Inge Sutanto

Improving the radical cure of vivax malaria (IMPROV): a study protocol for a multicentre randomised, placebo-controlled comparison of short and long course primaquine regimens

Plasmodium vivax malaria is a major cause of morbidity and recognised as an important contributor to mortality in some endemic areas. The current recommended treatment regimen for the radical cure of P. vivax includes a schizontocidal antimalarial, usually chloroquine, combined with a 14 day regimen of primaquine. The long treatment course frequently results in poor adherence and effectiveness. Shorter courses of higher daily doses of primaquine have the potential to improve adherence and thus effectiveness without compromising safety. The proposed multicentre randomised clinical trial aims to provide evidence across a variety of endemic settings on the safety and efficacy of high dose short course primaquine in glucose-6-phosphate-dehydrogenase (G6PD) normal patients. This study is designed as a placebo controlled, double blinded, randomized trial in four countries: Indonesia, Vietnam, Afghanistan and Ethiopia. G6PD normal patients diagnosed with vivax malaria are randomized to receive either 7 or 14 days high dose primaquine or placebo. G6PD deficient (G6PDd) patients are allocated to weekly primaquine doses for 8 weeks. All treatment is directly observed and recurrent episodes are treated with the same treatment than allocated at the enrolment episode. Patients are followed daily until completion of treatment, weekly until 8 weeks and then monthly until 1 year after initiation of the treatment. The primary endpoint is the incidence rate (per person year) of symptomatic recurrent P. vivax parasitaemia over 12 months of follow-up, for all individuals, controlling for site, comparing the 7 versus 14-day primaquine treatment arms. Secondary endpoints are other efficacy measures such as incidence risk at different time points. Further endpoints are risks of haemolysis and severe adverse events. This study has been approved by relevant institutional ethics committees in the UK and Australia, and all participating countries. Results will be disseminated to inform P. vivax malaria treatment policy through peer-reviewed publications and academic presentations. Findings will contribute to a better understanding of the risks and benefits of primaquine which is crucial in persuading policy makers as well as clinicians of the importance of radical cure of vivax malaria, contributing to decreased transmission and a reduce parasite reservoir. ClinicalTrials.gov Identifier: NCT01814683. Registered March 18, 2013

Does treatment of intestinal helminth infections influence malaria? Background and methodology of a longitudinal study of clinical, parasitological and immunological parameters in Nangapanda, Flores, Indonesia (ImmunoSPIN Study)

BackgroundGiven that helminth infections are thought to have strong immunomodulatory activity, the question whether helminth infections might affect responses to malaria antigens needs to be addressed. Different cross-sectional studies using diverse methodologies have reported that helminth infections might either exacerbate or reduce the severity of malaria attacks. The same discrepancies have been reported for parasitemia.Methods/DesignTo determine the effect of geohelminth infections and their treatment on malaria infection and disease outcome, as well as on immunological parameters, the area of Nangapanda on Flores Island, Indonesia, where malaria and helminth parasites are co-endemic was selected for a longitudinal study. Here a Double-blind randomized trial will be performed, incorporating repeated treatment with albendazole (400 mg) or placebo at three monthly intervals. Household characteristic data, anthropometry, the presence of intestinal helminth and Plasmodium spp infections, and the incidence of malaria episodes are recorded. In vitro cultures of whole blood, stimulated with a number of antigens, mitogens and toll like receptor ligands provide relevant immunological parameters at baseline and following 1 and 2 years of treatment rounds. The primary outcome of the study is the prevalence of Plasmodium falciparum and P. vivax infection. The secondary outcome will be incidence and severity of malaria episodes detected via both passive and active follow-up. The tertiary outcome is the inflammatory cytokine profile in response to parasite antigens. The project also facilitates the transfer of state of the art methodologies and technologies, molecular diagnosis of parasitic diseases, immunology and epidemiology from Europe to Indonesia.DiscussionThe study will provide data on the effect of helminth infections on malaria. It will also give information on anthelminthic treatment efficacy and effectiveness and could help develop evidence-based policymaking.Trial registrationThis study was approved by The Ethical Committee of Faculty of Medicine, University of Indonesia, ref:194/PT02.FK/Etik/2006 and has been filed by ethics committee of the Leiden University Medical Center. Clinical trial number:ISRCTN83830814. The study is reported in accordance with the CONSORT guidelines for cluster-randomized studies.

Identification of the Plasmodium vivax mdr-Like Gene (pvmdr1) and Analysis of Single-Nucleotide Polymorphisms among Isolates from Different Areas of Endemicity

Because of the lack of methods for continuous in vitro culture of Plasmodium vivax, little is known about drug-resistance mechanisms in this malaria-causing parasite. Therefore, identification of all the genes potentially involved in drug resistance and of molecular markers related to drug resistance would provide a framework for studying the incidence and spread of drug-resistant P. vivax strains. We have identified the P. vivax orthologue of the pfmdr1 gene (pvmdr1), which was shown to have a role in the drug resistance of Plasmodium falciparum. Comparison of the alignments of both nucleotide and amino acid sequences of pvmdr1 with those of other Plasmodium multidrug-resistance genes revealed an open-reading frame of 4392 base pairs encoding a deduced protein of 1464 amino acids. Nucleotide polymorphisms at 2 codons of the pvmdr1 gene--Y976F and F1076L--were found in 14 of 23 P. vivax isolates from different areas of endemicity, including Thailand, Indonesia, Turkey, Azerbaijan, and French Guyana.

Contrasting Transmission Dynamics of Co-endemic Plasmodium vivax and P. falciparum: Implications for Malaria Control and Elimination.

Background Outside of Africa, P. falciparum and P. vivax usually coexist. In such co-endemic regions, successful malaria control programs have a greater impact on reducing falciparum malaria, resulting in P. vivax becoming the predominant species of infection. Adding to the challenges of elimination, the dormant liver stage complicates efforts to monitor the impact of ongoing interventions against P. vivax. We investigated molecular approaches to inform the respective transmission dynamics of P. falciparum and P. vivax and how these could help to prioritize public health interventions. Methodology/ Principal Findings Genotype data generated at 8 and 9 microsatellite loci were analysed in 168 P. falciparum and 166 P. vivax isolates, respectively, from four co-endemic sites in Indonesia (Bangka, Kalimantan, Sumba and West Timor). Measures of diversity, linkage disequilibrium (LD) and population structure were used to gauge the transmission dynamics of each species in each setting. Marked differences were observed in the diversity and population structure of P. vivax versus P. falciparum. In Bangka, Kalimantan and Timor, P. falciparum diversity was low, and LD patterns were consistent with unstable, epidemic transmission, amenable to targeted intervention. In contrast, P. vivax diversity was higher and transmission appeared more stable. Population differentiation was lower in P. vivax versus P. falciparum, suggesting that the hypnozoite reservoir might play an important role in sustaining local transmission and facilitating the spread of P. vivax infections in different endemic settings. P. vivax polyclonality varied with local endemicity, demonstrating potential utility in informing on transmission intensity in this species. Conclusions/ Significance Molecular approaches can provide important information on malaria transmission that is not readily available from traditional epidemiological measures. Elucidation of the transmission dynamics circulating in a given setting will have a major role in prioritising malaria control strategies, particularly against the relatively neglected non-falciparum species.

Randomized, Open Label Trial of Primaquine Against Vivax Malaria Relapse in Indonesia

ABSTRACT Radical cure of Plasmodium vivax infection applies blood schizontocidal therapy against the acute attack and hypnozoitocidal therapy against later relapse. Chloroquine and primaquine have been used for 60 years in this manner. Resistance to chloroquine by the parasite now requires partnering other blood schizontocides with primaquine. However, the safety and efficacy of primaquine against relapse when combined with other drugs have not been demonstrated. This randomized, open-label, and relapse-controlled trial estimated the efficacy of primaquine against relapse when administered with quinine or dihydroartemisinin-piperaquine for treatment of the acute infection. Among 650 soldiers who had returned to their malaria-free base in Java, Indonesia, after 12 months in malarious Papua, Indonesia, 143 with acute P. vivax malaria were eligible for study. One hundred sixteen enrolled subjects were randomized to these treatments: artesunate (200-mg dose followed by 100 mg/day for 6 days), quinine (1.8 g/day for 7 days) plus concurrent primaquine (30 mg/day for 14 days), or dihydroartemisinin (120 mg) plus piperaquine (960 mg) daily for 3 days followed 25 days later by primaquine (30 mg/day for 14 days). Follow-up was for 12 months. One hundred thirteen subjects were analyzable. Relapse occurred in 32 of 41 (78%) subjects administered artesunate alone (2.71 attacks/person-year), 7 of 36 (19%) administered quinine plus primaquine (0.23 attack/person-year), and 2 of 36 (6%) administered dihydroartemisinin-piperaquine plus primaquine (0.06 attack/person-year). The efficacy of primaquine against relapse was 92% (95% confidence interval [CI] = 81% to 96%) for quinine plus primaquine and 98% (95% CI = 91% to 99%) for dihydroartemisinin-piperaquine plus primaquine. Antirelapse therapy with primaquine begun a month after treatment of the acute attack with dihydroartemisinin-piperaquine proved safe and highly efficacious against relapse by P. vivax acquired in Papua, Indonesia.

Secreted and circulating antigens of the filarial parasite Brugia pahangi: Analysis of in vitro released components and detection of parasite products in vivo

A range of excretory-secretory (ES) antigens have been characterised following in vitro culture of adult Brugia pahangi filarial nematodes in serum-free medium. Analysis by radioiodination, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and immunoprecipitation of purified macromolecules with antibodies from human and experimental animal infections reveals both host and parasite components. Two host molecules appear by molecular weight and immunoprecipitation analysis to be immunoglobulin and serum albumin, presumed to be taken up from the jird host from which adult worms were recovered. A further prominent component, of 19 kDa, reacts with neither anti-host nor anti-filarial antibodies, and may represent a non-immunogenic parasite product. Three additional bands, although less intensely radiolabelled, did prove to be consistently antigenic, with apparent molecular weights of 15, 29 and 40 kDa. A further ES antigen, which does not take up radio-iodine or lend itself to electrophoretic analysis, has also been detected. This molecule reacts in a immunoradiometric assay in which monoclonal antibody directed against a repetitive epitope acts both to capture and indicate antigen presence. The same antibody, Bp-1, may also be employed to detect circulating antigen in the serum of animals experimentally infected with Brugia pahangi, and in the serum of patients with each of the three species of human lymphatic filariasis, Brugia malayi, Brugia timori and Wuchereria bancrofti.

Real-Time PCR for Dihydrofolate Reductase Gene Single-Nucleotide Polymorphisms in Plasmodium vivax Isolates

ABSTRACT Mutations in the dhfr gene of Plasmodium vivax (pvdhfr) are associated with resistance to the antifolate antimalarial drugs. Polymorphisms in the pvdhfr gene were assessed by hybridization probe technology on the LightCycler instrument with 134 P. vivax-infected blood samples from Turkey (n = 24), Azerbaijan (n = 39), Thailand (n = 16), Indonesia (n = 53), and travelers (n = 19). Double mutations (S58R and S117N) or quadruple mutations (F57L/I, S58R, T61M, and S117N) in the pvdhfr genes were found in all Thai samples (100%). pvdhfr mutant-type alleles were significantly more common in samples from travelers (42%) than in those from patients from Indonesia (5%). Surprisingly, the pvdhfr single-mutation allele (S117N) was identified at a high frequency in parasites from Turkey and Azerbaijan (71 and 36%, respectively), where sulfadoxine-pyrimethamine is not recommended for the treatment of P. vivax malaria by the World Health Organization and the Malaria National Programs.

The effect of primaquine on gametocyte development and clearance in the treatment of uncomplicated falciparum malaria with dihydroartemisinin-piperaquine in South Sumatra, Western Indonesia: an open label randomized controlled trial

BACKGROUND Artemisinin-based combination therapy is very effective in clearing asexual stages of malaria and reduces gametocytemia, but may not affect mature gametocytes. Primaquine is the only commercially available drug that eliminates mature gametocytes. METHODS We conducted a 2-arm, open-label, randomized, controlled trial to evaluate the efficacy of single-dose primaquine (0.75 mg/kg) following treatment with dihydroartemisinin-piperaquine (DHP) on Plasmodium falciparum gametocytemia, in Indonesia. Patients aged ≥5 years with uncomplicated falciparum malaria, normal glucose-6-phosphate dehydrogenase enzyme levels, and hemoglobin levels ≥8 g/dL were assigned by computerized-generating sequence to a standard 3-day course of DHP alone (n = 178) or DHP combined with a single dose of primaquine on day 3 (n = 171). Patients were seen on days 1, 2, 3, and 7 and then weekly for 42 days to assess the presence of gametocytes and asexual parasites by microscopy. Survival analysis was stratified by the presence of gametocytes on day 3. RESULTS DHP prevented development of gametocytes in 277 patients without gametocytes on day 3. In the gametocytemic patients (n = 72), primaquine was associated with faster gametocyte clearance (hazard ratio = 2.42 [95% confidence interval, 1.39-4.19], P = .002) and reduced gametocyte densities (P = .018). The day 42 cure rate of asexual stages in the DHP + primaquine and DHP-only arms were: polymerase chain reaction (PCR) unadjusted, 98.7% vs 99.4%, respectively; PCR adjusted, 100% for both. Primaquine was well tolerated. CONCLUSIONS Addition of single-dose 0.75 mg/kg primaquine shortens the infectivity period of DHP-treated patients and should be considered in low-transmission regions that aim to control and ultimately eliminate falciparum malaria. Clinical Trials Registration. NCT01392014.

Secreted and surface antigens from larval stages of Wuchereria bancrofti, the major human lymphatic filarial parasite

Antigenic proteins of microfilariae and infective larvae of Wuchereria bancrofti have been identified by intrinsic and extrinsic radiolabelling, and specific immunoprecipitation with sera from filarial patients. From 125I surface-labelling experiments, the most prominent antigen on both stages is of relative molecular mass (Mr) 17 000, while a molecule of similar size is both synthesized and released in vitro following labelling with [35S]methionine. A second similarity between the two stages is the production and secretion of a Mr 21 000 component, which is, however, not detected on the worm surfaces. A series of additional proteins from larval W. bancrofti are described from each parasite compartment (secreted, surface and somatic) and the antigenicity and specificity of these components explored with serum from patients with filariasis due to W. bancrofti or Brugia species, and with onchocerciasis. Among additional molecules released in vitro we have found a Mr 51 000 antigen from both stages, and also several proteins which are not recognised by antibody from human filarial patients.

The Effect of Three-Monthly Albendazole Treatment on Malarial Parasitemia and Allergy: A Household-Based Cluster-Randomized, Double-Blind, Placebo-Controlled Trial

Background Helminth infections are proposed to have immunomodulatory activities affecting health outcomes either detrimentally or beneficially. We evaluated the effects of albendazole treatment, every three months for 21 months, on STH, malarial parasitemia and allergy. Methods and Findings A household-based cluster-randomized, double-blind, placebo-controlled trial was conducted in an area in Indonesia endemic for STH. Using computer-aided block randomization, 481 households (2022 subjects) and 473 households (1982 subjects) were assigned to receive placebo and albendazole, respectively, every three months. The treatment code was concealed from trial investigators and participants. Malarial parasitemia and malaria-like symptoms were assessed in participants older than four years of age while skin prick test (SPT) to allergens as well as reported symptoms of allergy in children aged 5–15 years. The general impact of treatment on STH prevalence and body mass index (BMI) was evaluated. Primary outcomes were prevalence of malarial parasitemia and SPT to any allergen. Analysis was by intention to treat. At 9 and 21 months post-treatment 80.8% and 80.1% of the study subjects were retained, respectively. The intensive treatment regiment resulted in a reduction in the prevalence of STH by 48% in albendazole and 9% in placebo group. Albendazole treatment led to a transient increase in malarial parasitemia at 6 months post treatment (OR 4.16(1.35–12.80)) and no statistically significant increase in SPT reactivity (OR 1.18(0.74–1.86) at 9 months or 1.37 (0.93–2.01) 21 months). No effect of anthelminthic treatment was found on BMI, reported malaria-like- and allergy symptoms. No adverse effects were reported. Conclusions The study indicates that intensive community treatment of 3 monthly albendazole administration for 21 months over two years leads to a reduction in STH. This degree of reduction appears safe without any increased risk of malaria or allergies. Trial Registration Controlled-Trials.com ISRCTN83830814