Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. 1,2 Here we describe methods for large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short term culture. Analysis of 86,158 exonic SNPs that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.
New treatment strategies are needed for artemisinin-resistant falciparum malaria. This randomized trial shows that neither increasing nor splitting the standard once-daily artesunate dose reverses the markedly reduced parasite clearance rate in patients with artemisinin-resistant falciparum malaria.
Background. The emergence of Plasmodium falciparum resistance to artemisinins on the Cambodian and Myanmar-Thai borders poses severe threats to malaria control. We investigated whether increasing or splitting the dose of the short-half-life drug artesunate improves parasite clearance in falciparum malaria in the 2 regions.
Methods. In Pailin, western Cambodia (from 2008 to 2010), and Wang Pha, northwestern Thailand (2009–2010), patients with uncomplicated falciparum malaria were randomized to oral artesunate 6 mg/kg/d as a once-daily or twice-daily dose for 7 days, or artesunate 8 mg/kg/d as a once-daily or twice-daily dose for 3 days, followed by mefloquine. Parasite clearance and recrudescence for up to 63 days of follow-up were assessed.
Results. A total of 159 patients were enrolled. Overall median (interquartile range [IQR]) parasitemia half-life (half-life) was 6.03 (4.89–7.28) hours in Pailin versus 3.42 (2.20–4.85) hours in Wang Pha (P = .0001). Splitting or increasing the artesunate dose did not shorten half-life in either site. Pharmacokinetic profiles of artesunate and dihydroartemisinin were similar between sites and did not correlate with half-life. Recrudescent infections occurred in 4 of 79 patients in Pailin and 5 of 80 in Wang Pha and was not different between treatment arms (P = .68).
Conclusions. Increasing the artesunate treatment dose up to 8 mg/kg/d or splitting the dose does not improve parasite clearance in either artemisinin resistant or more sensitive infections with P. falciparum.
Clinical Trials Registration. ISRCTN15351875.
artemisinins; drug resistance; Plasmodium falciparum; neutropenia; reticulocytopenia
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.
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.
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.
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.)
Apparent emerging artemisinin-resistant Plasmodium falciparum malaria in Southeast Asia requires development of practical tools to monitor for resistant parasites. Although in vitro anti-malarial susceptibility tests are widely used, uncertainties remain regarding interpretation of P. falciparum field isolate values.
Performance parameters of the W2 P. falciparum clone (considered artemisinin “sensitive”) were evaluated as a reference for the HRP-2 immediate ex vivo assay. Variability in W2 IC50s was assessed, including intra- and inter-assay variability among and between technicians in multiple experiments, over five freeze-thaw cycles, over five months of continuous culture, and before and after transport of drug-coated plates to remote field sites. Nominal drug plate concentrations of artesunate (AS) and dihydroartemisinin (DHA) were verified by LC-MS analysis. Plasmodium falciparum field isolate IC50s for DHA from subjects in an artemisinin-resistant area in Cambodia were compared with W2 susceptibility.
Plate drug concentrations and day-to-day technical assay performance among technicians were important sources of variability for W2 IC50s within and between assays. Freeze-thaw cycles, long-term continuous culture, and transport to and from remote sites had less influence. Despite variability in W2 susceptibility, the median IC50s for DHA for Cambodian field isolates were higher (p <0.0001) than the W2 clone (3.9 nM), both for subjects with expected (less than 72 hours; 6.3 nM) and prolonged (greater or equal to 72 hours; 9.6 nM) parasite clearance times during treatment with artesunate monotherapy.
The W2 reference clone improved the interpretability of field isolate susceptibility from the immediate ex vivo HRP-2 assay from areas of artemisinin resistance. Methods to increase the reproducibility of plate coating may improve overall assay interpretability and utility.
Malaria; Plasmodium falciparum; HRP-2; ELISA; Anti-malarial drugs; Drug resistance; Drug susceptibility test
Opisthorchis viverrini infection was found to be highly prevalent in 3 riverside villages (Ang Svay Chek A, B, and C) of the Prey Kabas District, Takeo Province. This area is located in the southern part of Cambodia, where the recovery of adult O. viverrini worms was recently reported. From May 2006 until May 2010, fecal examinations were performed on a total of 1,799 villagers using the Kato-Katz thick smear technique. In the 3 villages, the overall positive rate for helminth eggs ranged from 51.7 to 59.0% (av. 57.4%), and the percentage positive for O. viverrini was 46.4-50.6% (47.5%). Other helminths detected included hookworms (13.2%), echinostomes (2.9%), Trichuris trichiura (1.3%), Ascaris lumbricoides (0.6%), and Taenia spp. (0.06%). The prevalence of O. viverrini eggs appeared to reflect a lower infection in younger individuals (<20 years) than in the adult population (>20 years). Men (50.4%) revealed a significantly higher (P=0.02) prevalence than women (44.3%). The Ang Svay Chek villages of the Prey Kabas District, Takeo Province, Cambodia have been confirmed to be a highly endemic area for human O. viverrini infection.
Opisthorchis viverrini; opisthorchiasis; trematode; prevalence; Cambodia (Takeo)
Malaria elimination requires a variety of approaches individually optimized for different transmission settings. A recent field study in an area of low seasonal transmission in South West Cambodia demonstrated dramatic reductions in malaria parasite prevalence following both mass drug administration (MDA) and high treatment coverage of symptomatic patients with artemisinin-piperaquine plus primaquine. This study employed multiple combined strategies and it was unclear what contribution each made to the reductions in malaria.
Method and Findings
A mathematical model fitted to the trial results was used to assess the effects of the various components of these interventions, design optimal elimination strategies, and explore their interactions with artemisinin resistance, which has recently been discovered in Western Cambodia. The modelling indicated that most of the initial reduction of P. falciparum malaria resulted from MDA with artemisinin-piperaquine. The subsequent continued decline and near elimination resulted mainly from high coverage with artemisinin-piperaquine treatment. Both these strategies were more effective with the addition of primaquine. MDA with artemisinin combination therapy (ACT) increased the proportion of artemisinin resistant infections, although much less than treatment of symptomatic cases with ACT, and this increase was slowed by adding primaquine. Artemisinin resistance reduced the effectiveness of interventions using ACT when the prevalence of resistance was very high. The main results were robust to assumptions about primaquine action, and immunity.
The key messages of these modelling results for policy makers were: high coverage with ACT treatment can produce a long-term reduction in malaria whereas the impact of MDA is generally only short-term; primaquine enhances the effect of ACT in eliminating malaria and reduces the increase in proportion of artemisinin resistant infections; parasite prevalence is a better surveillance measure for elimination programmes than numbers of symptomatic cases; combinations of interventions are most effective and sustained efforts are crucial for successful elimination.
Genome and transcriptome studies of Plasmodium nucleic acids obtained from parasitized whole blood are greatly improved by depletion of human DNA or enrichment of parasite DNA prior to next-generation sequencing and microarray hybridization. The most effective method currently used is a two-step procedure to deplete leukocytes: centrifugation using density gradient media followed by filtration through expensive, commercially available columns. This method is not easily implemented in field studies that collect hundreds of samples and simultaneously process samples for multiple laboratory analyses. Inexpensive syringes, hand-packed with CF11 cellulose powder, were recently shown to improve ex vivo cultivation of Plasmodium vivax obtained from parasitized whole blood. This study was undertaken to determine whether CF11 columns could be adapted to isolate Plasmodium falciparum DNA from parasitized whole blood and achieve current quantity and purity requirements for Illumina sequencing.
The CF11 procedure was compared with the current two-step standard of leukocyte depletion using parasitized red blood cells cultured in vitro and parasitized blood obtained ex vivo from Cambodian patients with malaria. Procedural variations in centrifugation and column size were tested, along with a range of blood volumes and parasite densities.
CF11 filtration reliably produces 500 nanograms of DNA with less than 50% human DNA contamination, which is comparable to that obtained by the two-step method and falls within the current quality control requirements for Illumina sequencing. In addition, a centrifuge-free version of the CF11 filtration method to isolate P. falciparum DNA at remote and minimally equipped field sites in malaria-endemic areas was validated.
CF11 filtration is a cost-effective, scalable, one-step approach to remove human DNA from P. falciparum-infected whole blood samples.
CF11; Cellulose powder; Leukocyte depletion; Plasmodium falciparum; Malaria; Next-generation sequencing
Malaria control has been scaled up in many developing countries in their efforts to achieve the Millennium Development Goals. Cambodia recently scaled up their Village Malaria Worker (VMW) project by substantially increasing the number of VMWs and expanding the project's health services to include treatment of fever, diarrhoea, and Acute Respiratory Infections (ARI) in children under five. This study examined if the scale-up interfered with VMWs' service quality, actions, and knowledge of malaria control, and analysed VMWs' overall achievements and perceptions of the newly added health services.
Structured interviews were conducted pre scale-up in February-March 2008 with 251 VMWs and post scale-up in July-August 2010 with 252 VMWs. Comparing the pre and post scale-up survey results (n = 195), changes were examined in terms of VMWs' 1) service quality, 2) malaria prevention and vector control actions, and 3) knowledge of malaria epidemiology and vector ecology. In addition, VMWs' newly added health services were descriptively analysed based on the post scale-up survey (n = 252).
VMWs' service quality and actions significantly improved overall during the scale-up of the VMW project (mean index score: +0.805, p < 0.001; +2.923, p < 0.001; respectively). Although most of knowledge areas also showed significant improvement (between +0.256 and +0.499, p < 0.001), less than half (10.3%-47.7%) of the VMWs correctly answered a set of questions on malaria epidemiology and vector ecology, even in the post scale-up survey. About 70% of the respondents reported that their health services to control malaria remained the same or that they were more active after the scale-up. Two-thirds (66.3%) had become more enthusiastic about serving as a VMW since the scale-up, and all but one respondent reported being willing to continue the new services.
The Cambodian experience clearly demonstrated that a nationwide scale-up of community-based malaria control can be achieved without degrading community health workers' service quality. The government's strategy to expand VMWs' health services, while providing sufficient training to maintain the quality of their original malaria control services, could have contributed to the improvement of VMW's service quality, actions, and knowledge in spite of the rapid scale-up of the project.
Scale-up; Malaria control; Community health workers; Service quality; Cambodia
Continued progress towards global reduction in morbidity and mortality due to malaria requires scale-up of effective case management with artemisinin-combination therapy (ACT). The first case of artemisinin resistance in Plasmodium falciparum was documented in western Cambodia. Spread of artemisinin resistance would threaten recent gains in global malaria control. As such, the anti-malarial market and malaria case management practices in Cambodia have global significance.
Nationally-representative household and outlet surveys were conducted in 2009 among areas in Cambodia with malaria risk. An anti-malarial audit was conducted among all public and private outlets with the potential to sell anti-malarials. Indicators on availability, price and relative volumes sold/distributed were calculated across types of anti-malarials and outlets. The household survey collected information about management of recent "malaria fevers." Case management in the public versus private sector, and anti-malarial treatment based on malaria diagnostic testing were examined.
Most public outlets (85%) and nearly half of private pharmacies, clinics and drug stores stock ACT. Oral artemisinin monotherapy was found in pharmacies/clinics (9%), drug stores (14%), mobile providers (4%) and grocery stores (2%). Among total anti-malarial volumes sold/distributed nationally, 6% are artemisinin monotherapies and 72% are ACT. Only 45% of people with recent "malaria fever" reportedly receive a diagnostic test, and the most common treatment acquired is a drug cocktail containing no identifiable anti-malarial. A self-reported positive diagnostic test, particularly when received in the public sector, improves likelihood of receiving anti-malarial treatment. Nonetheless, anti-malarial treatment of reportedly positive cases is low among people who seek treatment exclusively in the public (61%) and private (42%) sectors.
While data on the anti-malarial market shows favourable progress towards replacing artemisinin monotherapies with ACT, the widespread use of drug cocktails to treat malaria is a barrier to effective case management. Significant achievements have been made in availability of diagnostic testing and effective treatment in the public and private sectors. However, interventions to improve case management are urgently required, particularly in the private sector. Evidence-based interventions that target provider and consumer behaviour are needed to support uptake of diagnostic testing and treatment with full-course first-line anti-malarials.
Malaria; Cambodia; ACT; artemisinin monotherapy; diagnosis; treatment-seeking behaviour; public sector; private sector
Whilst some populations have recently experienced dramatic declines in malaria, the majority of those most at risk of Plasmodium falciparum malaria still lack access to effective treatment with artemisinin combination therapy (ACT) and others are already facing parasites resistant to artemisinins.
In this context, there is a crucial need to improve both access to and targeting of ACT through greater availability of good quality ACT and parasitological diagnosis. This is an issue of increasing urgency notably in the private commercial sector, which, in many countries, plays an important role in the provision of malaria treatment. The Affordable Medicines Facility for malaria (AMFm) is a recent initiative that aims to increase the provision of affordable ACT in public, private and NGO sectors through a manufacturer-level subsidy. However, to date, there is little documented experience in the programmatic implementation of subsidized ACT in the private sector. Cambodia is in the unique position of having more than 10 years of experience not only in implementing subsidized ACT, but also rapid diagnostic tests (RDT) as part of a nationwide social marketing programme. The programme includes behaviour change communication and the training of private providers as well as the sale and distribution of Malarine, the recommended ACT, and Malacheck, the RDT. This paper describes and evaluates this experience by drawing on the results of household and provider surveys conducted since the start of the programme.
The available evidence suggests that providers' and consumers' awareness of Malarine increased rapidly, but that of Malacheck much less so. In addition, improvements in ACT and RDT availability and uptake were relatively slow, particularly in more remote areas.
The lack of standardization in the survey methods and the gaps in the data highlight the importance of establishing a clear system for monitoring and evaluation for similar initiatives. Despite these limitations, a number of important lessons can still be learnt. These include the importance of a comprehensive communications strategy and of a sustained and reliable supply of products, with attention to the geographical reach of both. Other important challenges relate to the difficulty in incentivising providers and consumers not only to choose the recommended drug, but to precede this with a confirmatory blood test and ensure that providers adhere to the test results and patients to the treatment regime. In Cambodia, this is particularly complicated due to problems inherent to the drug itself and the emergence of artemisinin resistance.
Drug resistance of falciparum malaria is a global problem. Sulphadoxine/pyrimethamine-resistant and mefloquine-resistant strains of falciparum malaria have spread in Southeast Asia at lightning speed in 1980s-1990s, and the Cambodia-Thailand border is one of the malaria epidemic areas with the most severe forms of multi-drug resistant falciparum malaria.
Artemisinin-piperaquine (AP), dihydroartemisinin-piperaquine phosphate (DHP) and artemether-lumefantrine (AL) were used to treat 110, 55 and 55 uncomplicated malaria patients, respectively. The total dosage for adults is 1,750 mg (four tablets, twice over 24 hours) of AP, 2,880 mg (eight tablets, four times over two days) of DHP, and 3,360 mg (24 tablets, six times over three days) of AL. The 28-day cure rate, parasite clearance time, fever clearance time, and drug tolerance of patients to the three drugs were compared. All of the above methods were consistent with the current national guidelines.
The mean parasite clearance time was similar in all three groups (66.7 ± 21.9 hrs, 65.6 ± 27.3 hrs, 65.3 ± 22.5 hrs in AP, DHP and AL groups, respectively), and there was no remarkable difference between them; the fever clearance time was also similar (31.6 ± 17.7 hrs, 34.6 ± 21.8 hrs and 36.9 ± 15.4 hrs, respectively). After following up for 28-days, the cure rate was 95.1%(97/102), 98.2%(54/55) and 82.4%(42/51); and the recrudescence cases was 4.9%(5/102), 1.8%(1/55) and 17.6%(9/51), respectively. Therefore, the statistical data showed that 28-day cure rate in AP and DHP groups was superior to AL group obviously.
The patients had good tolerance to all the three drugs, and some side effects (anoxia, nausea, vomiting, headache and dizziness) could be found in every group and they were self-limited; patients in control groups also had good tolerance to DHP and AL, there was no remarkable difference in the three groups.
AP, DHP and AL all remained efficacious treatments for the treatment of falciparum malaria in Cambodia-Thailand border area. However, in this particular setting, the AP regimen turned out to be favourable in terms of efficacy and effectiveness, simplicity of administration, cost and compliance.
The trial was registered at Chinese Clinical Trial Register under identifier 2005L01041.
Artemisinin resistance in Plasmodium falciparum malaria has emerged in Western Cambodia. This is a major threat to global plans to control and eliminate malaria as the artemisinins are a key component of antimalarial treatment throughout the world. To identify key features associated with the delayed parasite clearance phenotype, we employed DNA microarrays to profile the physiological gene expression pattern of the resistant isolates.
In the ring and trophozoite stages, we observed reduced expression of many basic metabolic and cellular pathways which suggests a slower growth and maturation of these parasites during the first half of the asexual intraerythrocytic developmental cycle (IDC). In the schizont stage, there is an increased expression of essentially all functionalities associated with protein metabolism which indicates the prolonged and thus increased capacity of protein synthesis during the second half of the resistant parasite IDC. This modulation of the P. falciparum intraerythrocytic transcriptome may result from differential expression of regulatory proteins such as transcription factors or chromatin remodeling associated proteins. In addition, there is a unique and uniform copy number variation pattern in the Cambodian parasites which may represent an underlying genetic background that contributes to the resistance phenotype.
The decreased metabolic activities in the ring stages are consistent with previous suggestions of higher resilience of the early developmental stages to artemisinin. Moreover, the increased capacity of protein synthesis and protein turnover in the schizont stage may contribute to artemisinin resistance by counteracting the protein damage caused by the oxidative stress and/or protein alkylation effect of this drug. This study reports the first global transcriptional survey of artemisinin resistant parasites and provides insight to the complexities of the molecular basis of pathogens with drug resistance phenotypes in vivo.
Plasmodium falciparum, in vivo artemisinin-resistance; field isolates; comparative genomics; comparative transcriptomics
Opisthorchis viverrini; adult liver flukes; trematodes; parasites; humans; Cambodia; letter
The diversity in the Plasmodium falciparum genome can be used to explore parasite population dynamics, with practical applications to malaria control. The ability to identify the geographic origin and trace the migratory patterns of parasites with clinically important phenotypes such as drug resistance is particularly relevant. With increasing single-nucleotide polymorphism (SNP) discovery from ongoing Plasmodium genome sequencing projects, a demand for high SNP and sample throughput genotyping platforms for large-scale population genetic studies is required. Low parasitaemias and multiple clone infections present a number of challenges to genotyping P. falciparum. We addressed some of these issues using a custom 384-SNP Illumina GoldenGate assay on P. falciparum DNA from laboratory clones (long-term cultured adapted parasite clones), short-term cultured parasite isolates and clinical (non-cultured isolates) samples from East and West Africa, Southeast Asia and Oceania. Eighty percent of the SNPs (n = 306) produced reliable genotype calls on samples containing as little as 2 ng of total genomic DNA and on whole genome amplified DNA. Analysis of artificial mixtures of laboratory clones demonstrated high genotype calling specificity and moderate sensitivity to call minor frequency alleles. Clear resolution of geographically distinct populations was demonstrated using Principal Components Analysis (PCA), and global patterns of population genetic diversity were consistent with previous reports. These results validate the utility of the platform in performing population genetic studies of P. falciparum.
Fecal examinations using the Kato Katz technique were performed on a total of 1,287 villagers (945 students and 342 general inhabitants) of Oddar Meanchey Province, Cambodia in May 2007 and November 2009. The overall intestinal helminth egg positive rate was 23.9%, and the most prevalent helminth species was hookworms (21.6%). Other helminth eggs detected included echinostomes (1.0%), Enterobius vermicularis (0.8%), small trematode eggs (0.7%), which may include Opisthorchis viverrini and Haplorchis spp., and Hymenolepis nana (0.4%). In order to recover adult echinostomes, we treated 2 patients with 10-15 mg/kg praziquantel and purged. Total 14 adult echinostomes, 1 and 13 worms from each patient, were collected. The echinostomes characteristically had 49-51 collar spines and 2 round or slightly lobated testes. They were identified as Echinostoma ilocanum (Garrison, 1908) Odhner, 1911. So far as literature are concerned, this is the first record on the discovery of human E. ilocanum infection in Cambodia.
Echinostoma ilocanum; worm recovery; trematode; echinostome; Cambodia
We collected fecal samples from 21 individuals infected with Taenia tapeworms in Koh Kong Province, Cambodia, and performed nucleotide sequencing of the cox1 gene and multiplex PCR on the eggs for DNA differential diagnosis of human Taenia tapeworms. Genomic DNA was extracted from the eggs of a minimum number of 10 isolated from fecal samples. Using oligonucleotide primers Ta7126F, Ts7313F, Tso7466F, and Rev7915, the multiplex PCR assay proved useful for differentially diagnosing Taenia solium, Taenia saginata, and Taenia asiatica based on 706, 629, and 474 bp bands, respectively. All of the Taenia specimens from Kho Kong, Cambodia, were identified as either T. saginata (n=19) or T. solium (n=2) by cox1 sequencing and multiplex PCR.
Taenia saginata; Taenia solium; molecular survey; egg DNA; multiplex PCR
The emergence of artemisinin resistance has raised concerns that the most potent antimalarial drug may be under threat. The currently recommended daily dose of artesunate (AS) is 4 mg/kg, and is administered for 3 days together with a partner antimalarial drug. This study investigated the impact of different AS doses on clinical and parasitological responses in malaria patients from an area of known artemisinin resistance in western Cambodia.
Adult patients with uncomplicated P. falciparum malaria were randomized into one of three 7-day AS monotherapy regimens: 2, 4 or 6 mg/kg/day (total dose 14, 28 and 42 mg/kg). Clinical, parasitological, pharmacokinetic and in vitro drug sensitivity data was collected over a 7-day inpatient period and during weekly follow-up to 42 days.
143 patients were enrolled (n = 75, 40 and 28 to receive AS 2, 4 and 6 mg/kg/day respectively). Cure rates were high in all treatment groups at 42 days despite almost half the patients remaining parasitemic on Day 3. There was no impact of increasing AS dose on median parasite clearance times, median parasite clearance rates or on the proportion of patients remaining parasitemic on Day 3. However at the lowest dose used (2 mg/kg/d) patients with parasitemia >10,000/µL had longer median (IQR) parasite clearance times than those with parasitemia <10,000/µL (63 (48–75) vs. 84 (66–96) hours, p<0.0001). 19% of patients in the high-dose arm developed neutropenia (absolute neutrophil count <1.0×109/L) by Day 14 and resulted in the arm being halted early.
There is no pharmacodynamic benefit of increasing the daily dose of AS (4mg/kg) currently recommended for short-course combination treatment of uncomplicated malaria, even in regions with emerging artemisinin resistance, as long as the partner drug retains high efficacy.
In Western Cambodia malaria parasites clear slowly from the blood following treatment with artemisinin derivatives, but it is unclear whether this results from parasite, host, or other factors specific to this population. We measured heritability of clearance rate (CR), by examining patients infected with identical or non-identical parasite genotypes, using methods analogous to human twin studies. A substantial proportion (56-58%) of the variation in CR is explained by parasite genetics. This has two important implications: (1) selection with artemisinin derivatives will tend to drive resistance spread, (2) because heritability is high, genes underlying CR may be identified by genome-wide association.
Artemisinin; clearance time; heritability; twin studies; resistance; microsatellite
Mixed P. falciparum/P. vivax infections are common in southeast Asia. When patients with P. falciparum malaria are treated and followed for several weeks, a significant proportion will develop P. vivax malaria. In a combined analysis of 243 patients recruited to two malaria treatment trials in western Cambodia, 20/43 (47%) of those with P. falciparum gametocytes on admission developed P. vivax malaria by Day 28 of follow-up. The presence of Pf gametocytes on an initial blood smear was associated with a 3.5-fold greater rate of vivax parasitemia post-treatment (IRR = 3.5, 95% CI 2.0–6.0, p<0.001). The increased rate of post-treatment P. vivax infection persisted when correlates of exposure and immunity such as a history of malaria, male gender, and age were controlled for (IRR = 3.0, 95% CI 1.9–4.7, p<0.001). Polymerase chain reaction (PCR) confirmed that only a low proportion of subjects (5/55 or 9.1%) who developed vivax during follow-up had detectable Pv parasites in the peripheral blood at baseline. Molecular detection of falciparum gametocytes by reverse transcriptase PCR in a subset of patients strengthened the observed association, while PCR detection of Pv parasitemia at follow-up was similar to microscopy results. These findings suggest that the majority of vivax infections arising after treatment of falciparum malaria originate from relapsing liver-stage parasites. In settings such as western Cambodia, the presence of both sexual and asexual forms of P. falciparum on blood smear at presentation with acute falciparum malaria serves as a marker for possible occult P. vivax coinfection and subsequent relapse. These patients may benefit from empiric treatment with an 8-aminoquinolone such as primaquine.
New antimalarials are needed for P. vivax and P. falciparum malaria. This study compared the efficacy and safety of pyronaridine-artesunate with that of chloroquine for the treatment of uncomplicated P. vivax malaria.
Methods and Findings
This phase III randomized, double-blind, non-inferiority trial included five centers across Cambodia, Thailand, India, and Indonesia. In a double-dummy design, patients (aged >3–≤60 years) with microscopically confirmed P. vivax mono-infection were randomized (1∶1) to receive pyronaridine-artesunate (target dose 7.2∶2.4 mg/kg to 13.8∶4.6 mg/kg) or chloroquine (standard dose) once daily for three days. Each treatment group included 228 randomized patients. Outcomes for the primary endpoint, Day-14 cure rate in the per-protocol population, were 99.5%, (217/218; 95%CI 97.5, 100) with pyronaridine-artesunate and 100% (209/209; 95%CI 98.3, 100) with chloroquine. Pyronaridine was non-inferior to chloroquine: treatment difference −0.5% (95%CI −2.6, 1.4), i.e., the lower limit of the 2-sided 95%CI for the treatment difference was greater than −10%. Pyronaridine-artesunate cure rates were non-inferior to chloroquine for Days 21, 28, 35 and 42. Parasite clearance time was shorter with pyronaridine-artesunate (median 23.0 h) versus chloroquine (32.0 h; p<0.0001), as was fever clearance time (median 15.9 h and 23.8 h, respectively; p = 0.0017). Kaplan-Meier estimates of post-baseline P. falciparum infection incidence until Day 42 were 2.5% with pyronaridine-artesunate, 6.1% with chloroquine (p = 0.048, log-rank test). Post-baseline P. vivax or P. falciparum infection incidence until Day 42 was 6.8% and 12.4%, respectively (p = 0.022, log rank test). There were no deaths. Adverse events occurred in 92/228 (40.4%) patients with pyronaridine-artesunate and 72/228 (31.6%) with chloroquine. Mild and transient increases in hepatic enzymes were observed for pyronaridine-artesunate.
Pyronaridine-artesunate efficacy in acute uncomplicated P. vivax malaria was at least that of chloroquine. As pyronaridine-artesunate is also efficacious against P. falciparum malaria, this combination has potential utility as a global antimalarial drug.
The reduced in vivo sensitivity of Plasmodium falciparum has recently been confirmed in western Cambodia. Identifying molecular markers for artemisinin resistance is essential for monitoring the spread of the resistant phenotype and identifying the mechanisms of resistance. Four candidate genes, including the P. falciparum mdr1 (pfmdr1) gene, the P. falciparum ATPase6 (pfATPase6) gene, the 6-kb mitochondrial genome, and ubp-1, encoding a deubiquitinating enzyme, of artemisinin-resistant P. falciparum strains from western Cambodia were examined and compared to those of sensitive strains from northwestern Thailand, where the artemisinins are still very effective. The artemisinin-resistant phenotype did not correlate with pfmdr1 amplification or mutations (full-length sequencing), mutations in pfATPase6 (full-length sequencing) or the 6-kb mitochondrial genome (full-length sequencing), or ubp-1 mutations at positions 739 and 770. The P. falciparum CRT K76T mutation was present in all isolates from both study sites. The pfmdr1 copy numbers in western Cambodia were significantly lower in parasite samples obtained in 2007 than in those obtained in 2005, coinciding with a local change in drug policy replacing artesunate-mefloquine with dihydroartemisinin-piperaquine. Artemisinin resistance in western Cambodia is not linked to candidate genes, as was suggested by earlier studies.
To determine the prevalence of helminthic infections in Pursat Province, Cambodia, we tested fecal specimens from 471 children, 10–14 years of age, in June 2007. The prevalence of infection with echinostome flukes ranged from 7.5% to 22.4% in 4 schools surveyed. Adult worms were identified as Echinostoma revolutum.
Parasites; Echinostoma revolutum; echinostomiasis; trematode; prevalence; children; Cambodia; dispatch
Unprecedented efforts are now underway to eliminate malaria from many regions. Despite the enormous financial resources committed, if malaria elimination is perceived as failing it is likely that this funding will not be sustained. It is imperative that methods are developed to use the limited data available to design site-specific, cost-effective elimination programmes. Mathematical modelling is a way of including mechanistic understanding to use available data to make predictions. Different strategies can be evaluated much more rapidly than is possible through trial and error in the field. Mathematical modelling has great potential as a tool to guide and inform current elimination efforts. Economic modelling weighs costs against characterised effects or predicted benefits in order to determine the most cost-efficient strategy but has traditionally used static models of disease not suitable for elimination. Dynamic mathematical modelling and economic modelling techniques need to be combined to contribute most effectively to ongoing policy discussions. We review the role of modelling in previous malaria control efforts as well as the unique nature of elimination and the consequent need for its explicit modelling, and emphasise the importance of good disease surveillance. The difficulties and complexities of economic evaluation of malaria control, particularly the end stages of elimination, are discussed.
Mathematical modelling; Model; Economic; Malaria; Policy; Control
Antimalarial drugs impose strong pressure on Plasmodium falciparum parasites and leave signatures of selection in the parasite genome 1,2. Search for signals of selection may lead to genes encoding drug or immune targets 3. The lack of high-throughput genotyping methods, inadequate knowledge of parasite population history, and time-consuming adaptations of parasites to in vitro culture have hampered genome-wide association studies (GWAS) of parasite traits. Here we report genotyping of DNA from 189 culture-adapted P. falciparum parasites using a custom-built array with thousands of single nucleotide polymorphisms (SNPs). Population structure, variation in recombination rate, and loci under recent positive selection were detected. Parasite half maximum inhibitory concentrations (IC50) to seven antimalarial drugs were obtained and used in GWAS to identify genes associated with drug responses. The SNP array and genome-wide parameters provide valuable tools and information for new advances in P. falciparum genetics.
malaria; single nucleotide polymorphism (SNP); genome-wide association study; recombination; drug resistance; population structure