Changing patterns of Plasmodium blood-stage infections in the Wosera region of Papua New Guinea monitored by light microscopy and high throughput PCR diagnosis.

In Papua New Guinea (PNG), complex patterns of malaria commonly include single and mixed infections of Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. Here, we assess recent epidemiologic characteristics of Plasmodium blood-stage infections in the Wosera region through four cross-sectional surveys (August 2001 to June 2003). Whereas previous studies performed here have relied on blood smear/light microscopy (LM) for diagnosing Plasmodium species infections, we introduce a newly developed, post-polymerase chain reaction (PCR), semi-quantitative, ligase detection reaction-fluorescent microsphere assay (LDR-FMA). A direct comparison of the two methods for > 1,100 samples showed that diagnosis was concordant for > 80% of the analyses performed for P. falciparum (PF), P. vivax (PV), and P. malariae (PM). Greater sensitivity of the LDR-FMA accounted for 75% of the discordance between diagnoses. Based on LM, the prevalence of blood-stage PF, PV, and PM infections was found to be markedly reduced compared with an early 1990s survey. In addition, there were significant shifts in age distribution of infections, with PV becoming the most common parasite in children < 4 years of age. Consistent with previous studies, prevalence of all Plasmodium species infections increased significantly in samples analyzed by the PCR-based LDR-FMA. This increase was most pronounced for PM, PO, and mixed infections and in adolescent (10-19 years) and adult age groups, suggesting that LM may lead to under-reported prevalence of less common Plasmodium species, infection complexity, and a skewed distribution of infections towards younger age groups. This study shows that the application of LDR-FMA diagnosis in large epidemiologic studies or malaria control interventions is feasible and may contribute novel insights regarding the epidemiology of malaria.

Placental malaria in women with South-East Asian ovalocytosis.

Malaria during pregnancy, which is characterized by the accumulation of infected erythrocytes in the placenta, often has severe consequences for the mother and newborn. We assessed the effect of the genetic trait South-East Asian ovalocytosis (SAO) on placental malaria in women from Papua New Guinea. In children, this trait confers protection against cerebral malaria, but not against mild malaria disease, malaria parasitemia, or severe malaria anemia. Using a case-control approach, we found that SAO women suffer from placental malaria, and SAO-infected erythrocytes can sequester in the placenta, but heavy placental infections tended to be less common in SAO than in control pregnant women. Reduced prevalence and severity of placental infection associated with SAO were observed only for primigravid women, who are the group at highest risk of suffering from severe manifestations of placental malaria. Furthermore, we found that the prevalence of the SAO trait was lower among pregnant women than among non-pregnant controls.

Artesunate suppositories versus intramuscular artemether for treatment of severe malaria in children in Papua New Guinea.

Drug treatment of severe malaria must be rapidly effective. Suppositories may be valuable for childhood malaria when circumstances prevent oral or parenteral therapy. We compared artesunate suppositories (n = 41; 8 to 16 mg/kg of body weight at 0 and 12 h and then daily) with intramuscular (i.m.) artemether (n = 38; 3.2 mg/kg at 0 h and then 1.6 mg/kg daily) in an open-label, randomized trial with children with severe Plasmodium falciparum malaria in Papua New Guinea (PNG). Parasite density and temperature were measured every 6 h for > or = 72 h. Primary endpoints included times to 50% and 90% parasite clearance (PCT50 and PCT90) and the time to per os status. In a subset of 29 patients, plasma levels of artemether, artesunate, and their common active metabolite dihydroartemisinin were measured during the first 12 h. One suppository-treated patient with multiple complications died within 2 h of admission, but the remaining 78 recovered uneventfully. Compared to the artemether-treated children, those receiving artesunate suppositories had a significantly earlier mean PCT50 (9.1 versus 13.8 h; P = 0.008) and PCT90 (15.6 versus 20.4 h; P = 0.011). Mean time to per os status was similar for each group. Plasma concentrations of primary drug plus active metabolite were significantly higher in the artesunate suppository group at 2 h postdose. The earlier initial fall in parasitemia with artesunate is clinically advantageous and mirrors higher initial plasma concentrations of active drug/metabolite. In severely ill children with malaria in PNG, artesunate suppositories were at least as effective as i.m. artemether and may, therefore, be useful in settings where parenteral therapy cannot be given.

Parasitological and clinical efficacy of standard treatment regimens against Plasmodium falciparum, P. vivax and P. malariae in Papua New Guinea.

Resistance of Plasmodium falciparum (Pf) and P. vivax (Pv) to standard antimalarials is widespread in Papua New Guinea (PNG). The objective of the study was to assess the rate of clinical treatment failure (TF) and parasite resistance to amodiaquine (AQ), chloroquine (CQ) and quinine+sulfadoxine/pyrimethamine (Q+SP) for malaria in a rural health centre of the East Sepik Province. 179 patients presenting with symptoms and signs of malaria and with Pf (144 patients), Pv (18 patients), P. malariae (Pm) (7 patients) or mixed infection (10 patients) were included. 86 were treated with AQ, 88 with CQ and 5 with Q+SP. 21/179 patients (12%) were not cured or had a recrudescence of symptoms associated with parasitaemia in the 28 days following treatment, 14% after AQ, 10% after CQ and 0% after Q+SP. The proportion of TF was higher (17%) when the analysis population included only the 108 subjects who had a complete follow-up, especially for failure with Pf following AQ treatment (26%). During the 28 days of follow-up, RII or RIII level of resistance in Pf was detected in 55% of the subjects treated with amodiaquine, 30% of those treated with chloroquine and 0% of those treated with quinine+SP. Of the Pv or Pm parasites only one Pv was found to be RII resistant to CQ in the 28-day test. In vitro resistance of Pf to CQ was higher than to AQ (50% versus 27% of 36 parasite samples that grew successfully). The level of TF and parasitological resistance to standard antimalarial drugs was lower in this area than in urban settings, where drugs are more easily available. AQ performed less well than CQ but the difference is likely to be due to the age of the users, ie, their level of immunity, AQ being the first-line drug for young children. These results provided support for the recent change in the policy for the standard treatment of uncomplicated malaria in PNG from AQ or CQ to the combination of AQ+SP or CQ+SP, a recommendation aimed at slowing down the spread of multidrug resistance.

Plasmodium falciparum: distribution of msp2 genotypes among symptomatic and asymptomatic individuals from the Wosera region of Papua New Guinea.

The merozoite surface protein 2 (MSP2) is a leading asexual-stage malaria vaccine candidate that has already proven to have an effect in phase I/IIb vaccine trials, where it was tested in combination with other antigens. Alleles of msp2 fall within two major allelic families, 3D7 and FC27. We analyzed the msp2 genotype in 306 asymptomatic and 63 symptomatic infections from the Wosera region of Papua New Guinea. The multiplicity of infection and the distribution of msp2 alleles was similar to that found in previous studies in the region, but there was no association found between FC27-type or 3D7-type forms of MSP2 and clinical malaria.

Safety and therapeutic efficacy of artesunate suppositories for treatment of malaria in children in Papua New Guinea.

BACKGROUND: Although suppositories of artemisinin derivatives may be a valuable option for treatment of malaria in children when circumstances prevent oral and parenteral therapy, few confirmatory data have been published. METHODS: We assessed the safety and efficacy of rectal artesunate in 47 children ages 5 to 10 years with uncomplicated malaria acquired in a hyperendemic area of Papua New Guinea. Thirty were symptomatic and had Plasmodium falciparum parasitemia >2000/microl (Group 1), 12 had and either a parasitemia <2000/microl or minimal/no symptoms (Group 2) and 5 had Plasmodium vivax (Group 3). Each child received rectal artesunate 10 to 15 mg/kg at 0 and 12 h. After monitoring for 24 h, chloroquine plus sulfadoxine/pyrimethamine was given, and the patient discharged. RESULTS: Artesunate suppositories were well-tolerated. After 24 h only one child (from Group 1) had persistent parasitemia, and only one (from Group 3) had not defervesced. These two children received intramuscular quinine and recovered uneventfully. Three Group 2 children redeveloped fever and tachycardia at 24 h, but each responded to simple supportive measures and remained aparasitemic. CONCLUSIONS: Intrarectal artesunate is safe, effective initial treatment for uncomplicated malaria in children. A transient fever spike can sometimes occur after parasite clearance. We recommend that children with uncomplicated malaria receive two doses of > or =10 mg/kg rectal artesunate within the first 24 h.

Antibodies to Plasmodium falciparum glycosylphosphatidylinositols: inverse association with tolerance of parasitemia in Papua New Guinean children and adults.

Individuals living in regions of intense malaria transmission exhibit natural immunity that facilitates persistence of parasitemia at controlled densities for much of the time without symptoms. This aspect of immunity has been referred to as malarial "tolerance" and is thought to partly involve inhibition of the chain of events initiated by a parasite toxin(s) that may otherwise result in cytokine release and symptoms such as fever. Antibodies to the candidate Plasmodium falciparum glycosylphosphatidylinositol (GPI) toxin have been viewed as likely mediators of such tolerance. In this study, the relationship between antibodies to P. falciparum GPIs, age, and parasitemia was determined in asymptomatic children and adults living in Madang, Papua New Guinea. The prevalence and intensity of antibody responses increased with age and were lowest in children 1 to 4 years old with the highest-density parasitemias. In children of this age group who were tolerant of parasitemia during the study, only 8.3% had detectable immunoglobulin G (IgG) and none had IgM antibodies to GPI. This suggests that anti-GPI antibodies are unlikely to be the sole mediator of malarial tolerance, especially in children younger than 5 years. Following antimalarial treatment, clearance of parasitemia led to a fall in anti-GPI IgG response in children and adolescents within 6 weeks. As anti-GPI antibodies potentially play a role in protecting against disease progression, our results caution against the treatment of asymptomatic parasitemia and suggest that generation of a sustained antibody response in children poses a challenge to novel antitoxic vaccination strategies.

A recombinant blood-stage malaria vaccine reduces Plasmodium falciparum density and exerts selective pressure on parasite populations in a phase 1-2b trial in Papua New Guinea.

The malaria vaccine Combination B comprises recombinant Plasmodium falciparum ring-infected erythrocyte surface antigen and 2 merozoite surface proteins (MSP1 and MSP2) formulated in oil-based adjuvant. A phase 1-2b double-blind, randomized, placebo-controlled trial in 120 children (5-9 years old) in Papua New Guinea demonstrated a 62% (95% confidence limits: 13%, 84%) reduction in parasite density in children not pretreated with sulfadoxine-pyrimethamine. Vaccinees had a lower prevalence of parasites carrying the MSP2-3D7 allelic form (corresponding to that in the vaccine) and a higher incidence of morbid episodes associated with FC27-type parasites. These results demonstrate functional activity of Combination B against P. falciparum in individuals with previous malaria exposure. The specific effects on parasites with particular msp2 genotypes suggest that the MSP2 component, at least in part, accounted for the activity. The vaccine-induced selection pressure exerted on the parasites and its consequences for morbidity strongly argue for developing vaccines comprising conserved antigens and/or multiple components covering all important allelic types.

Malaria infections are randomly distributed in diverse holoendemic areas of Papua New Guinea.

Malaria is holoendemic in the lowlands of Papua New Guinea (PNG), and interactions among Plasmodium species may influence prevalence of mixed infections. Previously, field samples from a cross-sectional survey in Dreikikir, East Sepik Province, analyzed by blood smear and polymerase chain reaction (PCR), showed that mixed infections were common and randomly distributed in this malaria endemic region. To evaluate further whether Plasmodium species distribution is random, blood smear- and PCR/sequence-specific oligonucleotide probe hybridization-based analyses of cross-sectional survey samples were conducted in 2 additional malaria holoendemic regions of northern PNG. Despite ecologic, species prevalence, and transmission season differences in these new surveys, all 4 Plasmodium species were found to be randomly distributed in each area; random distribution patterns also were observed when study populations were divided into age groups. These findings provide consistent evidence that Plasmodium species infections occur independently of one another in PNG malaria holoendemic sites. This independent occurrence suggests that age-dependent, acquired malaria immunity has limited influence on the distribution pattern of Plasmodium species infections in endemic human populations; infection by 1 human malaria parasite species does not reduce susceptibility to infection by others; and malaria vaccines would exhibit limited protection against blood-stage infection by heterologous Plasmodium species.