to support malaria control programs.

Assessment of the neurotoxicity of parenteral artemisinin derivatives in mice.

In all experimental mammals tested (rats, dogs, primates), intramuscular injections of the oil-soluble antimalarial artemisinin derivatives artemether and arteether have produced an unusual pattern of selective damage to brain stem centers predominantly involved in auditory processing and vestibular reflexes. Artesunate, the most widely used of these compounds, is a water soluble hemisuccinate derivative given parenterally either by intravenous or intramuscular injection. The neurotoxic potential of parenteral artesunate and artemether was compared in a murine model. Adult Swiss albino mice were assigned randomly to 28-day regimens of intramuscular artemether or artesunate in doses ranging from 30 to 100 mg/kg/day. At 30 mg/kg/day, no abnormalities were detected with either drug. At 50 mg/kg/day, abnormalities were observed in six of 12 artemether recipients and two of 12 artesunate recipients. These were reversible in all but one (artemether) mouse. At 100 mg/kg/day, eight of 36 artemether recipients, two of 36 artesunate recipients, and one of 18 control mice died. All but four surviving mice in the artemether group (86%) showed obvious and usually irreversible abnormalities of balance and equilibrium, whereas only four artesunate recipients (11%) exhibited abnormalities, and these were reversible in each case (P &lt; 0.001). At this dose the relative risk (95% confidence interval) for death or disability was 5.3 (2.6-11.2) for artemether recipients. Intramuscular artemether is significantly more neurotoxic than intramuscular artesunate in this murine model.

The maintenance of genetic polymorphism in small island populations: large mammals in the Hebrides

Conventionally, small populations living on islands are expected to lose genetic variation by drift. Fluctuations in population size, combined with polygynous mating systems, are expected to contribute to the process by increasing sampling effects on genetic variation. However, in individually monitored populations of Red deer on Rum and Soay sheep on St. Kilda, which experience fluctuations in population size, two processes have been identified which mitigate loss of genetic variation. First, in a number of examples, population reductions are associated with selection. Selection may be in favour of heterozygotes, or, as we have documented in several cases, it may fluctuate in direction temporally. Second, in Soay sheep, in which mortality over population crashes is male-biased, ostensibly leading to low effective numbers of males, molecular studies show that there are systematic changes in the reproductive success of young males, and in variance in male success, that broaden genetic representation compared with expectation.

DRUG-RESISTANCE - CHANGING PATTERNS - WORKSHOP REPORT AND RECOMMENDATIONS

No abstract is provided for this article.

CLINICAL IMPORTANCE OF ANTIMALARIAL PHARMACOKINETICS

No abstract is provided for this article.

Development of temporal modelling for forecasting and prediction of malaria infections using time-series and ARIMAX analyses: A case study in endemic districts of Bhutan

Malaria still remains a public health problem in some districts of Bhutan despite marked reduction of cases in last few years. To strengthen the country's prevention and control measures, this study was carried out to develop forecasting and prediction models of malaria incidence in the endemic districts of Bhutan using time series and ARIMAX.This study was carried out retrospectively using the monthly reported malaria cases from the health centres to Vector-borne Disease Control Programme (VDCP) and the meteorological data from Meteorological Unit, Department of Energy, Ministry of Economic Affairs. Time series analysis was performed on monthly malaria cases, from 1994 to 2008, in seven malaria endemic districts. The time series models derived from a multiplicative seasonal autoregressive integrated moving average (ARIMA) was deployed to identify the best model using data from 1994 to 2006. The best-fit model was selected for each individual district and for the overall endemic area was developed and the monthly cases from January to December 2009 and 2010 were forecasted. In developing the prediction model, the monthly reported malaria cases and the meteorological factors from 1996 to 2008 of the seven districts were analysed. The method of ARIMAX modelling was employed to determine predictors of malaria of the subsequent month.It was found that the ARIMA (p, d, q) (P, D, Q)s model (p and P representing the auto regressive and seasonal autoregressive; d and D representing the non-seasonal differences and seasonal differencing; and q and Q the moving average parameters and seasonal moving average parameters, respectively and s representing the length of the seasonal period) for the overall endemic districts was (2,1,1)(0,1,1)12; the modelling data from each district revealed two most common ARIMA models including (2,1,1)(0,1,1)12 and (1,1,1)(0,1,1)12. The forecasted monthly malaria cases from January to December 2009 and 2010 varied from 15 to 82 cases in 2009 and 67 to 149 cases in 2010, where population in 2009 was 285,375 and the expected population of 2010 to be 289,085. The ARIMAX model of monthly cases and climatic factors showed considerable variations among the different districts. In general, the mean maximum temperature lagged at one month was a strong positive predictor of an increased malaria cases for four districts. The monthly number of cases of the previous month was also a significant predictor in one district, whereas no variable could predict malaria cases for two districts.The ARIMA models of time-series analysis were useful in forecasting the number of cases in the endemic areas of Bhutan. There was no consistency in the predictors of malaria cases when using ARIMAX model with selected lag times and climatic predictors. The ARIMA forecasting models could be employed for planning and managing malaria prevention and control programme in Bhutan.

Primaquine-induced haemolysis in females heterozygous for G6PD deficiency

Oxidative agents can cause acute haemolytic anaemia in persons with G6PD deficiency. Understanding the relationship between G6PD genotype and the phenotypic expression of the enzyme deficiency is necessary so that severe haemolysis can be avoided. The patterns of oxidative haemolysis have been well described in G6PD deficient hemizygous males and homozygous females; and haemolysis in the proportionally more numerous heterozygous females has been documented mainly following consumption of fava beans and more recently dapsone. It has long been known that 8-aminoquinolines, notably primaquine and tafenoquine, cause acute haemolysis in G6PD deficiency. To support wider use of primaquine in Plasmodium vivax elimination, more data are needed on the haemolytic consequences of 8-aminoquinolines in G6PD heterozygous females. Two recent studies (in 2017) have provided precisely such data; and the need has emerged for the development of point of care quantitative testing of G6PD activity. Another priority is exploring alternative 8-aminoquinoline dosing regimens that are practical and improve safety in G6PD deficient individuals.

Primate socioecology at the crossroads: Past, present, and future

Attempts to explain differences in the size and structure of primate groups have argued that they are a consequence of variation in the intensity of feeding competition caused by contrasts in food distribution. However, although feeding competition can limit the size of female groups, many other factors affect the costs and the benefits of sociality to females and contribute to differences in group size. Moreover, interspecific differences in social relationships between females, in female philopatry, and in kinship between group members appear to be more closely associated with variation in life‐history parameters, reproductive strategies, and phylogeny than with contrasts in food distribution or feeding competition. The mismatch between predictions of socioecological theory and observed variation in primate social behavior has led to protracted arguments about the future of primate socioecology. We argue that future attempts to understand the diversity of primate societies need to be based on an approach that explores separate explanations for different components of social organization, combines ecological and phylogenetic information, and integrates research on primates with similar studies of other groups of mammals. © 2012 Wiley Periodicals, Inc.

Life history trade-offs at a single locus maintain sexually selected genetic variation

No abstract is provided for this article.

Predicting the Clinical Outcome of Severe Falciparum Malaria in African Children: Findings From a Large Randomized Trial

Background. Data from the largest randomized, controlled trial for the treatment of children hospitalized with severe malaria were used to identify such predictors of a poor outcome from severe malaria. Methods. African children (<15 years) with severe malaria participated in a randomized comparison of parenteral artesunate and parenteral quinine in 9 African countries. Detailed clinical assessment was performed on admission. Parasite densities were assessed in a reference laboratory. Predictors of death were examined using a multivariate logistic regression model. Results. Twenty indicators of disease severity were assessed, out of which 5 (base deficit, impaired consciousness, convulsions, elevated blood urea, and underlying chronic illness) were associated independently with death. Tachypnea, respiratory distress, deep breathing, shock, prostration, low pH, hyperparasitemia, severe anemia, and jaundice were statistically significant indicators of death in the univariate analysis but not in the multivariate model. Age, glucose levels, axillary temperature, parasite density, heart rate, blood pressure, and blackwater fever were not related to death in univariate models. Conclusions. Acidosis, cerebral involvement, renal impairment, and chronic illness are key independent predictors for a poor outcome in African children with severe malaria. Mortality is markedly increased in cerebral malaria combined with acidosis. Clinical Trial Registration. ISRCTN50258054.

Low-density genetically diverse natural Wolbachia infections in malaria vectors in Kayin state, Myanmar

<ns4:p><ns4:bold>Background</ns4:bold><ns4:bold>:</ns4:bold> Natural <ns4:italic>Wolbachia </ns4:italic>infections in malaria mosquitoes were recently reported in Africa, and negatively correlated with the development of <ns4:italic>Plasmodium falciparum</ns4:italic> in the vectors. The occurrence and effects of <ns4:italic>Wolbachia </ns4:italic>infections outside Africa have not been described and may have been underestimated.</ns4:p><ns4:p> <ns4:bold>Methods</ns4:bold><ns4:bold>:</ns4:bold> Mosquitoes were collected by human-landing catch during May and June 2017 in ten villages in Kayin state, Myanmar. Closely related species of malaria vectors were identified with molecular assays. <ns4:italic>Wolbachia</ns4:italic> infection rates were assessed with quantitative real-time PCR.</ns4:p><ns4:p> <ns4:bold>Results:</ns4:bold> Low titer of <ns4:italic>Wolbachia </ns4:italic>DNA was detected in 13/370 samples in six malaria vector species. Phylogenetic analysis based on 16S rRNA sequences revealed a high diversity of <ns4:italic>Wolbachia </ns4:italic>strains and identified lineages different from those described in the African malaria mosquitoes.</ns4:p><ns4:p> <ns4:bold>Conclusion:</ns4:bold> These low-density genetically diverse natural <ns4:italic>Wolbachia </ns4:italic>infections question the ecology and biology of <ns4:italic>Wolbachia</ns4:italic>-<ns4:italic>Anopheles</ns4:italic> interactions<ns4:italic> </ns4:italic>in Southeast Asia. Their effects on malaria transmission and mosquito vectors are yet to be determined.</ns4:p>