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1.  Laboratory Detection of Artemisinin-Resistant Plasmodium falciparum 
Conventional 48-h in vitro susceptibility tests have low sensitivity in identifying artemisinin-resistant Plasmodium falciparum, defined phenotypically by low in vivo parasite clearance rates. We hypothesized originally that this discrepancy was explained by a loss of ring-stage susceptibility and so developed a simple field-adapted 24-h trophozoite maturation inhibition (TMI) assay focusing on the ring stage and compared it to the standard 48-h schizont maturation inhibition (WHO) test. In Pailin, western Cambodia, where artemisinin-resistant P. falciparum is prevalent, the TMI test mean (95% confidence interval) 50% inhibitory concentration (IC50) for artesunate was 6.8 (5.2 to 8.3) ng/ml compared with 1.5 (1.2 to 1.8) ng/ml for the standard 48-h WHO test (P = 0.001). TMI IC50s correlated significantly with the in vivo responses to artesunate (parasite clearance time [r = 0.44, P = 0.001] and parasite clearance half-life [r = 0.46, P = 0.001]), whereas the standard 48-h test values did not. On continuous culture of two resistant isolates, the artemisinin-resistant phenotype was lost after 6 weeks (IC50s fell from 10 and 12 ng/ml to 2.7 and 3 ng/ml, respectively). Slow parasite clearance in falciparum malaria in western Cambodia results from reduced ring-stage susceptibility.
doi:10.1128/AAC.01924-13
PMCID: PMC4068498  PMID: 24663013
2.  Uncomplicated Plasmodium vivax malaria in pregnancy associated with mortality from acute respiratory distress syndrome 
Malaria Journal  2014;13:191.
The association between severe malaria and Plasmodium vivax species is contentious. On the Thai-Myanmar border, all pregnant women are followed systematically with active weekly malaria screening. Over a 27-year period of providing antenatal care, 48,983 have been prospectively followed until pregnancy outcome (miscarriage or delivery) and 4,298 women have had P. vivax detected at least once. Reported here is the first known P. vivax-associated death amongst these women. The initial patient presentation was of uncomplicated P. vivax (0.5% parasitaemia) in a term, multigravida woman who responded rapidly to oral artesunate and mefloquine treatment, clearing her blood stage parasites within 48 hours. The patient appeared well, was ambulatory and due to be discharged but became unwell with acute respiratory distress syndrome (ARDS) requiring ventilation three days (67 hours) into treatment. Despite induction and delivery of a stillborn foetus, ventilatory requirements increased and the patient died on day 7. The patient had a low body mass index. Sensitive detection with nested PCR confirmed only the presence of P. vivax species and concomitant infections such as tuberculosis and human immunodeficiency virus (HIV) were also ruled out. The contemporaneous treatment of acute uncomplicated P. vivax and the onset of ARDS on day 3 in this patient implies a possible but unconfirmed association with death in this patient. Assuming this death was caused by P. vivax, the risk of ARDS-related maternal mortality in this setting did not differ significantly between Plasmodium falciparum and P. vivax (0.24 per 1,000 (1/4,158) versus 0.23 per 1,000 (1/4,298), contrary to the increased risk of maternal mortality from P. falciparum compared to P. vivax, 2.89 per 1,000 (12/4,158) versus 0.23 per 1,000 (1/4,298), P = 0.003.
doi:10.1186/1475-2875-13-191
PMCID: PMC4046059  PMID: 24886559
Acute respiratory distress syndrome; Maternal mortality; Plasmodium vivax; Severe malaria
3.  Randomized Controlled Trial of Levamisole Hydrochloride as Adjunctive Therapy in Severe Falciparum Malaria With High Parasitemia 
The Journal of Infectious Diseases  2013;209(1):120-129.
Background. Cytoadherence and sequestration of erythrocytes containing mature stages of Plasmodium falciparum are central to the pathogenesis of severe malaria. The oral anthelminthic drug levamisole inhibits cytoadherence in vitro and reduces sequestration of late-stage parasites in uncomplicated falciparum malaria treated with quinine.
Methods. Fifty-six adult patients with severe malaria and high parasitemia admitted to a referral hospital in Bangladesh were randomized to receive a single dose of levamisole hydrochloride (150 mg) or no adjuvant to antimalarial treatment with intravenous artesunate.
Results. Circulating late-stage parasites measured as the median area under the parasite clearance curves were 2150 (interquartile range [IQR], 0–28 025) parasites/µL × hour in patients treated with levamisole and 5489 (IQR, 192–25 848) parasites/µL × hour in controls (P = .25). The “sequestration ratios” at 6 and 12 hours for all parasite stages and changes in microvascular blood flow did not differ between treatment groups (all P > .40). The median time to normalization of plasma lactate (<2 mmol/L) was 24 (IQR, 12–30) hours with levamisole vs 28 (IQR, 12–36) hours without levamisole (P = .15).
Conclusions. There was no benefit of a single-dose of levamisole hydrochloride as adjuvant to intravenous artesunate in the treatment of adults with severe falciparum malaria. Rapid parasite killing by intravenous artesunate might obscure the effects of levamisole.
doi:10.1093/infdis/jit410
PMCID: PMC3864382  PMID: 23943850
malaria; severe; falciparum; sequestration; artesunate; levamisole
4.  Effect of High-Dose or Split-Dose Artesunate on Parasite Clearance in Artemisinin-Resistant Falciparum Malaria 
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.
doi:10.1093/cid/cis958
PMCID: PMC3563392  PMID: 23175556
artemisinins; drug resistance; Plasmodium falciparum; neutropenia; reticulocytopenia
5.  Artemisinin Resistance in Plasmodium falciparum Malaria 
The New England journal of medicine  2009;361(5):455-467.
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.)
doi:10.1056/NEJMoa0808859
PMCID: PMC3495232  PMID: 19641202
6.  Defining Falciparum-Malaria-Attributable Severe Febrile Illness in Moderate-to-High Transmission Settings on the Basis of Plasma PfHRP2 Concentration 
The Journal of Infectious Diseases  2012;207(2):351-361.
Background. In malaria-endemic settings, asymptomatic parasitemia complicates the diagnosis of malaria. Histidine-rich protein 2 (HRP2) is produced by Plasmodium falciparum, and its plasma concentration reflects the total body parasite burden. We aimed to define the malaria-attributable fraction of severe febrile illness, using the distributions of plasma P. falciparum HRP2 (PfHRP2) concentrations from parasitemic children with different clinical presentations.
Methods. Plasma samples were collected from and peripheral blood slides prepared for 1435 children aged 6−60 months in communities and a nearby hospital in northeastern Tanzania. The study population included children with severe or uncomplicated malaria, asymptomatic carriers, and healthy control subjects who had negative results of rapid diagnostic tests. The distributions of plasma PfHRP2 concentrations among the different groups were used to model severe malaria-attributable disease.
Results. The plasma PfHRP2 concentration showed a close correlation with the severity of infection. PfHRP2 concentrations of >1000 ng/mL denoted a malaria-attributable fraction of severe disease of 99% (95% credible interval [CI], 96%–100%), with a sensitivity of 74% (95% CI, 72%–77%), whereas a concentration of <200 ng/mL denoted severe febrile illness of an alternative diagnosis in >10% (95% CI, 3%–27%) of patients. Bacteremia was more common among patients in the lowest and highest PfHRP2 concentration quintiles.
Conclusions. The plasma PfHRP2 concentration defines malaria-attributable disease and distinguishes severe malaria from coincidental parasitemia in African children in a moderate-to-high transmission setting.
doi:10.1093/infdis/jis675
PMCID: PMC3532834  PMID: 23136222
case definition; severe malaria; Plasmodium falciparum; histidine-rich protein 2; malaria-attributable disease; asymptomatic parasitemia; bacteremia; Tanzania
7.  Antimalarial Drug Susceptibility of Plasmodium vivax in the Republic of Korea 
The antimalarial susceptibility of ring stage (> 80%) Plasmodium vivax from the Republic of Korea, where long incubation-period strains are prevalent, was evaluated using the schizont maturation inhibition technique. During 2005–2007, susceptibility to seven antimalarial drugs was evaluated with 24 fresh isolates. The geometric mean (95% confidence interval) 50% inhibition concentration (IC50) were quinine 60 (54-75) ng/mL, chloroquine 39 (22–282) ng/mL, piperaquine 27 (17–58) ng/mL, mefloquine 39 (35–67) ng/mL, pyrimethamine 138 (89–280) ng/mL, artesunate 0.6 (0.5–0.8) ng/mL, and primaquine 122 (98-232) ng/mL. Positive correlations were found between quinine and mefloquine (r = 0.6, P = 0.004), piperaquine and chloroquine (r = 0.6, P = 0.008), and piperaquine and primaquine IC50 values (r = 0.5, P = 0.01). Compared with P. vivax in Thailand, P. vivax in the Republic of Korea was more sensitive to quinine and mefloquine, but equally sensitive to chloroquine and artesunate.
PMCID: PMC3444524  PMID: 19478246
8.  Ultrastructural and Real-time Microscopic Changes in P. falciparum-infected Red Blood Cells Following Treatment with Antimalarial Drugs 
Ultrastructural pathology  2011;35(5):214-225.
Ultrastructural changes to P. falciparum-infected red blood cells were examined in vitro after treatment with antimalarial drugs. Artesunate had the most rapid parasitocidal effect. All three drugs caused structural changes within the parasite, including dilatation of the parasitophorus vacuole membrane, depletion of ribosomes, mitochondrial swelling, and decreased formation of hemozoin crystals. The structure of surface knobs and Maurer’s clefts were similar to controls but reduced in number. Only depletion of free ribosomes correlated with antimalarial drug exposure. Drug treatment decreased movement of hemozoin granules within parasites on real-time microscopy, before recognizable morphological changes of parasite death.
doi:10.3109/01913123.2011.601405
PMCID: PMC3444525  PMID: 21910567
drug treatment; malaria; Plasmodium falciparum; real-time microscopy; ultrastructural changes
9.  Diagnosing Severe Falciparum Malaria in Parasitaemic African Children: A Prospective Evaluation of Plasma PfHRP2 Measurement 
PLoS Medicine  2012;9(8):e1001297.
Arjen Dondorp and colleagues investigate whether the plasma level of Plasmodium falciparum histidine-rich protein 2 can be used to distinguish between severe malaria and other severe febrile illness in African children with malaria.
Background
In African children, distinguishing severe falciparum malaria from other severe febrile illnesses with coincidental Plasmodium falciparum parasitaemia is a major challenge. P. falciparum histidine-rich protein 2 (PfHRP2) is released by mature sequestered parasites and can be used to estimate the total parasite burden. We investigated the prognostic significance of plasma PfHRP2 and used it to estimate the malaria-attributable fraction in African children diagnosed with severe malaria.
Methods and Findings
Admission plasma PfHRP2 was measured prospectively in African children (from Mozambique, The Gambia, Kenya, Tanzania, Uganda, Rwanda, and the Democratic Republic of the Congo) aged 1 month to 15 years with severe febrile illness and a positive P. falciparum lactate dehydrogenase (pLDH)-based rapid test in a clinical trial comparing parenteral artesunate versus quinine (the AQUAMAT trial, ISRCTN 50258054). In 3,826 severely ill children, Plasmadium falciparum PfHRP2 was higher in patients with coma (p = 0.0209), acidosis (p<0.0001), and severe anaemia (p<0.0001). Admission geometric mean (95%CI) plasma PfHRP2 was 1,611 (1,350–1,922) ng/mL in fatal cases (n = 381) versus 1,046 (991–1,104) ng/mL in survivors (n = 3,445, p<0.0001), without differences in parasitaemia as assessed by microscopy. There was a U-shaped association between log10 plasma PfHRP2 and risk of death. Mortality increased 20% per log10 increase in PfHRP2 above 174 ng/mL (adjusted odds ratio [AOR] 1.21, 95%CI 1.05–1.39, p = 0.009). A mechanistic model assuming a PfHRP2-independent risk of death in non-malaria illness closely fitted the observed data and showed malaria-attributable mortality less than 50% with plasma PfHRP2≤174 ng/mL. The odds ratio (OR) for death in artesunate versus quinine-treated patients was 0.61 (95%CI 0.44–0.83, p = 0.0018) in the highest PfHRP2 tertile, whereas there was no difference in the lowest tertile (OR 1.05; 95%CI 0.69–1.61; p = 0.82). A limitation of the study is that some conclusions are drawn from a mechanistic model, which is inherently dependent on certain assumptions. However, a sensitivity analysis of the model indicated that the results were robust to a plausible range of parameter estimates. Further studies are needed to validate our findings.
Conclusions
Plasma PfHRP2 has prognostic significance in African children with severe falciparum malaria and provides a tool to stratify the risk of “true” severe malaria-attributable disease as opposed to other severe illnesses in parasitaemic African children.
Please see later in the article for the Editors' Summary.
Editors' Summary
Background
Malaria is a life-threatening disease caused by parasites that are transmitted to people through the bites of infected mosquitoes. In 2010, malaria caused an estimated 655,000 deaths worldwide, mostly in Africa, where according to the World Health Organization, one African child dies every minute from the disease. There are four Plasmodium parasite species that cause malaria in humans, with one species, Plasmodium falciparum, causing the most severe disease. However, diagnosing severe falciparum malaria in children living in endemic areas is problematic, as many semi-immune children may have the malaria parasites in their blood (described as being parasitaemic) but do not have clinical disease. Therefore, a positive malaria blood smear may be coincidental and not be diagnostic of severe malaria, and unfortunately, neither are the clinical symptoms of severe malaria, such as shock, acidosis, or coma, which can also be caused by other childhood infections. For these reasons, the misdiagnosis of falciparum malaria in severely ill children is an important problem in sub-Saharan Africa, and may result in unnecessary child deaths.
Why Was This Study Done?
Previous studies have suggested that a parasite protein—P. falciparum histidine-rich protein-2 (PfHRP2)—is a measure of the total number of parasites in the patient. Unlike the circulating parasites detected on a blood film, which do not represent the parasites that get stuck in vital organs, PfHRP2 is distributed equally through the total blood plasma volume, and so can be considered a measure of the total parasite burden in the previous 48 hours. So in this study, the researchers assessed the prognostic value of plasma PfHRP2 in African children with severe malaria and whether this protein could distinguish children who really do have severe malaria from those who have severe febrile illness but coincidental parasitaemia, who may have another infection.
What Did the Researchers Do and Find?
The researchers assessed levels of plasma PfHRP2 in 3,826 out of a possible 5,425 African children who participated in a large multinational trial (in Mozambique, The Gambia, Rwanda, Tanzania, Kenya, Uganda, and the Democratic Republic of Congo) that compared the anti-malarial drugs quinine and artesunate for the treatment of severe malaria. All children had a clinical diagnosis of severe malaria confirmed by a rapid diagnostic test, and the researchers used clinical signs to define the severity of malaria. The researchers assessed the relationship between plasma PfHRP2 concentrations and risk of death taking other well established predictors of death, such as coma, convulsions, hypoglycaemia, respiratory distress, and shock, into account.
The researchers found that PfHRP2 was detectable in 3,800/3,826 (99%) children with severe malaria and that the average plasma PfHRP2 levels was significantly higher in the 381 children who died from malaria than in children who survived (1,611 ng/mL versus 1,046 ng/mL). Plasma PfHRP2 was also significantly higher in children with severe malaria signs and symptoms such as coma, acidosis, and severe anaemia. Importantly, the researchers found that high death rates were associated with either very low or very high values of plasma PfHRP2: the odds (chance) of death were 20% higher per unit increase in PfHRP2 above a specific threshold (174 ng/ml), but below this concentration, the risk of death increased with decreasing levels, probably because at lower levels disease was caused by a severe febrile disease other than malaria, like septicemia. Finally, the researchers found that in children within the highest PfHRP2 tertile, the chance of death when treated with the antimalarial drug artesunate versus quinine was 0.61 but that there was no difference in death rates in the lowest tertile, which supports that patients with very low plasma PfHRP2 have a different severe febrile illness than malaria. The researchers use mathematical modeling to provide cut-off values for plasma PfHRP2 denoting the proportion of patients with a diagnosis other than severe malaria.
What Do These Findings Mean?
These findings suggest that in areas of moderate or high malaria transmission where a high proportion of children are parasitaemic, plasma PfHRP2 levels taken on admission to hospital can differentiate children at highest risk of death from severe falciparum malaria from those likely to have alternative causes of severe febrile illness. Therefore, plasma PfHRP2 could be considered a valuable additional diagnostic tool and prognostic indicator in African children with severe falciparum malaria. This finding is important for clinicians treating children with severe febrile illnesses in malaria-endemic countries: while high levels of plasma PfHRP2 is indicative of severe malaria which needs urgent antimalarial treatment, low levels suggest that another severe infective disease should be considered, warranting additional investigations and urgent treatment with antibiotics.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001297.
A previous small study in PLOS ONE explores the relationship between plasma PfHRP2 and severe malaria in Tanzanian children
The WHO website and the website of Malaria No More have comprehensive information about malaria
doi:10.1371/journal.pmed.1001297
PMCID: PMC3424256  PMID: 22927801
10.  Sequence variation does not confound the measurement of plasma PfHRP2 concentration in African children presenting with severe malaria 
Malaria Journal  2012;11:276.
Background
Plasmodium falciparum histidine-rich protein PFHRP2 measurement is used widely for diagnosis, and more recently for severity assessment in falciparum malaria. The Pfhrp2 gene is highly polymorphic, with deletion of the entire gene reported in both laboratory and field isolates. These issues potentially confound the interpretation of PFHRP2 measurements.
Methods
Studies designed to detect deletion of Pfhrp2 and its paralog Pfhrp3 were undertaken with samples from patients in seven countries contributing to the largest hospital-based severe malaria trial (AQUAMAT). The quantitative relationship between sequence polymorphism and PFHRP2 plasma concentration was examined in samples from selected sites in Mozambique and Tanzania.
Results
There was no evidence for deletion of either Pfhrp2 or Pfhrp3 in the 77 samples with lowest PFHRP2 plasma concentrations across the seven countries. Pfhrp2 sequence diversity was very high with no haplotypes shared among 66 samples sequenced. There was no correlation between Pfhrp2 sequence length or repeat type and PFHRP2 plasma concentration.
Conclusions
These findings indicate that sequence polymorphism is not a significant cause of variation in PFHRP2 concentration in plasma samples from African children. This justifies the further development of plasma PFHRP2 concentration as a method for assessing African children who may have severe falciparum malaria. The data also add to the existing evidence base supporting the use of rapid diagnostic tests based on PFHRP2 detection.
doi:10.1186/1475-2875-11-276
PMCID: PMC3480887  PMID: 22898068
Malaria; Falciparum; Severe; Africa; Histidine-rich protein; Tandem repeat
11.  In vitro Anti-Malarial Drug Susceptibility of Temperate Plasmodium vivax from Central China 
In the face of recent increase of Plasmodium vivax malaria in central China, we conducted a study to evaluate in vitro susceptibility of temperate-zone P. vivax parasites to antimalarial drugs. During 2005–2006, in vitro drug susceptibility was measured for 42 clinical P. vivax isolates by using a schizont maturation inhibition technique. Geometric means of 50% inhibitory concentrations (IC50s) and 95% confidence intervals (CIs) were 10.87 (4.50–26.26) ng/mL for chloroquine, 4.21 (1.88–9.42–8) ng/mL for mefloquine, 11.82 (6.20–22.56) ng/mL for quinine, 0.13 (0.09–0.20) ng/mL for artesunate, 18.32 (8.08–41.50) ng/mL for pyrimethamine, and 17.73 (10.29–30.57) ng/mL for piperaquine. The IC50 for chloroquine was lower than those obtained from isolates from Thailand and South Korea, suggesting that chloroquine remained effective against P. vivax malaria in central China. The results further indicated that temperate-zone P. vivax isolates from China were more susceptible to chloroquine, quinine, and mefloquine than isolates from Thailand.
doi:10.4269/ajtmh.2011.10-0070
PMCID: PMC3144812  PMID: 21813834
12.  Diagnosis, Clinical Presentation, and In-Hospital Mortality of Severe Malaria in HIV-Coinfected Children and Adults in Mozambique 
Background. Severe falciparum malaria with human immunodeficiency virus (HIV) coinfection is common in settings with a high prevalence of both diseases, but there is little information on whether HIV affects the clinical presentation and outcome of severe malaria.
Methods. HIV status was assessed prospectively in hospitalized parasitemic adults and children with severe malaria in Beira, Mozambique, as part of a clinical trial comparing parenteral artesunate versus quinine (ISRCTN50258054). Clinical signs, comorbidity, complications, and disease outcome were compared according to HIV status.
Results. HIV-1 seroprevalence was 11% (74/655) in children under 15 years and 72% (49/68) in adults with severe malaria. Children with HIV coinfection presented with more severe acidosis, anemia, and respiratory distress, and higher peripheral blood parasitemia and plasma Plasmodium falciparum histidine-rich protein-2 (PfHRP2). During hospitalization, deterioration in coma score, convulsions, respiratory distress, and pneumonia were more common in HIV-coinfected children, and mortality was 26% (19/74) versus 9% (53/581) in uninfected children (P < .001). In an age- and antimalarial treatment–adjusted logistic regression model, significant, independent predictors for death were renal impairment, acidosis, parasitemia, and plasma PfHRP2 concentration.
Conclusions. Severe malaria in HIV-coinfected patients presents with higher parasite burden, more complications, and comorbidity, and carries a higher case fatality rate. Early identification of HIV coinfection is important for the clinical management of severe malaria.
doi:10.1093/cid/cis590
PMCID: PMC3447636  PMID: 22752514
13.  The effects of serum lipids on the in vitro activity of lumefantrine and atovaquone against Plasmodium falciparum 
Malaria Journal  2012;11:177.
Background
Lumefantrine and atovaquone are highly lipophilic anti-malarial drugs. As a consequence absorption is increased when the drugs are taken together with a fatty meal, but the free fraction of active drug decreases in the presence of triglyceride-rich plasma lipoproteins. In this study, the consequences of lipidaemia on anti-malarial drug efficacy were assessed in vitro.
Methods
Serum was obtained from non-immune volunteers under fasting conditions and after ingestion of a high fat meal and used in standard Plasmodium falciparum in-vitro susceptibility assays. Anti-malarial drugs, including lumefantrine, atovaquone and chloroquine in five-fold dilutions (range 0.05 ng/ml – 1 ug/mL) were diluted in culture medium supplemented with fasting or post-prandial 10% donor serum. The in-vitro drug susceptibility of parasite isolates was determined using the 3H-hypoxanthine uptake inhibition method and expressed as the concentration which gave 50% inhibition of hypoxanthine uptake (IC50).
Results
Doubling plasma triglyceride concentrations (from 160 mg/dL to 320 mg/dL), resulted in an approximate doubling of the IC50 for lumefantrine (191 ng/mL to 465 ng/mL, P < 0.01) and a 20-fold increase in the IC50 for atovaquone (0.5 ng/mL to 12 ng/ml; P < 0.01). In contrast, susceptibility to the hydrophilic anti-malarial chloroquine did not change in relation to triglyceride content of the medium.
Conclusions
Lipidaemia reduces the anti-malarial activity of lipophilic anti-malarial drugs. This is an important confounder in laboratory in vitro testing and it could have therapeutic relevance.
doi:10.1186/1475-2875-11-177
PMCID: PMC3426466  PMID: 22640826
Malaria; Anti-malarial drugs; In vitro-susceptibility
14.  Plasmodium vivax Adherence to Placental Glycosaminoglycans 
PLoS ONE  2012;7(4):e34509.
Background
Plasmodium vivax infections seldom kill directly but do cause indirect mortality by reducing birth weight and causing abortion. Cytoadherence and sequestration in the microvasculature are central to the pathogenesis of severe Plasmodium falciparum malaria, but the contribution of cytoadherence to pathology in other human malarias is less clear.
Methodology
The adherence properties of P. vivax infected red blood cells (PvIRBC) were evaluated under static and flow conditions.
Principal Findings
P. vivax isolates from 33 patients were studied. None adhered to immobilized CD36, ICAM-1, or thrombospondin, putative ligands for P. falciparum vascular cytoadherence, or umbilical vein endothelial cells, but all adhered to immobilized chondroitin sulphate A (CSA) and hyaluronic acid (HA), the receptors for adhesion of P. falciparum in the placenta. PvIRBC also adhered to fresh placental cells (N = 5). Pre-incubation with chondroitinase prevented PvIRBC adherence to CSA, and reduced binding to HA, whereas preincubation with hyaluronidase prevented adherence to HA, but did not reduce binding to CSA significantly. Pre-incubation of PvIRBC with soluble CSA and HA reduced binding to the immobilized receptors and prevented placental binding. PvIRBC adhesion was prevented by pre-incubation with trypsin, inhibited by heparin, and reduced by EGTA. Under laminar flow conditions the mean (SD) shear stress reducing maximum attachment by 50% was 0.06 (0.02) Pa but, having adhered, the PvIRBC could then resist detachment by stresses up to 5 Pa. At 37°C adherence began approximately 16 hours after red cell invasion with maximal adherence at 30 hours. At 39°C adherence began earlier and peaked at 24 hours.
Significance
Adherence of P. vivax-infected erythrocytes to glycosaminoglycans may contribute to the pathogenesis of vivax malaria and lead to intrauterine growth retardation.
doi:10.1371/journal.pone.0034509
PMCID: PMC3328474  PMID: 22529919
15.  Circulating Red Cell–derived Microparticles in Human Malaria 
The Journal of Infectious Diseases  2011;203(5):700-706.
In patients with falciparum malaria, plasma concentrations of cell-derived microparticles correlate with disease severity. Using flow cytometry, we quantified red blood cell–derived microparticles (RMPs) in patients with malaria and identified the source and the factors associated with production. RMP concentrations were increased in patients with Plasmodium falciparum (n = 29; median, 457 RMPs/μL [range, 13–4,342 RMPs/μL]), Plasmodium vivax (n = 5; median, 409 RMPs/μL [range, 281–503/μL]), and Plasmodium malariae (n = 2; median, 163 RMPs/μL [range, 127–200 RMPs/μL]) compared with those in healthy subjects (n = 11; median, 8 RMPs/μL [range, 3–166 RMPs/μL]; P = .01). RMP concentrations were highest in patients with severe falciparum malaria (P = .01). Parasitized red cells produced >10 times more RMPs than did unparasitized cells, but the overall majority of RMPs still derived from uninfected red blood cells (URBCs). In cultures, RMP production increased as the parasites matured. Hemin and parasite products induced RMP production in URBCs, which was inhibited by N-acetylcysteine, suggesting heme-mediated oxidative stress as a pathway for the generation of RMPs.
doi:10.1093/infdis/jiq104
PMCID: PMC3072726  PMID: 21282195
16.  Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription 
BMC Genomics  2011;12:391.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2164-12-391
PMCID: PMC3163569  PMID: 21810278
Plasmodium falciparum, in vivo artemisinin-resistance; field isolates; comparative genomics; comparative transcriptomics
17.  High heritability of malaria parasite clearance rate indicates a genetic basis for artemisinin resistance in Western Cambodia 
The Journal of infectious diseases  2010;201(9):1326-1330.
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.
doi:10.1086/651562
PMCID: PMC2853733  PMID: 20350192
Artemisinin; clearance time; heritability; twin studies; resistance; microsatellite
18.  Modulating effects of plasma containing anti-malarial antibodies on in vitro anti-malarial drug susceptibility in Plasmodium falciparum 
Malaria Journal  2010;9:326.
Background
The efficacy of anti-malarial drugs is determined by the level of parasite susceptibility, anti-malarial drug bioavailability and pharmacokinetics, and host factors including immunity. Host immunity improves the in vivo therapeutic efficacy of anti-malarial drugs, but the mechanism and magnitude of this effect has not been characterized. This study characterized the effects of 'immune' plasma to Plasmodium falciparumon the in vitro susceptibility of P. falciparum to anti-malarial drugs.
Methods
Titres of antibodies against blood stage antigens (mainly the ring-infected erythrocyte surface antigen [RESA]) were measured in plasma samples obtained from Thai patients with acute falciparum malaria. 'Immune' plasma was selected and its effects on in vitro parasite growth and multiplication of the Thai P. falciparum laboratory strain TM267 were assessed by light microscopy. The in vitro susceptibility to quinine and artesunate was then determined in the presence and absence of 'immune' plasma using the 3H-hypoxanthine uptake inhibition method. Drug susceptibility was expressed as the concentrations causing 50% and 90% inhibition (IC50 and IC90), of 3H-hypoxanthine uptake.
Results
Incubation with 'immune' plasma reduced parasite maturation and decreased parasite multiplication in a dose dependent manner. 3H-hypoxanthine incorporation after incubation with 'immune' plasma was decreased significantly compared to controls (median [range]; 181.5 [0 to 3,269] cpm versus 1,222.5 [388 to 5,932] cpm) (p= 0.001). As a result 'immune' plasma reduced apparent susceptibility to quinine substantially; median (range) IC50 6.4 (0.5 to 23.8) ng/ml versus 221.5 (174.4 to 250.4) ng/ml (p = 0.02), and also had a borderline effect on artesunate susceptibility; IC50 0.2 (0.02 to 0.3) ng/ml versus 0.8 (0.2 to 2.3) ng/ml (p = 0.08). Effects were greatest at low concentrations, changing the shape of the concentration-effect relationship. IC90 values were not significantly affected; median (range) IC90 448.0 (65 to > 500) ng/ml versus 368.8 (261 to 501) ng/ml for quinine (p > 0.05) and 17.0 (0.1 to 29.5) ng/ml versus 7.6 (2.3 to 19.5) ng/ml for artesunate (p = 0.4).
Conclusions
'Immune' plasma containing anti-malarial antibodies inhibits parasite development and multiplication and increases apparent in vitro anti-malarial drug susceptibility of P. falciparum. The IC90 was much less affected than the IC50 measurement.
doi:10.1186/1475-2875-9-326
PMCID: PMC2993733  PMID: 21078202
19.  Plasmodium falciparum genome-wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs 
Nature genetics  2010;42(3):268-271.
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.
doi:10.1038/ng.528
PMCID: PMC2828519  PMID: 20101240
malaria; single nucleotide polymorphism (SNP); genome-wide association study; recombination; drug resistance; population structure
20.  A Comparison of Two Short-Course Primaquine Regimens for the Treatment and Radical Cure of Plasmodium vivax Malaria in Thailand 
Thai adult males (N = 85) with acute Plasmodium vivax malaria and normal glucose-6-phosphate dehydrogenase screening were randomized to receive 30 mg or 60 mg primaquine daily for 7 days (N = 43 and 42, respectively). The regimens were well tolerated and all patients recovered fully. Median fever clearance (47 hours; range 4 to 130 hours), mean ± SD parasite clearance times (87.7 ± 25.3 hours), gametocyte clearance, and adverse effects were similar in the 2 groups. Two patients, 1 from each group, had a 30% reduction in hematocrit. The cumulative 28 day relapse rate (95% confidence interval) by Kaplan Meier survival analysis was 29% (16–49%) in the 30 mg group compared with 7% (2–24%) in the 60 mg group; P = 0.027. Comparison with previous data obtained at this same site suggests that the recurrences comprised approximately 17% recrudescences and 12% relapses in the 30 mg/day group compared with 3% recrudescences and 4% relapses in the 60 mg/day group. These data suggest that the dose-response relationships for primaquine's asexual stage and hypnozoitocidal activities in-vivo are different. A 1 week course of primaquine 60 mg daily is an effective treatment of vivax malaria in this region.
doi:10.4269/ajtmh.2010.09-0428
PMCID: PMC2844579  PMID: 20348496
21.  Plasmodium vivax parasites alter the balance of myeloid and plasmacytoid dendritic cells and induction of regulatory T cells 
European journal of immunology  2008;38(10):2697-2705.
Summary
Immunity induced by Plasmodium vivax infections leads to memory T cell recruitment and activation during subsequent infections. Here, we investigated the role of regulator T cells (Treg) in coordination with the host immune response during P. vivax infection. Our results showed a significant increase in the percentage of FOXP3+ Treg, IL-10 secreting Type I Treg (Tr1) and IL-10 levels in patients with acute P. vivax infection as compared to those found in either naïve or immune controls. The concurrent increase in the Treg population could also be reproduced in vitro using PBMC from naïve controls stimulated with crude antigens extracted from P. vivax-infected red blood cells. Acute P. vivax infections were associated with a significant decrease in the numbers of DC, indicating a general immunosuppression during P.vivax infections. However, unlike P. falciparum infections, we found that the ratio between myeloid DC (MDC) and plamacytoid (PDC) was significantly lower in acute P. vivax patients than that of naïve and immune controls. Moreover, the reduction of PDC may be partly responsible for the poor antibody responses during P. vivax infections. Taken together, these results suggest that P. vivax parasites interact with DC, which alters MDC/PDC ratio that potentially leads to Treg activation and IL-10 release.
doi:10.1002/eji.200838186
PMCID: PMC2757553  PMID: 18825754
Plasmodium vivax; malaria; immunity to malaria; regulatory T cell; IL-10; dendritic cell
22.  Plasmodium falciparum pfmdr1 Amplification, Mefloquine Resistance, and Parasite Fitness▿ † 
Mefloquine is widely used in combination with artemisinin derivatives for the treatment of falciparum malaria. Mefloquine resistance in Plasmodium falciparum has been related to increased copy numbers of multidrug-resistant gene 1 (pfmdr1). We studied the ex vivo dynamics of pfmdr1 gene amplification in culture-adapted P. falciparum in relation to mefloquine resistance and parasite fitness. A Thai P. falciparum isolate (isolate TM036) was assessed by the use of multiple genetic markers as a single genotype. Resistance was selected by exposure to stepwise increasing concentrations of mefloquine up to 30 ng/ml in continuous culture. The pfmdr1 gene copy numbers increased as susceptibility to mefloquine declined (P = 0.03). No codon mutations at positions 86, 184, 1034, 1042, and 1246 in the pfmdr1 gene were detected. Two subclones of selected parasites (average copy numbers, 2.3 and 3.1, respectively) showed a fitness disadvantage when they were grown together with the original parasites containing a single pfmdr1 gene copy in the absence of mefloquine; the multiplication rates were 6.3% and 8.7% lower, respectively (P < 0.01). Modeling of the dynamics of the pfmdr1 copy numbers over time in relation to the relative fitness of the parasites suggested that net pfmdr1 gene amplification from one to two copies occurs once in every 108 parasites and that amplification from two to three copies occurs once in every 103 parasites. pfmdr1 gene amplification in P. falciparum is a frequent event and confers mefloquine resistance. Parasites with multiple copies of the pfmdr1 gene have decreased survival fitness in the absence of drug pressure.
doi:10.1128/AAC.00241-08
PMCID: PMC2663078  PMID: 19164150
23.  Genetic Analysis of the Dihydrofolate Reductase-Thymidylate Synthase Gene from Geographically Diverse Isolates of Plasmodium malariae▿  
Antimicrobial Agents and Chemotherapy  2007;51(10):3523-3530.
Plasmodium malariae, the parasite responsible for quartan malaria, is transmitted in most areas of malaria endemicity and is associated with significant morbidity. The sequence of the gene coding for the enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) was obtained from field isolates of P. malariae and from the closely related simian parasite Plasmodium brasilianum. The two sequences were nearly 100% homologous, adding weight to the notion that they represent genetically distinct lines of the same species. A survey of polymorphisms of the dhfr sequences in 35 isolates of P. malariae collected from five countries in Asia and Africa revealed a low number of nonsynonymous mutations in five codons. In five of the isolates collected from southeast Asia, a nonsynonymous mutation was found at one of the three positions known to be associated with antifolate resistance in other Plasmodium species. Five isolates with the wild-type DHFR could be assayed for drug susceptibility in vitro and were found to be sensitive to pyrimethamine (mean 50% inhibitory concentration, 2.24 ng/ml [95% confidence interval, 0.4 to 3.1]).
doi:10.1128/AAC.00234-07
PMCID: PMC2043249  PMID: 17682097
24.  Overestimating resistance in field testing of malaria parasites: simple methods for estimating high EC50 values using a Bayesian approach 
Malaria Journal  2007;6:4.
Conventional methods of assessing in-vitro antimalarial drug-concentration effect relationships in field testing of fresh isolates assess each parasite isolate individually. This leads to systematic overestimation of EC50 values for the most resistant isolates, and thus overestimation of the degree of resistance. In antimalarial drug-susceptibility studies conducted on the north-western border of Thailand the overestimation of EC50 for the most resistant isolate ranged from 15% for artesunate to 43% for mefloquine. If isolates cannot be stored for re-testing, more accurate estimations of the degree of resistance can be obtained using a Bayesian approach to data analysis which is described here.
doi:10.1186/1475-2875-6-4
PMCID: PMC1794247  PMID: 17229314
25.  Transmission-Blocking Activities of Quinine, Primaquine, and Artesunate 
The infectivity of Plasmodium falciparum gametocytes after exposure in vitro to quinine, artesunate, and primaquine was assessed in Anopheles dirus, a major vector of malaria in Southeast Asia. Mature gametocytes (stage 5) of a Thai isolate of P. falciparum were exposed to the drugs for 24 h in vitro before membrane feeding to A. dirus. After 10 days, the mosquito midguts were dissected and the oocysts were counted. In this system, artesunate showed the most potent transmission-blocking activity; the mean (standard deviation [SD]) 50% and 90% effective concentrations (EC50, and EC90, respectively, in nanograms per milliliter) were 0.1 (0.02) and 0.4 (0.15), respectively. Transmission-blocking activity of quinine and primaquine was observed at relatively high concentrations (SDs): EC50 of quinine, 642 (111) ng/ml; EC50 of primaquine, 181 (23) ng/ml; EC90 of quinine, 816 (96) ng/ml; EC90 of primaquine, 543 (43) ng/ml. Artesunate both prevents the maturation of immature P. falciparum gametocytes and reduces the transmission potential of mature gametocytes. Both of these effects may contribute to reducing malaria transmission.
doi:10.1128/AAC.01472-05
PMCID: PMC1479118  PMID: 16723547

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