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Publication Open Access Evaluation of the phenotypic test and genetic analysis in the detection of glucose-6-phosphate dehydrogenase deficiency(2013-08-21) Duangdao Nantakomol; ดวงดาว นันทโกมล; Paul, Rick; Attakorn Palasuwa; อรรถกร ปาละสุวรรณ; Day, Nicholas P.J.; White, Nicholas J.; Mallika Imwong; มัลลิกา อิ่มวงศ์; Mallika Imwong; Mahidol University. Faculty of Tropical Medicine. Department of Molecular Tropical Medicine and Genetics; Mahidol University. Faculty of Tropical Medicine. Mahidol Oxford Research UnitBACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is particularly prevalent in historically malaria-endemic countries. Although most individuals with G6PD deficiency are asymptomatic, deficiency can result in acute haemolytic anaemia after exposure to oxidative agents. A reliable test is necessary for diagnosing the deficiency to prevent an acute haemolytic crisis following, for example, anti-malarial treatment. The aim of this study was to investigate which method was the best predictor of this disorder. METHODS: The present study investigated four G6PD activity detections (fluorescence spot (FS), methaemoglobin reduction (MR), biochemical and cytochemical test). These methods accompanied with mutation analysis of blood samples were taken from 295 apparently healthy individuals with unknown G6PD deficiency status. RESULTS: Molecular characterization of 295 Thai adults revealed an overall prevalence of 14.2%. The G6PD Viangchan (871 G>A) was the most common (83.3%), followed by G6PD Mahidol (487G>A) (11.9%), and G6PD Union (1360 C>T) (4.8%). There were two cases of G6PD deficiency carrying the double mutations of Viangchan (871G > A)-Mahidol (487G > A) and Viangchan (871G > A)-Union (1360C > T). In comparison, the prevalence of G6PD deficiency was 6.1% by FS test and 7.1% by MR test. G6PD activity was 11 ± 2.5 IU/gHb in non-deficient females (mean ± SD), and 10.9 ± 0.6 IU/gHb in non-deficient males. The upper and lower limit cut-off points for partial and severe deficiency in adults were 5.7 IU/gHb (60% of the normal mean) and 0.95 IU/gHb (10% of the normal mean), respectively. All hemizygote, homozygote and double mutations were associated with severe enzyme deficiency (the residual enzyme activity <10% of the normal mean), whereas only 14.3% of the heterozygote mutations showed severe enzyme deficiency. Based on the cut-off value <5.7 IU/gHb, the quantitative G6PD assay diagnosed 83% of cases as G6PD-deficient. Using a cut-off number of negative cell >20% in the cytochemical assay to define G6PD deficiency, the prevalence of G6PD deficiency was closest to the molecular analysis (12.9% G6PD-deficient) compared to the others methods. CONCLUSION: The cytochemical method is a significant predictor of this disease, while FS and MR test are recommended for the detection of severe G6PD deficiency in developing countries.Publication Open Access Thiamin supplementation does not reduce the frequency of adverse events after anti-malarial therapy among patients with falciparum malaria in southern Laos(2014-07-15) Mayxay, Mayfong; Khanthavong, Maniphone; Cox, Lorna; Sichanthongthip, Odai; Mallika Imwong; มัลลิกา อิ่มวงศ์; Pongvongsa, Tiengkham; Hongvanthong, Bouasy; Phompida, Samlane; Vanisaveth, Viengxay; White, Nicholas J; Newton, Paul N; Mayxay, Mayfong; Mahidol University. Faculty of Tropical Medicine. Department of Molecular Tropical Medicine and Genetics.Background: In a recent study one third of Lao patients presenting with uncomplicated Plasmodium falciparum malaria had biochemical evidence of thiamin deficiency, which was associated with a higher incidence of adverse events. Thiamin supplementation might, therefore, reduce adverse events in this population. Methods: An exploratory, double-blind, parallel group, placebo-controlled, superiority trial of thiamin supplementation in patients of all ages with uncomplicated and severe falciparum malaria was conducted in Xepon District, Savannakhet Province, southern Laos. Patients were randomly assigned to either oral thiamin 10 mg/day for 7 days immediately after standard anti-malarial treatment then 5 mg daily until day 42, or identical oral placebo. Results: After interim analyses when 630 patients (314 in thiamin and 316 in placebo groups) had been recruited, the trial was discontinued on the grounds of futility. On admission biochemical thiamin deficiency (alpha ≥ 25%) was present in 27% of patients and 9% had severe deficiency (alpha > 31%). After 42 days of treatment, the frequency of thiamin deficiency was lower in the thiamin (2%, 1% severe) compared to the placebo (11%, 3% severe) groups (p < 0.001 and p = 0.05), respectively. Except for diarrhoea, 7% in the placebo compared to 3% in the thiamin group (p = 0.04), and dizziness on day 1 (33% vs 25%, p = 0.045), all adverse events were not significantly different between the groups (p > 0.05). Clinical, haematological, and parasitological responses to treatment did not differ significantly between the two groups. Conclusion: Thiamin supplementation reduced biochemical thiamin deficiency among Lao malaria patients following anti-malarial drug treatment, but it did not reduce the frequency of adverse events after anti-malarial therapy or have any detected clinical or parasitological impact.Publication Open Access Sequence variation does not confound the measurement of plasma PfHRP2 concentration in African children presenting with severe malaria(2012-08-16) Thiranut Ramutton; Hendriksen, Ilse CE; Mwanga-Amumpaire, Juliet; Mtove, George; Olaosebikan, Rasaq; Tshefu, Antoinette K.; Onyamboko, Marie A.; Karema, Corine; Maitland, Kathryn; Gomes, Ermelinda; Gesase, Samwel; Reyburn, Hugh; Kamolrat Silamut; Kesinee Chotivanich; เกศินี โชติวานิช; Kamoltip Promnares; Fanello, Caterina I.; Seidlein, Lorenz von; Day, Nicholas P.J.; White, Nicholas J.; Dondorp, Arjen M.; Mallika Imwong; มัลลิกา อิ่มวงศ์; Woodrow, Charles J.; Woodrow, Charles J.; Mahidol University. Faculty of Tropical Medicine. Department of Clinical Tropical Medicine.; Mahidol University. Faculty of Tropical Medicine. Department of Molecular Tropical Medicine and Genetics.; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit.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.Publication Open Access Chloroquine resistant vivax malaria in a pregnant woman on the western border of Thailand(2011-05-05) Rijken, Marcus J.; Boel, Machteld E.; Russell, Bruce; Mallika Imwong; มัลลิกา อิ่มวงศ์; Leimanis, Mara L.; Phyo, Aung Pyae; Muehlenbachs, Atis; Lindegardh, Niklas; McGready, Rose; Rénia, Laurent; Snounou, Georges; Pratap Singhasivanon; ประตาป สิงหศิวานนท์; Nosten, François; Rijken, Marcus J.; Mahidol University. Faculty of Tropical Medicine.Department of Molecular Tropical Medicine and GeneticsChloroquine (CQ) resistant vivax malaria is spreading. In this case, Plasmodium vivax infections during pregnancy and in the postpartum period were not satisfactorily cleared by CQ, despite adequate drug concentrations. A growth restricted infant was delivered. Poor susceptibility to CQ was confirmed in-vitro and molecular genotyping was strongly suggestive of true recrudescence of P. vivax. This is the first clinically and laboratory confirmed case of two high-grade CQ resistant vivax parasite strains from Thailand.Publication Open Access Long-term storage limits PCR-based analyses of malaria parasites in archival dried blood spots(2012-10) Hwang, Joyce; Juthamas Jaroensuk; Leimanis, Mara L.; Russell, Bruce; McGready, Rose; Day, Nicholas; Snounou, George; Nosten, Francois; Mallika Imwong; มัลลิกา อิ่มวงศ์; Snounou, George; Mahidol University. Faculty of Tropical Medicine. Department of Molecular Tropical Medicine and GeneticsBACKGROUND: Blood samples collected in epidemiological and clinical investigations and then stored, often at room temperature, as blood spots dried on a filter paper have become one of the most popular source of material for further molecular analyses of malaria parasites. The dried blood spots are often archived so that they can be used for further retrospective investigations of parasite prevalence, or as new genetic markers come to the fore. However, the suitability of the template obtained from dried blood spots that have been stored for long periods for DNA amplification is not known. METHODS: DNA from 267 archived blood spots collected over a period of 12 years from persons with microscopically confirmed Plasmodium falciparum infection was purified by one of two methods, Chelex and Qiagen columns. These templates were subjected to highly sensitive nested PCR amplification targeting three parasite loci that differ in length and/or copy number. RESULTS: When a 1.6 kb fragment of the parasites' small subunit ribosomal RNA was targeted (primary amplification), the efficiency of P. falciparum detection decreased in samples archived for more than six years, reaching very low levels for those stored for more than 10 years. Positive amplification was generally obtained more often with Qiagen-extracted templates. P. falciparum could be detected in 32 of the 40 negative Qiagen-extracted templates when a microsatellite of about 180 bp was targeted. The remaining eight samples gave a positive amplification when a small region of 238 bp of the higher copy number (20 to 200) mitochondrial genome was targeted. CONCLUSIONS: The average length of DNA fragments that can be recovered from dried blood spots decreases with storage time. Recovery of the DNA is somewhat improved, especially in older samples, by the use of a commercial DNA purification column, but targets larger than 1.5 kb are unlikely to be present 10 years after the initial blood collection, when the average length of the DNA fragments present is likely to be around a few hundred bp. In conclusion, the utility of archived dried blood spots for molecular analyses decreases with storage time.Publication Open Access Dihydroartemisinin-piperaquine versus chloroquine to treat vivax malaria in Afghanistan: an open randomized,non-inferiority, trial(2010-04) Awab, Ghulam Rahim; Sasithon Pukrittayakamee; ศศิธร ผู้กฤตยาคามี; Mallika Imwong; มัลลิกา อิ่มวงศ์; Dondorp, Arjen M.; Woodrow, Charles J.; Lee, Sue Jean; Day, Nicholas P.J.; Pratap Singhasivanon; ประตาป สิงหศิวานนท์; White, Nicholas J.; Kaker, Faizullah; White, Nicholas J.; Mahidol University. Faculty of Tropical MediicneBACKGROUND: Afghanistan's national guidelines recommend chloroquine for the treatment of Plasmodium vivax infection, the parasite responsible for the majority of its malaria burden. Chloroquine resistance in P. vivax is emerging in Asia. Therapeutic responses across Afghanistan have not been evaluated in detail. METHODS: Between July 2007 and February 2009, an open-label, randomized controlled trial of chloroquine and dihydroartemisinin-piperaquine in patients aged three months and over with slide-confirmed P. vivax mono-infections was conducted. Consistent with current national guidelines, primaquine was not administered. Subjects were followed up daily during the acute phase of illness (days 0-3) and weekly until day 56. The primary endpoint was the overall cumulative parasitological failure rate at day 56 after the start of treatment, with the hypothesis being that dihydroartemisinin-piperaquine was non-inferior compared to chloroquine (Delta = 5% difference in proportion of failures). RESULTS: Of 2,182 individuals with positive blood films for P. vivax, 536 were enrolled in the trial. The day 28 cure rate was 100% in both treatment groups. Parasite clearance was more rapid with dihydroartemisinin-piperaquine than chloroquine. At day 56, there were more recurrent infections in the chloroquine arm (8.9%, 95% CI 6.0-13.1%) than the dihydroartemisinin-piperaquine arm (2.8%, 95% CI 1.4-5.8%), a difference in cumulative recurrence rate of 6.1% (2-sided 90%CI +2.6 to +9.7%). The log-rank test comparing the survival curves confirmed the superiority of dihydroartemisinin-piperaquine over chloroquine (p = 0.003). Multivariate analysis showed that a lower initial haemoglobin concentration was also independently associated with recurrence. Both regimens were well tolerated and no serious adverse events were reported. CONCLUSIONS: Chloroquine remains an efficacious treatment for the treatment of vivax malaria in Afghanistan. In a setting where radical therapy cannot be administered, dihydroartemisinin-piperaquine provides additional benefit in terms of post-treatment prophylaxis, reducing the incidence of recurrence from 4-8 weeks after treatment.Publication Open Access Efficacy of artemether-lumefantrine, the nationally-recommended artemisinin combination for the treatment of uncomplicated falciparum malaria, in southern Laos.(2012-06-8) Mayfong Mayxay; Maniphone Khanthavong; Odai Chanthongthip; Mallika Imwong; มัลลิกา อิ่มวงศ์; Tiengkham Pongvongsa; Bouasy Hongvanthong; Samalane Phompida; Viengxay Vanisaveth; White, Nicholas J.; Newton, Paul N; Mayfong Mayxay; Mahidol University. Faculty of Tropical Medicine. Department of Molecular Tropical Medicine and GeneticsBACKGROUND: The Lao Government changed the national policy for uncomplicated Plasmodium falciparum malaria from chloroquine to artemether-lumefantrine (AL) in 2005. Since then, no information on AL efficacy has been reported. With evidence of resistance to artemisinin derivatives in adjacent Cambodia, there has been a concern as to AL efficacy. Monitoring of AL efficacy would help the Lao Government to make decisions on appropriate malaria treatment. METHODS: The efficacy of a three-day, twice daily oral artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in Xepon District, Savannakhet Province, southern Laos was studied over 42 days follow-up. This was part of a trial of thiamin supplementation in falciparum malaria. RESULTS: Of 630 patients with P. falciparum enrolled in the trial of thiamin treatment, 549 (87%, 357 children ≤15 years and 192 adults) were included in this study. The per protocol 42-day cure rates were 97% (524/541) [96% (337/352) for children and 99% (187/189) for adults, p = 0.042]. By conventional intention-to-treat analysis, the 42-day cure rates adjusted for re-infection, were 97% (532/549) [96% (342/357) in children and 99% (190/192) in adults, p = 0.042]. The proportion of patients who remained parasitaemic at day 1 after treatment was significantly higher in children [33% (116/356)] compared to adults [15% (28/192)] (p < 0.001) and only one adult patient had detectable parasitaemia on day 2. There were no serious adverse events. Potential side effects after treatment were reported more commonly in adults (32%) compared to children (15%) (p < 0.001). Patients with recrudescent infections were significantly younger, had longer mean time to fever clearance, and had longer median time to parasite clearance compared to those who were cured. CONCLUSIONS: The current nationally-recommended anti-malarial treatment (artemether-lumefantrine) remains highly efficacious for the treatment of uncomplicated falciparum malaria five years after introduction in Laos. Regular monitoring is required in case artemisinin-resistant P. falciparum parasites should appear.Publication Open Access A review of mixed malaria species infections in anopheline mosquitoes(2011-08-31) Mallika Imwong; มัลลิกา อิ่มวงศ์; Supatchara Nakeesathit; Day, Nicholas P.J.; White, Nicholas J.; White, Nicholas J.; Mahidol University. Faculty of Tropical Medicine. Department of Molecular Tropical Medicine and Genetics.; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit (MORU)BACKGROUND: In patients with malaria mixed species infections are common and under reported. In PCR studies conducted in Asia mixed infection rates often exceed 20%. In South-East Asia, approximately one third of patients treated for falciparum malaria experience a subsequent Plasmodium vivax infection with a time interval suggesting relapse. It is uncertain whether the two infections are acquired simultaneously or separately. To determine whether mixed species infections in humans are derived from mainly from simultaneous or separate mosquito inoculations the literature on malaria species infection in wild captured anopheline mosquitoes was reviewed. METHODS: The biomedical literature was searched for studies of malaria infection and species identification in trapped wild mosquitoes and artificially infected mosquitoes. The study location and year, collection methods, mosquito species, number of specimens, parasite stage examined (oocysts or sporozoites), and the methods of parasite detection and speciation were tabulated. The entomological results in South East Asia were compared with mixed infection rates documented in patients in clinical studies. RESULTS: In total 63 studies were identified. Individual anopheline mosquitoes were examined for different malaria species in 28 of these. There were 14 studies from Africa; four with species evaluations in individual captured mosquitoes (SEICM). One study, from Ghana, identified a single mixed infection. No mixed infections were identified in Central and South America (seven studies, two SEICM). 42 studies were conducted in Asia and Oceania (11 from Thailand; 27 SEICM). The proportion of anophelines infected with Plasmodium falciparum parasites only was 0.51% (95% CI: 0.44 to 0.57%), for P. vivax only was 0.26% (95% CI: 0.21 to 0.30%), and for mixed P. falciparum and P. vivax infections was 0.036% (95% CI: 0.016 to 0.056%). The proportion of mixed infections in mosquitoes was significantly higher than expected by chance (P < 0.001), but was one fifth of that sufficient to explain the high rates of clinical mixed infections by simultaneous inoculation. CONCLUSIONS: There are relatively few data on mixed infection rates in mosquitoes from Africa. Mixed species malaria infections may be acquired by simultaneous inoculation of sporozoites from multiply infected anopheline mosquitoes but this is relatively unusual. In South East Asia, where P. vivax infection follows P. falciparum malaria in one third of cases, the available entomological information suggests that the majority of these mixed species malaria infections are acquired from separate inoculations.Publication Open Access Investigations on anopheline mosquitoes close to the nest sites of chimpanzees subject to malaria infection in Ugandan highlands(2012-04-17) Krief, Sabrina; Levrero, Florence; Krief, Jean-Michel; Supinya Thanapongpichat; สุภิญญา ธนาพงษ์ภิชาติ; Mallika Imwong; มัลลิกา อิ่มวงศ์; Snounou, Georges; Kasenene, John M.; Cibot, Marie; Gantier, Jean-Charles; Krief, Sabrina; Mahidol University. Faculty of Tropical MedicineBACKGROUND: Malaria parasites (Plasmodium sp.), including new species, have recently been discovered as low grade mixed infections in three wild chimpanzees (Pan troglodytes schweinfurthii) sampled randomly in Kibale National Park, Uganda. This suggested a high prevalence of malaria infection in this community. The clinical course of malaria in chimpanzees and the species of the vectors that transmit their parasites are not known. The fact that these apes display a specific behaviour in which they consume plant parts of low nutritional value but that contain compounds with anti-malarial properties suggests that the apes health might be affected by the parasite. The avoidance of the night-biting anopheline mosquitoes is another potential behavioural adaptation that would lead to a decrease in the number of infectious bites and consequently malaria. METHODS: Mosquitoes were collected over two years using suction-light traps and yeast-generated CO(2) traps at the nesting and the feeding sites of two chimpanzee communities in Kibale National Park. The species of the female Anopheles caught were then determined and the presence of Plasmodium was sought in these insects by PCR amplification. RESULTS: The mosquito catches yielded a total of 309 female Anopheles specimens, the only known vectors of malaria parasites of mammalians. These specimens belonged to 10 species, of which Anopheles implexus, Anopheles vinckei and Anopheles demeilloni dominated. Sensitive DNA amplification techniques failed to detect any Plasmodium-positive Anopheles specimens. Humidity and trap height influenced the Anopheles capture success, and there was a negative correlation between nest numbers and mosquito abundance. The anopheline mosquitoes were also less diverse and numerous in sites where chimpanzees were nesting as compared to those where they were feeding. CONCLUSIONS: These observations suggest that the sites where chimpanzees build their nests every night might be selected, at least in part, in order to minimize contact with anopheline mosquitoes, which might lead to a reduced risk in acquiring malaria infections.