Development and evaluation of a multiplex quantitative real-time Polymerase Chain Reaction for hookworm species in human stool

Hookworm disease caused by; Necator americanus; ,; Ancylostoma duodenale; , and; Ancylostoma ceylanicum; affects half a billion people worldwide. The prevalence and intensity of infection of individual hookworm species are vital for assessing morbidity and generating targeted intervention programs for their control. The present study aims to evaluate a multiplex real-time quantitative PCR (qPCR) assay to determine the prevalence and egg intensity of all three hookworm species and compare this with standard microscopy and published genus-based conventional and real-time multiplex qPCRs. Performance of the diagnostic assays was evaluated using DNA extracted from 192 fecal samples collected as part of a soil-transmitted helminth (STH) survey in northern Cambodia. The prevalence of hookworms as detected by the multiplex hookworm qPCR of 84/192 (43.8%) was significantly higher than that using microscopy of 49/192 (25.5%). The hookworm multiplex qPCR showed very good agreement for the detection of both; N. americanus; (Kappa 0.943) and; Ancylostoma; spp. (Kappa 0.936) with a multiplex STH qPCR. A strong and moderate quantitative correlation between cycle threshold and eggs per gram (EPG) feces was obtained for the hookworm qPCR for seeded DNA egg extracts (; R; 2; ≥ 0.9004) and naturally egg-infected individuals (; R; 2; = 0.6848), respectively. The newly developed hookworm quantitative multiplex qPCR has the potential for application in anthelmintic efficacy trials and for monitoring the success of mass deworming programs targeting individual species of anthroponotic and zoonotic hookworms

Cluster-randomized trial of monthly malaria prophylaxis versus focused screening and treatment: a study protocol to define malaria elimination strategies in Cambodia

Abstract Background Malaria remains a critical public health problem in Southeast Asia despite intensive containment efforts. The continued spread of multi-drug-resistant Plasmodium falciparum has led to calls for malaria elimination on the Thai-Cambodian border. However, the optimal approach to elimination in difficult-to-reach border populations, such as the Military, remains unclear. Methods/design A two-arm, cluster-randomized controlled, open-label pilot study is being conducted in military personnel and their families at focal endemic areas on the Thai-Cambodian border. The primary objective is to compare the effectiveness of monthly malaria prophylaxis (MMP) with dihydroartemisinin-piperaquine and weekly primaquine for 12 weeks compared with focused screening and treating (FSAT) following current Cambodian national treatment guidelines. Eight separate military encampments, making up approximately 1000 military personnel and their families, undergo randomization to the MMP or FSAT intervention for 3 months, with an additional 3 months’ follow-up. In addition, each treatment cluster of military personnel and civilians is also randomly assigned to receive either permethrin- or sham (water)-treated clothing in single-blind fashion. The primary endpoint is risk reduction for malaria infection in geographically distinct military encampments based on their treatment strategy. Monthly malaria screening in both arms is done via microscopy, PCR, and rapid diagnostic testing to compare both the accuracy and cost-effectiveness of diagnostic modalities to detect asymptomatic infection. Universal glucose-6-phosphate dehydrogenase (G6PD) deficiency screening is done at entry, comparing the results from a commercially available rapid diagnostic test, the fluorescence spot test, and quantitative testing for accuracy and cost-effectiveness. The comparative safety of the interventions chosen is also being evaluated. Discussion Despite the apparent urgency, the key operational elements of proposed malaria elimination strategies in Southeast Asian mobile and migrant populations, including the Military, have yet to be rigorously tested in a well-controlled clinical study. Here, we present a protocol for the primary evaluation of two treatment paradigms – monthly malaria prophylaxis and focused screening and treatment – to achieve malaria elimination in a Cambodian military population. We will also assess the feasibility and incremental benefit of outdoor-biting vector intervention – permethrin-treated clothing. In the process, we aim to define the cost-effectiveness of the inputs required for success including a responsive information system, skilled human resource and laboratory infrastructure requirements, and quality management. Despite being a relatively low transmission area, the complexities of multi-drug-resistant malaria and the movement of vulnerable populations require an approach that is not only technically sound, but simple enough to be achievable. Trial registration ClinicalTrials.gov, ID: NCT02653898 . Registered on 13 January 2016

Dynamics of G6PD activity in patients receiving weekly primaquine for therapy of Plasmodium vivax malaria

International audienceBackground Acute Plasmodium vivax malaria is associated with haemolysis, bone marrow suppression, reticulocytopenia, and post-treatment reticulocytosis leading to haemoglobin recovery. Little is known how malaria affects glucose-6-phosphate dehydrogenase (G6PD) activity and whether changes in activity when patients present may lead qualitative tests, like the fluorescent spot test (FST), to misdiagnose G6PD deficient (G6PDd) patients as G6PD normal (G6PDn). Giving primaquine or tafenoquine to such patients could result in severe haemolysis.Methods We investigated the G6PD genotype, G6PD enzyme activity over time and the baseline FST phenotype in Cambodians with acute P. vivax malaria treated with 3-day dihydroartemisinin piperaquine and weekly primaquine, 0.75 mg/kg x8 doses.Results: Of 75 recruited patients (males 63), aged 5–63 years (median 24), 15 were G6PDd males (14 Viangchan, 1 Canton), 3 were G6PD Viangchan heterozygous females, and 57 were G6PDn; 6 patients had α/β-thalassaemia and 26 had HbE.Median (range) Day0 G6PD activities were 0·85 U/g Hb (0·10–1·36) and 11·4 U/g Hb (6·67–16·78) in G6PDd and G6PDn patients, respectively, rising significantly to 1·45 (0·36–5·54, p<0.01) and 12·0 (8·1–17·4, p = 0.04) U/g Hb on Day7, then falling to ~Day0 values by Day56. Day0 G6PD activity did not correlate (p = 0.28) with the Day0 reticulocyte counts but both correlated over time. The FST diagnosed correctly 17/18 G6PDd patients, misclassifying one heterozygous female as G6PDn.Conclusions: In Cambodia, acute P. vivax malaria did not elevate G6PD activities in our small sample of G6PDd patients to levels that would result in a false normal qualitative test. Low G6PDd enzyme activity at disease presentation increases upon parasite clearance, parallel to reticulocytosis. More work is needed in G6PDd heterozygous females to ascertain the effect of P. vivax on their G6PD activities

Tolerability and safety of weekly primaquine against relapse of \u3ci\u3ePlasmodium vivax\u3c/i\u3e in Cambodians with glucose-6-phosphate dehydrogenase deficiency

Background: Primaquine is used to prevent Plasmodium vivax relapse; however, it is not implemented in many malaria-endemic countries, including Cambodia, for fear of precipitating primaquine-induced acute haemolytic anaemia in patients with glucose-6-phosphate dehydrogenase deficiency (G6PDd). Reluctance to use primaquine is reinforced by a lack of quality safety data. This study was conducted to assess the tolerability of a primaquine regimen in Cambodian severely deficient G6PD variants to ascertain whether a weekly primaquine could be given without testing for G6PDd. Methods: From January 2013 to January 2014, Cambodians with acute vivax malaria were treated with dihydroartemisinin/piperaquine on days (D) 0, 1 and 2 with weekly doses of primaquine 0.75 mg/kg for 8 weeks (starting on D0, last dose on D49), and followed until D56. Participants’ G6PD status was confirmed by G6PD genotype and measured G6PD activity. The primary outcome was treatment completion without primaquine toxicity defined as any one of: (1) severe anaemia (haemoglobin [Hb]/dL), (2) a \u3e25 % fractional fall in Hb from D0, (3) the need for a blood transfusion, (4) haemoglobinuria, (5) acute kidney injury (an increase in baseline serum creatinine \u3e50 %) or (6) methaemoglobinaemia \u3e20 %. Results: We enrolled 75 patients with a median age of 24 years (range 5–63); 63 patients (84 %) were male. Eighteen patients were G6PDd (17/18 had the Viangchan variant) and had D0 G6PD activity ranging from 0.1 to 1.5 U/g Hb (median 0.85 U/g Hb). In the 57 patients with normal G6PD (G6PDn), D0 G6PD activity ranged from 6.9 to 18.5 U/g Hb (median 12 U/g Hb). Median D0 Hb concentrations were similar (P = 0.46) between G6PDd (13 g/dL, range 9.6–16) and G6PDn (13.5 g/dL, range 9–16.3) and reached a nadir on D2 in both groups: 10.8 g/dL (8.2–15.3) versus 12.4 g/dL (8.8–15.2) (P = 0.006), respectively. By D7, five G6PDd patients (27.7 %) had a \u3e25 % fall in Hb, compared to 0 G6PDn patients (P = 0.00049). One of these G6PDd patients required a blood transfusion (D0–D5 Hb, 10.0–7.2 g/dL). No patients developed severe anaemia, haemoglobinuria, a methaemoglobin concentration \u3e4.9 %, or acute kidney injury

Cluster-randomized trial of monthly malaria prophylaxis versus focused screening and treatment: a study protocol to define malaria elimination strategies in Cambodia

Abstract Background Malaria remains a critical public health problem in Southeast Asia despite intensive containment efforts. The continued spread of multi-drug-resistant Plasmodium falciparum has led to calls for malaria elimination on the Thai-Cambodian border. However, the optimal approach to elimination in difficult-to-reach border populations, such as the Military, remains unclear. Methods/design A two-arm, cluster-randomized controlled, open-label pilot study is being conducted in military personnel and their families at focal endemic areas on the Thai-Cambodian border. The primary objective is to compare the effectiveness of monthly malaria prophylaxis (MMP) with dihydroartemisinin-piperaquine and weekly primaquine for 12 weeks compared with focused screening and treating (FSAT) following current Cambodian national treatment guidelines. Eight separate military encampments, making up approximately 1000 military personnel and their families, undergo randomization to the MMP or FSAT intervention for 3 months, with an additional 3 months’ follow-up. In addition, each treatment cluster of military personnel and civilians is also randomly assigned to receive either permethrin- or sham (water)-treated clothing in single-blind fashion. The primary endpoint is risk reduction for malaria infection in geographically distinct military encampments based on their treatment strategy. Monthly malaria screening in both arms is done via microscopy, PCR, and rapid diagnostic testing to compare both the accuracy and cost-effectiveness of diagnostic modalities to detect asymptomatic infection. Universal glucose-6-phosphate dehydrogenase (G6PD) deficiency screening is done at entry, comparing the results from a commercially available rapid diagnostic test, the fluorescence spot test, and quantitative testing for accuracy and cost-effectiveness. The comparative safety of the interventions chosen is also being evaluated. Discussion Despite the apparent urgency, the key operational elements of proposed malaria elimination strategies in Southeast Asian mobile and migrant populations, including the Military, have yet to be rigorously tested in a well-controlled clinical study. Here, we present a protocol for the primary evaluation of two treatment paradigms – monthly malaria prophylaxis and focused screening and treatment – to achieve malaria elimination in a Cambodian military population. We will also assess the feasibility and incremental benefit of outdoor-biting vector intervention – permethrin-treated clothing. In the process, we aim to define the cost-effectiveness of the inputs required for success including a responsive information system, skilled human resource and laboratory infrastructure requirements, and quality management. Despite being a relatively low transmission area, the complexities of multi-drug-resistant malaria and the movement of vulnerable populations require an approach that is not only technically sound, but simple enough to be achievable. Trial registration ClinicalTrials.gov, ID: NCT02653898. Registered on 13 January 2016

Hemolytic Dynamics of Weekly Primaquine Antirelapse Therapy Among Cambodians With Acute Plasmodium vivax Malaria With or Without Glucose-6-Phosphate Dehydrogenase Deficiency

International audienceBackground: Hemoglobin (Hb) data are limited in Southeast Asian glucose-6-phosphate dehydrogenase (G6PD) deficient (G6PD−) patients treated weekly with the World Health Organization–recommended primaquine regimen (ie, 0.75 mg/kg/week for 8 weeks [PQ 0.75]).Methods: We treated Cambodians who had acute Plasmodium vivax infection with PQ0.75 and a 3-day course of dihydroartemisinin/piperaquine and determined the Hb level, reticulocyte count, G6PD genotype, and Hb type.Results: Seventy-five patients (male sex, 63) aged 5–63 years (median, 24 years) were enrolled. Eighteen were G6PD deficient (including 17 with G6PD Viangchan) and 57 were not G6PD deficient; 26 had HbE (of whom 25 were heterozygous), and 6 had α-/β-thalassemia. Mean Hb concentrations at baseline (ie, day 0) were similar between G6PD deficient and G6PD normal patients (12.9 g/dL [range, 9‒16.3 g/dL] and 13.26 g/dL [range, 9.6‒16 g/dL], respectively; P = .46). G6PD deficiency (P = <.001), higher Hb concentration at baseline (P = <.001), higher parasitemia level at baseline (P = .02), and thalassemia (P = .027) influenced the initial decrease in Hb level, calculated as the nadir level minus the baseline level (range, −5.8–0 g/dL; mean, −1.88 g/dL). By day 14, the mean difference from the day 7 level (calculated as the day 14 level minus the day 7 level) was 0.03 g/dL (range, −0.25‒0.32 g/dL). Reticulocyte counts decreased from days 1 to 3, peaking on day 7 (in the G6PD normal group) and day 14 (in the G6PD deficient group); reticulocytemia at baseline (P = .001), G6PD deficiency (P = <.001), and female sex (P = .034) correlated with higher counts. One symptomatic, G6PD-deficient, anemic male patient was transfused on day 4.Conclusions: The first PQ0.75 exposure was associated with the greatest decrease in Hb level and 1 blood transfusion, followed by clinically insignificant decreases in Hb levels. PQ0.75 requires monitoring during the week after treatment. Safer antirelapse regimens are needed in Southeast Asia

An optimised age-based dosing regimen for single low-dose primaquine for blocking malaria transmission in Cambodia

BACKGROUND: In 2012, the World Health Organization recommended the addition of single low-dose primaquine (SLDPQ, 0.25 mg base/kg body weight) to artemisinin combination therapies to block the transmission of Plasmodium falciparum without testing for glucose-6-phosphate dehydrogenase deficiency. The targeted group was non-pregnant patients aged ≥ 1 year (later changed to ≥ 6 months) with acute uncomplicated falciparum malaria, primarily in countries with artemisinin-resistant P. falciparum (ARPf). No dosing regimen was suggested, leaving malaria control programmes and clinicians in limbo. Therefore, we designed a user-friendly, age-based SLDPQ regimen for Cambodia, the country most affected by ARPf. METHODS: By reviewing primaquine's pharmacology, we defined a therapeutic dose range of 0.15-0.38 mg base/kg (9-22.5 mg in a 60-kg adult) for a therapeutic index of 2.5. Primaquine doses (1-20 mg) were tested using a modelled, anthropometric database of 28,138 Cambodian individuals (22,772 healthy, 4119 with malaria and 1247 with other infections); age distributions were: 0.5-4 years (20.0 %, n = 5640), 5-12 years (9.1 %, n = 2559), 13-17 years (9.1 %, n = 2550), and ≥ 18 years (61.8 %, n = 17,389). Optimal age-dosing groups were selected according to calculated mg base/kg doses and proportions of individuals receiving a therapeutic dose. RESULTS: Four age-dosing bands were defined: (1) 0.5-4 years, (2) 5-9 years, (3) 10-14 years, and (4) ≥15 years to receive 2.5, 5, 7.5, and 15 mg of primaquine base, resulting in therapeutic doses in 97.4 % (5494/5640), 90.5 % (1511/1669), 97.7 % (1473/1508), and 95.7 % (18,489/19,321) of individuals, respectively. Corresponding median (1st-99th centiles) mg base/kg doses of primaquine were (1) 0.23 (0.15-0.38), (2) 0.29 (0.18-0.45), (3) 0.27 (0.15-0.39), and (4) 0.29 (0.20-0.42). CONCLUSIONS: This age-based SLDPQ regimen could contribute substantially to malaria elimination and requires urgent evaluation in Cambodia and other countries with similar anthropometric characteristics. It guides primaquine manufacturers on suitable tablet strengths and doses for paediatric-friendly formulations. Development of similar age-based dosing recommendations for Africa is needed