The Incidence and Differential Seasonal Patterns of Plasmodium vivax Primary Infections and Relapses in a Cohort of Children in Papua New Guinea.

Plasmodium vivax has the ability to relapse from dormant parasites in the liver weeks or months after inoculation, causing further blood-stage infection and potential onward transmission. Estimates of the force of blood-stage infections arising from primary infections and relapses are important for designing intervention strategies. However, in endemic settings their relative contributions are unclear. Infections are frequently asymptomatic, many individuals harbor multiple infections, and while high-resolution genotyping of blood samples enables individual infections to be distinguished, primary infections and relapses cannot be identified. We develop a model and fit it to longitudinal genotyping data from children in Papua New Guinea to estimate the incidence and seasonality of P vivax primary infection and relapse. The children, aged one to three years at enrolment, were followed up over 16 months with routine surveys every two months. Blood samples were taken at the routine visits and at other times if the child was ill. Samples positive by microscopy or a molecular method for species detection were genotyped using high-resolution capillary electrophoresis for P vivax MS16 and msp1F3, and P falciparum msp2. The data were summarized as longitudinal patterns of success or failure to detect a genotype at each routine time-point (eg 001000001). We assume that the seasonality of P vivax primary infection is similar to that of P falciparum since they are transmitted by the same vectors and, because P falciparum does not have the ability to relapse, the seasonality can be estimated. Relapses occurring during the study period can be a consequence of infections occurring prior to the study: we assume that the seasonal pattern of primary infections repeats over time. We incorporate information from parasitological and entomology studies to gain leverage for estimating the parameters, and take imperfect detection into account. We estimate the force of P vivax primary infections to be 11.5 (10.5, 12.3) for a three-year old child per year and the mean number of relapses per infection to be 4.3 (4.0, 4.6) over 16 months. The peak incidence of relapses occurred in the two month interval following the peak interval for primary infections: the contribution to the force of blood-stage infection from relapses is between 71% and 90% depending on the season. Our estimates contribute to knowledge of the P vivax epidemiology and have implications for the timing of intervention strategies targeting different stages of the life cycle.

Estimating the numbers of malaria infections in blood samples using high-resolution genotyping data.

People living in endemic areas often habour several malaria infections at once. High-resolution genotyping can distinguish between infections by detecting the presence of different alleles at a polymorphic locus. However the number of infections may not be accurately counted since parasites from multiple infections may carry the same allele. We use simulation to determine the circumstances under which the number of observed genotypes are likely to be substantially less than the number of infections present and investigate the performance of two methods for estimating the numbers of infections from high-resolution genotyping data. The simulations suggest that the problem is not substantial in most datasets: the disparity between the mean numbers of infections and of observed genotypes was small when there was 20 or more alleles, 20 or more blood samples, a mean number of infections of 6 or less and where the frequency of the most common allele was no greater than 20%. The issue of multiple infections carrying the same allele is unlikely to be a major component of the errors in PCR-based genotyping. Simulations also showed that, with heterogeneity in allele frequencies, the observed frequencies are not a good approximation of the true allele frequencies. The first method that we proposed to estimate the numbers of infections assumes that they are a good approximation and hence did poorly in the presence of heterogeneity. In contrast, the second method by Li et al estimates both the numbers of infections and the true allele frequencies simultaneously and produced accurate estimates of the mean number of infections.

Force of infection is key to understanding the epidemiology of Plasmodium falciparum malaria in Papua New Guinean children.

Genotyping Plasmodium falciparum parasites in longitudinal studies provides a robust approach to estimating force of infection (FOI) in the presence of superinfections. The molecular parameter (mol)FOI, defined as the number of new P. falciparum clones acquired over time, describes basic malaria epidemiology and is suitable for measuring outcomes of interventions. This study was designed to test whether (mol)FOI influenced the risk of clinical malaria episodes and how far (mol)FOI reflected environmental determinants of transmission, such as seasonality and small-scale geographical variation or effects of insecticide-treated nets (ITNs). Two hundred sixty-four children 1-3 y of age from Papua New Guinea were followed over 16 mo. Individual parasite clones were tracked longitudinally by genotyping. On average, children acquired 5.9 (SD 9.6) new P. falciparum infections per child per y. (mol)FOI showed a pronounced seasonality, was strongly reduced in children using ITNs (incidence rate ratio, 0.49; 95% confidence interval, [0.38, 0.61]), increased with age, and significantly varied within villages (P = 0.001). The acquisition of new parasite clones was the major factor determining the risk of clinical illness (incidence rate ratio, 2.12; 95% confidence interval, [1.93, 2.31]). Adjusting for individual differences in (mol)FOI completely explained spatial variation, age trends, and the effect of ITN use. This study highlights the suitability of (mol)FOI as a measure of individual exposure and its central role in malaria epidemiology. It has substantial advantages over entomological measures in studies of transmission patterns, and could be used in analyses of host variation in susceptibility, in field efficacy trials of novel interventions or vaccines, and for evaluating intervention effects.

How much remains undetected? Probability of molecular detection of human Plasmodia in the field.

BACKGROUND: In malaria endemic areas, most people are simultaneously infected with different parasite clones. Detection of individual clones is hampered when their densities fluctuate around the detection limit and, in case of P. falciparum, by sequestration during part of their life cycle. This has important implications for measures of levels of infection or for the outcome of clinical trials. This study aimed at measuring the detectability of individual P. falciparum and P. vivax parasite clones in consecutive samples of the same patient and at investigating the impact of sampling strategies on basic epidemiological measures such as multiplicity of infection (MOI). METHODS: Samples were obtained in a repeated cross-sectional field survey in 1 to 4.5 years old children from Papua New Guinea, who were followed up in 2-monthly intervals over 16 months. At each follow-up visit, two consecutive blood samples were collected from each child at intervals of 24 hours. Samples were genotyped for the polymorphic markers msp2 for P. falciparum and msp1F3 and MS16 for P. vivax. Observed prevalence and mean MOI estimated from single samples per host were compared to combined data from sampling twice within 24 h. FINDINGS AND CONCLUSION: Estimated detectability was high in our data set (0.79 [95% CI 0.76-0.82] for P. falciparum and, depending on the marker, 0.61 [0.58-0.63] or 0.73 [0.71-0.75] for P. vivax). When genotyping data from sequential samples, collected 24 hours apart, were combined, the increase in measured prevalence was moderate, 6 to 9% of all infections were missed on a single day. The effect on observed MOI was more pronounced, 18 to 31% of all individual clones were not detected in a single bleed. Repeated sampling revealed little difference between detectability of P. falciparum and P. vivax.

Plasmodium falciparum msp1, msp2 and glurp allele frequency and diversity in sub-Saharan Africa.

BACKGROUND: The efficacy of anti-malarial drugs is assessed over a period of 28-63 days (depending on the drugs' residence time) following initiation of treatment in order to capture late failures. However, prolonged follow-up increases the likelihood of new infections depending on transmission intensity. Therefore, molecular genotyping of highly polymorphic regions of Plasmodium falciparum msp1, msp2 and glurp loci is usually carried out to distinguish recrudescence (true failures) from new infections. This tool has now been adopted as an integral part of anti-malarial efficacy studies and clinical trials. However, there are concerns over its utility and reliability because conclusions drawn from molecular typing depend on the genetic profile of the respective parasite populations, but this profile is not systematically documented in most endemic areas. This study presents the genetic diversity of P. falciparum msp1, msp2 and glurp markers in selected sub-Saharan Africa countries with varying levels of endemicity namely Malawi, Tanzania, Uganda, Burkina Faso and São Tomé. METHODS: A total 780 baseline (Day 0) blood samples from children less than seven years, recruited in a randomized controlled clinical trials done between 1996 and 2000 were genotyped. DNA was extracted; allelic frequency and diversity were investigated by PCR followed by capillary electrophoresis for msp2 and fragment sizing by a digitalized gel imager for msp1 and glurp. RESULTS AND CONCLUSION: Plasmodium falciparum msp1, msp2 and glurp markers were highly polymorphic with low allele frequencies. A total of 17 msp1 genotypes [eight MAD20-, one RO33- and eight K1-types]; 116 msp2 genotypes [83 3D7 and 33 FC27- types] and 14 glurp genotypes were recorded. All five sites recorded very high expected heterozygosity (HE) values (0.68 - 0.99). HE was highest in msp2 locus (HE=0.99), and lowest for msp1 (HE=0.68) (P<0.0001). The genetic diversity and allelic frequency recorded were independent of transmission intensity (P=0.84, P=0.25 respectively. A few genotypes had particularly high frequencies; however the most abundant showed only a 4% probability that a new infection would share the same genotype as the baseline infection. This is unlikely to confound the distinction of recrudescence from new infection, particularly if more than one marker is used for genotyping. Hence, this study supports the use of msp1, msp2 and glurp in malaria clinical trials in sub-Saharan Africa to discriminate new from recrudescent infections.

Treatment with coartem (artemether-lumefantrine) in Papua New Guinea.

A recent drug efficacy trial reported Coartem (artemether-lumefantrine) to be highly effective against Plasmodium falciparum in children less than 5 years of age in Papua New Guinea (PNG). In contrast, we have observed high levels of treatment failures in non-trial conditions in a longitudinal cohort study in the same age group in PNG. Recrudescences were confirmed by genotyping of three different marker genes to provide optimal discrimination power between parasite clones. After excluding genetic host factors by genotyping potentially relevant cytochrome P450 loci, the high number of treatment failures in our study is best explained by poor adherence to complex dosing regimens in combination with insufficient fat supplementation, which are both crucial parameters for the outcome of Coartem treatment. In contrast to the situation in classic drug trials with ideal treatment conditions, our field survey highlights potential problems with unsupervised usage of Coartem in routine clinical practice and under program conditions.

Comparison of Plasmodium falciparum allelic frequency distribution in different endemic settings by high-resolution genotyping.

BACKGROUND: The diversity of genotyping markers of Plasmodium falciparum depends on transmission intensity. It has been reported that the diversity of the merozoite surface protein 2 (msp2) is greater in areas of high compared to low endemicity, however, results for msp1 were inconsistent. These previous reports relied on low resolution genotyping techniques. METHODS: In the present study, a high-resolution capillary electrophoresis-based technique was applied to genotype samples from areas of different endemicity in Papua New Guinea and Tanzania. For both endemic settings, the diversity of msp1 and msp2 was investigated; the mean multiplicity of infection (MOI) and the FST values were determined to investigate whether more accurate sizing generates different results. RESULTS AND CONCLUSION: The results of the present study confirmed previous reports of a higher mean MOI for both marker genes and increased genetic diversity in areas of higher endemicity as estimated by the total number of distinct alleles for msp2. For msp1 a minor increase in diversity was observed. Measures of between population variance in allele frequencies (FST) indicated little genetic differentiation for both marker genes between the two populations from different endemic settings. MOI adjusted for the probability of multiple infections sharing the same allele was estimated by using the msp2 allele frequency distribution and the distribution of observed numbers of concurrent infections. For the high-resolution typing technique applied in this study, this adjustment made little difference to the estimated mean MOI compared to the observed mean MOI.

Immunoglobulin G subclass-specific responses against Plasmodium falciparum merozoite antigens are associated with control of parasitemia and protection from symptomatic illness.

Substantial evidence indicates that antibodies to Plasmodium falciparum merozoite antigens play a role in protection from malaria, although the precise targets and mechanisms mediating immunity remain unclear. Different malaria antigens induce distinct immunoglobulin G (IgG) subclass responses, but the importance of different responses in protective immunity from malaria is not known and the factors determining subclass responses in vivo are poorly understood. We examined IgG and IgG subclass responses to the merozoite antigens MSP1-19 (the 19-kDa C-terminal region of merozoite surface protein 1), MSP2 (merozoite surface protein 2), and AMA-1 (apical membrane antigen 1), including different polymorphic variants of these antigens, in a longitudinal cohort of children in Papua New Guinea. IgG1 and IgG3 were the predominant subclasses of antibodies to each antigen, and all antibody responses increased in association with age and exposure without evidence of increasing polarization toward one subclass. The profiles of IgG subclasses differed somewhat for different alleles of MSP2 but not for different variants of AMA-1. Individuals did not appear to have a propensity to make a specific subclass response irrespective of the antigen. Instead, data suggest that subclass responses to each antigen are generated independently among individuals and that antigen properties, rather than host factors, are the major determinants of IgG subclass responses. High levels of AMA-1-specific IgG3 and MSP1-19-specific IgG1 were strongly predictive of a reduced risk of symptomatic malaria and high-density P. falciparum infections. However, no antibody response was significantly associated with protection from parasitization per se. Our findings have major implications for understanding human immunity and for malaria vaccine development and evaluation.

Heterogeneous distribution of Plasmodium falciparum drug resistance haplotypes in subsets of the host population.

BACKGROUND: The emergence of drug resistance is a major problem in malaria control. For mathematical modelling of the transmission and spread of drug resistance the determinant parameters need to be identified and measured. The underlying hypothesis is that mutations associated with drug resistance incur fitness costs to the parasite in absence of drug pressure. The distribution of drug resistance haplotypes in different subsets of the host population was investigated. In particular newly acquired haplotypes after radical cure were characterized and compared to haplotypes from persistent infections. METHODS: Mutations associated with antimalarial drug resistance were analysed in parasites from children, adults, and new infections occurring after treatment. Twenty-five known single nucleotide polymorphisms from four Plasmodium falciparum genes associated with drug resistance were genotyped by DNA chip technology. RESULTS: Haplotypes were found to differ between subsets of the host population. A seven-fold mutated haplotype was significantly reduced in adults compared to children and new infections, whereas parasites harbouring fewer mutations were more frequent in adults. CONCLUSION: The reduced frequency of highly mutated parasites in chronic infections in adults is likely a result of fitness costs of drug resistance that increases with number of mutations and is responsible for reduced survival of mutant parasites.

The risk of malarial infections and disease in Papua New Guinean children.

In a treatment re-infection study of 206 Papua New Guinean school children, we examined risk of reinfection and symptomatic malaria caused by different Plasmodium species. Although children acquired a similar number of polymerase chain reaction-detectable Plasmodium falciparum and P. vivax infections in six months of active follow-up (P. falciparum = 5.00, P. vivax = 5.28), they were 21 times more likely to develop symptomatic P. falciparum malaria (1.17/year) than P. vivax malaria (0.06/year). Children greater than nine years of age had a reduced risk of acquiring P. vivax infections of low-to-moderate (>150/microL) density (adjusted hazard rate [AHR] = 0.65 and 0.42), whereas similar reductions in risk with age of P. falciparum infection was only seen for parasitemias > 5,000/microL (AHR = 0.49) and symptomatic episodes (AHR = 0.51). Infection and symptomatic episodes with P. malariae and P. ovale were rare. By nine years of age, children have thus acquired almost complete clinical immunity to P. vivax characterized by a very tight control of parasite density, whereas the acquisition of immunity to symptomatic P. falciparum malaria remained incomplete. These observations suggest that different mechanisms of immunity may be important for protection from these malaria species.