Overestimating resistance in field testing of malaria parasites: simple methods for estimating high EC(50 )values using a Bayesian approach

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 EC(50 )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 EC(50 )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

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

Modulating effects of plasma containing anti-malarial antibodies on in vitro anti-malarial drug susceptibility in Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>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 <it>in vivo </it>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 <it>Plasmodium falciparum</it>on the <it>in vitro </it>susceptibility of <it>P. falciparum </it>to anti-malarial drugs.</p> <p>Methods</p> <p>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 <it>in vitro </it>parasite growth and multiplication of the Thai <it>P. falciparum </it>laboratory strain TM267 were assessed by light microscopy. The <it>in vitro </it>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 (IC₅₀and IC₉₀), of ³H-hypoxanthine uptake.</p> <p>Results</p> <p>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) (<it>p</it>= 0.001). As a result 'immune' plasma reduced apparent susceptibility to quinine substantially; median (range) IC₅₀6.4 (0.5 to 23.8) ng/ml versus 221.5 (174.4 to 250.4) ng/ml (<it>p </it>= 0.02), and also had a borderline effect on artesunate susceptibility; IC₅₀0.2 (0.02 to 0.3) ng/ml versus 0.8 (0.2 to 2.3) ng/ml (<it>p </it>= 0.08). Effects were greatest at low concentrations, changing the shape of the concentration-effect relationship. IC₉₀values were not significantly affected; median (range) IC₉₀448.0 (65 to > 500) ng/ml versus 368.8 (261 to 501) ng/ml for quinine (<it>p </it>> 0.05) and 17.0 (0.1 to 29.5) ng/ml versus 7.6 (2.3 to 19.5) ng/ml for artesunate (<it>p </it>= 0.4).</p> <p>Conclusions</p> <p>'Immune' plasma containing anti-malarial antibodies inhibits parasite development and multiplication and increases apparent <it>in vitro </it>anti-malarial drug susceptibility of <it>P. falciparum</it>. The IC₉₀was much less affected than the IC₅₀measurement.</p

Vivax malaria in pregnancy and lactation: a long way to health equity

The Sustainable Development Goals (SDG) call for increased gender equity and reduction in malaria-related mortality and morbidity. Plasmodium vivax infections in pregnancy are associated with maternal anaemia and increased adverse perinatal outcomes. Providing radical cure for women with 8-aminoquinolines (e.g., primaquine) is hindered by gender-specific complexities.; A symptomatic episode of vivax malaria at 18 weeks of gestation in a primigravid woman was associated with maternal anaemia, a recurrent asymptomatic P. vivax episode, severe intra-uterine growth restriction with no other identifiable cause and induction to reduce the risk of stillbirth. At 5 months postpartum a qualitative glucose-6-phosphate dehydrogenase (G6PD) point-of-care test was normal and radical cure with primaquine was prescribed to the mother. A 33% fractional decrease in haematocrit on day 7 of primaquine led to further testing which showed intermediate phenotypic G6PD activity; the G6PD genotype could not be identified. Her infant daughter was well throughout maternal treatment and found to be heterozygous for Mahidol variant.; Adverse effects of vivax malaria in pregnancy, ineligibility of radical cure for pregnant and postpartum women, and difficulties in diagnosing intermediate levels of G6PD activity multiplied morbidity in this woman. Steps towards meeting the SDG include prevention of malaria in pregnancy, reducing unnecessary exclusion of women from radical cure, and accessible quantitative G6PD screening in P. vivax-endemic settings

Allele-Specific Isothermal Amplification Method Using Unmodified Self-Stabilizing Competitive Primers.

Rapid and specific detection of single nucleotide polymorphisms (SNPs) related to drug resistance in infectious diseases is crucial for accurate prognostics, therapeutics and disease management at point-of-care. Here, we present a novel amplification method and provide universal guidelines for the detection of SNPs at isothermal conditions. This method, called USS-sbLAMP, consists of SNP-based loop-mediated isothermal amplification (sbLAMP) primers and unmodified self-stabilizing (USS) competitive primers that robustly delay or prevent unspecific amplification. Both sets of primers are incorporated into the same reaction mixture, but always targeting different alleles; one set specific to the wild type allele and the other to the mutant allele. The mechanism of action relies on thermodynamically favored hybridization of totally complementary primers, enabling allele-specific amplification. We successfully validate our method by detecting SNPs, C580Y and Y493H, in the Plasmodium falciparum kelch 13 gene that are responsible for resistance to artemisinin-based combination therapies currently used globally in the treatment of malaria. USS-sbLAMP primers can efficiently discriminate between SNPs with high sensitivity (limit of detection of 5 × 101 copies per reaction), efficiency, specificity and rapidness (<35 min) with the capability of quantitative measurements for point-of-care diagnosis, treatment guidance, and epidemiological reporting of drug-resistance

Genetic diversity of Plasmodium vivax in Kolkata, India

BACKGROUND: Plasmodium vivax malaria accounts for approximately 60% of malaria cases in Kolkata, India. There has been limited information on the genotypic polymorphism of P. vivax in this malaria endemic area. Three highly polymorphic and single copy genes were selected for a study of genetic diversity in Kolkata strains. METHODS: Blood from 151 patients with P. vivax infection diagnosed in Kolkata between April 2003 and September 2004 was genotyped at three polymorphic loci: the P. vivax circumsporozoite protein (pvcs), the merozoite surface protein 1 (pvmsp1) and the merozoite surface protein 3-alpha (pvmsp3-alpha). RESULTS: Analysis of these three genetic markers revealed that P. vivax populations in Kolkata are highly diverse. A large number of distinguishable alleles were found from three genetic markers: 11 for pvcs, 35 for pvmsp1 and 37 for pvmsp3-alpha. These were, in general, randomly distributed amongst the isolates. Among the 151 isolates, 142 unique genotypes were detected the commonest genotype at a frequency of less than 2% (3/151). The overall rate of mixed genotype infections was 10.6%. CONCLUSION: These results indicate that the P. vivax parasite population is highly diverse in Kolkata, despite the low level of transmission. The genotyping protocols used in this study may be useful for differentiating re-infection from relapse and recrudescence in studies assessing of malarial drug efficacy in vivax malaria

Activity of Ivermectin and Its Metabolites against Asexual Blood Stage Plasmodium falciparum and Its Interactions with Antimalarial Drugs

Ivermectin is an endectocide used widely to treat a variety of internal and external parasites. Field trials of ivermectin mass drug administration for malaria transmission control have demonstrated a reduction of Anopheles mosquito survival and human malaria incidence. Ivermectin will mostly be deployed together with artemisinin-based combination therapies (ACT), the first-line treatment of falciparum malaria. It has not been well established if ivermectin has activity against asexual stage Plasmodium falciparum or if it interacts with the parasiticidal activity of other antimalarial drugs. This study evaluated antimalarial activity of ivermectin and its metabolites in artemisinin-sensitive and artemisinin-resistant P. falciparum isolates and assessed in vitro drug-drug interaction with artemisinins and its partner drugs. The concentration of ivermectin causing half of the maximum inhibitory activity (IC50) on parasite survival was 0.81 μM with no significant difference between artemisinin-sensitive and artemisinin-resistant isolates (P = 0.574). The ivermectin metabolites were 2-fold to 4-fold less active than the ivermectin parent compound (P &lt; 0.001). Potential pharmacodynamic drug-drug interactions of ivermectin with artemisinins, ACT-partner drugs, and atovaquone were studied in vitro using mixture assays providing isobolograms and derived fractional inhibitory concentrations. There were no synergistic or antagonistic pharmacodynamic interactions when combining ivermectin and antimalarial drugs. In conclusion, ivermectin does not have clinically relevant activity against the asexual blood stages of P. falciparum. It also does not affect the in vitro antimalarial activity of artemisinins or ACT-partner drugs against asexual blood stages of P. falciparum.</p

Artemisinin resistance in Plasmodium falciparum malaria.

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

Estimation of the Total Parasite Biomass in Acute Falciparum Malaria from Plasma PfHRP2

BACKGROUND: In falciparum malaria sequestration of erythrocytes containing mature forms of Plasmodium falciparum in the microvasculature of vital organs is central to pathology, but quantitation of this hidden sequestered parasite load in vivo has not previously been possible. The peripheral blood parasite count measures only the circulating, relatively non-pathogenic parasite numbers. P. falciparum releases a specific histidine-rich protein (PfHRP2) into plasma. Quantitative measurement of plasma PfHRP2 concentrations may reflect the total parasite biomass in falciparum malaria. METHODS AND FINDINGS: We measured plasma concentrations of PfHRP2, using a quantitative antigen-capture enzyme-linked immunosorbent assay, in 337 adult patients with falciparum malaria of varying severity hospitalised on the Thai–Burmese border. Based on in vitro production rates, we constructed a model to link this measure to the total parasite burden in the patient. The estimated geometric mean parasite burden was 7 × 10(11) (95% confidence interval [CI] 5.8 × 10(11) to 8.5 × 10(11)) parasites per body, and was over six times higher in severe malaria (geometric mean 1.7 × 10(12), 95% CI 1.3 × 10(12) to 2.3 × 10(12)) than in patients hospitalised without signs of severity (geometric mean 2.8 × 10(11), 95% CI 2.3 × 10(11) to 3.5 × 10(11); p < 0.001). Parasite burden was highest in patients who died (geometric mean 3.4 × 10(12), 95% CI 1.9 × 10(12) to 6.3 × 10(12); p = 0.03). The calculated number of sequestered parasites increased with disease severity and was higher in patients with late developmental stages of P. falciparum present on peripheral blood smears. Comparing model and laboratory estimates of the time of sequestration suggested that admission to hospital with uncomplicated malaria often follows schizogony—but in severe malaria is unrelated to stage of parasite development. CONCLUSION: Plasma PfHRP2 concentrations may be used to estimate the total body parasite biomass in acute falciparum malaria. Severe malaria results from extensive sequestration of parasitised erythrocytes