Publications

Relation of the stage of parasite development in the peripheral blood to prognosis in severe falciparum malaria

Admission blood films from 72 patients who died of severe falciparum malaria (50 Thai adults, 22 Gambian children) were matched retrospectively for parasitaemia with equal numbers of survivors. The peripheral blood parasites from fatal cases were more mature than those from survivors. Tiny rings (TR) comprised >50% of parasites in 47/72 (65%) survivors but only 12/72 (17%) of fatal cases (P < 0.001). Parasites containing visible pigment (MTS: mature trophozoites and schizonts) comprised <20% of the total parasite count in 10/72 (14%) survivors compared with 31/72 (43%) fatal cases (P < 0.001). Of the 39 patients with >104 MTS/μL, 30 (81%) died. These findings were confirmed in a prospective study of 279 adult Thai patients admitted sequentially with acute falciparum malaria. Only 4 of the 19 fatal cases (21%) had >50% TR, compared with 130 of 260 (50%) survivors, whereas >20% MTS were found in 10/19 (53%) fatal cases, compared with 28/108 (27%) severe malaria survivors, and 26/155 (17%) patients with moderately severe malaria (P = 0·001). As a predictor of fatal outcome, the finding of either >104 MTS/μL or >5× 105 parasites/μL in severe malaria had a sensitivity of 90% (95% confidence interval [CI] = 75–97%) and a specificity of 72% (95%CI = 59–86%). These observations are consistent with the hypothesis that a predominance of mature parasites in the peripheral blood reflects a greater sequestered biomass, and thus more severe disease. Simple microscopical assessment of parasite maturity on an admission blood slide provides important pathophysiological and prognostic information in severe falciparum malaria.

Sun-Earth, Sun-Earth-Moon, and idealized system datasets paper_c390_sunearthmoon_log2aubound06_meshlevel02_ic_rednoise_randseed9.mat

:unav

Characterization of Solid Counterfeit Drug Samples by Desorption Electrospray Ionization and Direct‐analysis‐in‐real‐time Coupled to Time‐of‐flight Mass Spectrometry

Vacuum is not the limit: direct analysis in real time (DART), a new MS ionization method, allows probing of pharmaceuticals under atmospheric pressure, thus bypassing lengthy sample preparation steps and enhancing throughput. DART allows rapid screening of solid drugs at almost constant temperature which decreases errors in mass measurement and improves the identification of potentially harmful unknowns. This is important for identifying counterfeit drugs (fake hologram shown).

High throughput assay for the determination of piperaquine in plasma

A high throughput assay for the determination of the antimalarial piperaquine in plasma has been developed and validated. The assay utilises 96-wellplate formats throughout the whole procedure, and easily enables a throughput of 192 samples a day using a single LC system. Buffer (pH 2.0; 0.05M) containing internal standard was added to 0.25mL plasma in a 96-wellplate (2mL wells). The samples were extracted on a MPC solid phase extraction deep well 96-wellplate (3M Empore). Piperaquine and internal standard were analysed by liquid chromatography with UV detection on a Chromolith Performance (100mm×4.6mm) column with a mobile phase containing acetonitrile–phosphate buffer (pH 2.5; 0.1M) (8:92, v/v) at a flow rate of 3.0mL/min. The within-day precisions for piperaquine were 3.3 and 2.3% at 40 and 1250ng/mL, respectively. The between-day precisions for piperaquine were 5.8 and 1.3% at 40 and 1250ng/mL, respectively. The total assay precisions using 29 replicates over 5 days were 6.7, 4.5 and 2.7% at 40, 200 and 1250ng/mL, respectively. The lower limit of quantification (LLOQ) and the limit of detection (LOD) were 10 and 5ng/mL, respectively using 0.25mL plasma. Using 1mL of plasma, it was possible to decrease LLOQ and LOD to 2.5 and 1.25ng/mL, respectively.

Why is it that antimalarial drug treatments do not always work

The objective of antimalarial drug treatment in severe malaria is to save the patient's life. In uncomplicated malaria it is to reduce the parasite biomass to zero, or down to a level where host defences can deal with the remainder. Treatment regimens with rapidly eliminated drugs must generally cover four asexual life-cycles (i.e. > 6 days for Plasmodium falciparum and P. vivax) to eradicate all the parasites in the blood. For slowly eliminated drugs, blood concentrations must exceed the parasites' minimum inhibitory concentration (and preferably the minimum parasiticidal concentration) until all parasites have been eradicated. Resistance means that there is a right shift in the concentration-effect relationship. This may be large and abrupt, as with the point mutations that confer pyrimethamine, sulphonamide or atovaquone resistance, or slow and gradual, as with the processes that determine resistance to chloroquine, quinine or mefloquine. Although treatment failure in malaria usually results from poor compliance, inadequate dosing, pharmacokinetic factors or resistance, some infections will recrudesce when none of these factors operates. How parasites persist despite apparently adequate antimalarial treatment remains unresolved.

Odour-based kin discrimination in the cooperatively breeding meerkat

Kin recognition is a useful ability for animals, facilitating cooperation among relatives and avoidance of excessive kin competition or inbreeding. In meerkats, Suricata suricatta, encounters between unfamiliar kin are relatively frequent, and kin recognition by phenotype matching is expected to avoid inbreeding with close relatives. Here, we investigate whether female meerkats are able to discriminate the scent of unfamiliar kin from unfamiliar non-kin. Dominant females were presented with anal gland secretion from unfamiliar individuals that varied in their relatedness. Our result indicates that females spent more time investigating the scent of related than unrelated unfamiliar individuals, suggesting that females may use a phenotype matching mechanism (or recognition alleles) to discriminate the odour of their kin from the odour of their non-kin. Our study provides a key starting point for further investigations into the use of kin recognition for inbreeding avoidance in the widely studied meerkat.

ACT PARTNER DRUG EROSION: EVIDENCE OF PIPERAQUINE-RESISTANT PLASMODIUM FALCIPARUM IN CAMBODIA

No abstract is provided for this article.

Rectal alternatives to existing oral and injectable pediatric antibiotherapies: Going beyond a wax suppository

No abstract is provided for this article.

Dihydroartemisinin-piperaquine against multidrug-resistant Plasmodium falciparum malaria in Vietnam: randomised clinical trial

No abstract is provided for this article.

Genetic Variability of Plasmodium malariae dihydropteroate synthase (dhps) in Four Asian Countries

The dihydropteroate synthase (dhps) genes of 44 P. malariae strains from four Asian countries were isolated. Only a limited number of polymorphisms were observed. Comparison with homologous mutations in other Plasmodium species showed that these polymorphisms are unlikely to be associated with sulfadoxine resistance.

Comparison of Antibody Responses and Parasite Clearance in Artemisinin Therapeutic Efficacy Studies in the Democratic Republic of Congo and Asia

Background Understanding the effect of immunity on Plasmodium falciparum clearance is essential for interpreting therapeutic efficacy studies designed to monitor emergence of artemisinin drug resistance. In low-transmission areas of Southeast Asia, where resistance has emerged, P. falciparum antibodies confound parasite clearance measures. However, variation in naturally acquired antibodies across Asian and sub-Saharan African epidemiological contexts and their impact on parasite clearance re yet to be quantified. Methods In an artemisinin therapeutic efficacy study, antibodies to 12 pre-erythrocytic and erythrocytic P. falciparum antigens were measured in 118 children with uncomplicated P. falciparum malaria in the Democratic Republic of Congo (DRC) and compared with responses in patients from Asian sites, described elsewhere. Results Parasite clearance half-life was shorter in DRC patients (median, 2 hours) compared with most Asian sites (median, 2–7 hours), but P. falciparum antibody levels and seroprevalences were similar. There was no evidence for an association between antibody seropositivity and parasite clearance half-life (mean difference between seronegative and seropositive, −0.14 to +0.40 hour) in DRC patients. Conclusions In DRC, where artemisinin remains highly effective, the substantially shorter parasite clearance time compared with Asia was not explained by differences in the P. falciparum antibody responses studied.

Tafenoquine — A Radical Improvement?

Plasmodium vivax is a major cause of malaria in the Americas, the Horn of Africa, and Asia. P. vivax malaria has a propensity to recur (relapse), often multiple times, after resolution of the initial illness. This may cause substantial medical complications and contribute to death.1 Relapses arise from the dormant parasite stage in the liver (the hypnozoite). The only available drug that clears hypnozoites, thereby providing “radical cure,” is the 8-aminoquinoline primaquine. In this issue of the Journal, two studies report on the radical curative efficacy of tafenoquine, a newly registered, slowly eliminated, single-dose 8-aminoquinoline.2,3 It’s been a long time . . .

Polymorphic markers for identification of parasite population in Plasmodium malariae

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 P. 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 ( H d ) 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 ( I A S = 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 0.092, 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 and pmmsp1 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.

Responding to the evidence for the management of severe malaria

Humanity owes a great debt to quinine. Cinchona alkaloids have been used to treat malaria for hundreds of years after the arrival of cinchona bark in Europe in the 17th century, where it was mixed with rose leaves, lemon juice and wine to treat the malarious patients of Essex (Butler et al. 2010). While quinine has been largely replaced by more effective and better-tolerated drugs in the treatment of uncomplicated malaria, it remains the standard treatment for severe malaria in many countries. In Africa, where over 90% of the estimated 781 000 malaria-related deaths in 2009 occurred, quinine is first-line therapy for severe malaria in almost all countries (WHO 2010). Recent evidence shows that it is time to replace quinine for severe malaria as well. A large randomized trial conducted in Asia in 2005 found that parenteral artesunate reduced overall mortality by 39% compared with quinine (Dondorp et al. 2005). As a result, in 2006 WHO recommended artesunate as the treatment of choice for adults, but considered there was insufficient evidence to extend this recommendation to children in Africa. That evidence came in late 2010, from the largest ever study of severe malaria (5425 children across Africa), which found that artesunate reduced mortality by 22.5% compared with quinine (Dondorp et al. 2010). Importantly, there was no evidence in this or previous studies of an increase in neurological sequelae in survivors. These results were confirmed by a recent Cochrane meta-analysis that found an overall mortality reduction of 39% among adults and 24% among children compared to quinine (Sinclair et al. 2011), an identical finding to the meta-analysis accompanying the African trial (Dondorp et al. 2010). So what now? WHO has just changed its guidelines to put artesunate as the treatment of choice for severe malaria everywhere (WHO 2011), and these recommendations need to be disseminated to all relevant malaria actors to support national guideline change where needed. To date, only one African country (Nigeria) has revised its guidelines to include artesunate for severe malaria, and only as an alternative treatment (WHO 2010). Given the long history of quinine use, the dissemination of national guidelines will need to be accompanied by training to help shift health provider habits and personal convictions. All this needs to be properly supported, both by international donors and through technical advice from WHO’s regional offices. The challenge in translating evidence into practice should never be underestimated, particularly when it comes to malaria treatment, as recent history shows. In 2000, WHO recommended a policy shift in the management of uncomplicated malaria from chloroquine to artemisinin-based combination therapies (ACTs) because of high levels of chloroquine resistance. Yet, despite substantial evidence supporting the superior efficacy of ACTs, international donors and ministries of health in malaria-endemic countries continued to support chloroquine use for several years, mainly because chloroquine was a much cheaper drug (Attaran et al. 2004). Similarly, the higher unit price of artesunate is likely to be a barrier. Cost effectiveness studies, however, show that when mortality and associated costs such as reduced side-effect management and hospitalization are considered, artesunate is cost effective (Lubell et al. 2009, 2011). Furthermore, the cost of artesunate may fall as demand increases. Nevertheless, for policy and practice to change in the short-term, additional international funding support is needed. Another important potential concern is the limited availability of quality-assured sources of artesunate. Currently, only one source of injectable artesunate has been pre-qualified by WHO, and malaria control programmes may be reluctant to make the switch as long as consistency in supply is uncertain. In addition, quinine production represents an important economic activity in a number of malaria-endemic countries; in Burundi, for example, quinine is one of the few drugs manufactured in-country, making it more popular and more easily accessible than imported antimalarial drugs (Amuasi et al. 2011). However, artemisinin, the raw material for artesunate, is increasingly being produced in Africa, including in Kenya, Tanzania and Uganda (http://www.artepal.org), and as demand grows, supply should logically follow. Finally, it can be anticipated that calls for local evidence may be made by national governments out of concern that studies performed in other contexts may not apply to their setting. MSF faced similar challenges when trying to move from chloroquine to ACT (Guthmann et al. 2008). While this may initially appear reasonable, the evidence to date is broadly generalizable and the latest Cochrane review concludes that further research to assess the efficacy of artesunate versus quinine is unnecessary (Sinclair et al. 2011). Patients should only be subjected to experimental trials if there is real uncertainty about which drug is better (CIOMS 2002), and while operational research may help guide implementation, it would clearly be unethical to delay implementation and subject patients to further drug effectiveness studies. Global funding for malaria control is already insufficient (Snow et al. 2010), and in the current economic climate, donors may be reluctant to support a switch to a more expensive treatment. However, replacing quinine with artesunate is a clear cut intervention that has the potential to save nearly 200 000 lives each year and the total annual cost of providing artesunate for treating all cases of severe malaria worldwide would likely be less than $US 50 million (MSF 2011). For African countries to make the switch, strong international support will be required to provide additional funds to support drug procurement and training costs and send a clear message to manufacturers that quality sources of artesunate are needed.

Immunofluorescence microscopy for the rapid diagnosis of melioidosis.

A direct immunofluorescent antibody test (DIF) was developed for the rapid diagnosis of melioidosis, a potentially fatal infection caused by Pseudomonas pseudomallei. In a clinical evaluation of 369 sputum, pus, or urine specimens from 272 patients with suspected melioidosis, the DIF had a sensitivity of 73% and a specificity of 99% compared with culture. Using this DIF, a confident diagnosis of melioidosis can now be made within two hours of admission to hospital, compared with the delay of two to four days required for culture results. Consequent early institution of specific antimicrobial therapy may help to save lives.

Protective effect of Mediterranean-type glucose-6-phosphate dehydrogenase deficiency against Plasmodium vivax malaria

X-linked glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy. The severe Mediterranean variant (G6PD Med) found across Europe and Asia is thought to confer protection against malaria, but its effect is unclear. We fitted a Bayesian statistical model to observed G6PD Med allele frequencies in 999 Pashtun patients presenting with acute Plasmodium vivax malaria and 1408 population controls. G6PD Med was associated with reductions in symptomatic P. vivax malaria incidence of 76% (95% credible interval [CI], 58–88) in hemizygous males and homozygous females combined and 55% (95% CI, 38–68) in heterozygous females. Unless there is very large population stratification within the Pashtun (confounding these results), the G6PD Med genotype confers a very large and gene-dose proportional protective effect against acute vivax malaria. The proportion of patients with vivax malaria at risk of haemolysis following 8-aminoquinoline radical cure is substantially overestimated by studies measuring G6PD deficiency prevalence in healthy subjects.

Acute Management of Dengue Shock Syndrome: A Randomized Double-Blind Comparison of 4 Intravenous Fluid Regimens in the First Hour

Dengue hemorrhagic fever is an important cause of morbidity among Asian children, and the more severe dengue shock syndrome (DSS) causes a significant number of childhood deaths. DSS is characterized by a massive increase in systemic capillary permeability with consequent hypovolemia. Fluid resuscitation is critical, but as yet there have been no large trials to determine the optimal fluid regimen. We undertook a randomized blinded comparison of 4 fluids (dextran, gelatin, lactated Ringer's, and "normal" saline) for initial resuscitation of 230 Vietnamese children with DSS. All the children survived, and there was no clear advantage to using any of the 4 fluids, but the longest recovery times occurred in the lactated Ringer's group. The most significant factor determining clinical response was the pulse pressure at presentation. A comparison of the colloid and crystalloid groups suggested benefits in children presenting with lower pulse pressures who received one of the colloids. Further large-scale studies, stratified for admission pulse pressure, are indicated.

Contributors