2. Genotypes of merozoite surface protein 2 of Plasmodium falciparum in Tanzania

The merozoite surface protein 2 (MSP2) of Plasmodium falciparum is extremely polymorphic: 82 different msp2 alleles were found in 4 studies of molecular epidemiology conducted in Tanzania. This diversity renders msp2 suitable as a marker gene for the genotyping of P. falciparum infections. Amplification of msp2 by the polymerase chain reaction (PCR), and subsequent restriction digests of the PCR product (PCR-restriction fragment length polymorphism genotyping), has proved to be an informative tool for enumerating multiple concurrent infections in a blood sample, and distinguishing individual alleles. Depending on the specific questions asked in a genotyping study, analytical techniques of different degrees of complexity are employed. The restriction fragments resulting from a single HinfI digest generally allow the enumeration of multiple concurrent infections and the determination of their allelic families. When a restriction pattern is too complex to be resolved, owing to the high number of concurrent infections, or due to the appearance of previously undescribed alleles, one or more additional digests (DdeI, RsaI, ScrfI) may be necessary. To determine individual alleles unequivocally, in particular in longitudinal studies, when several consecutive samples need to be compared with each other, a more detailed analysis involving all 3 additional digests is applied. The methodological experience and results gained in 4 epidemiological field studies involving msp2 genotyping are summarized. We also provide the HinfI restriction patterns and some nucleotide sequences of the alleles found so far in our studies in Tanzani

11. Premunition in Plasmodium falciparum infection: insights from the epidemiology of multiple infections

Epidemiological studies of multiple clone infections by Plasmodium falciparum in highly endemic areas have demonstrated age dependence in both the multiplicity of infection and the relationships between this multiplicity and the risk of acute illness. We hypothesize that, in infants, host defence against blood-stage infections with P falciparum relies mainly on fever and cytokine activities, and the infections are of short duration. In older children, a high multiplicity of infection is characteristic of low-level chronic parasitaemia. This appears to confer cross-protection against newly inoculated parasites, via partially genotype-specific responses which are short-term, lasting little longer than the infections themselves. This has important implications for our understanding of immunity against P. falciparum, its ecological niche, and the epidemiological impact of interventions against i

10. Effect of insecticide-treated bed nets on the dynamics of multiple Plasmodium falciparum infections

The rates of acquisition and loss of individual genotypes belonging to the FC27 family of the Plasmodium falciparum merozoite surface protein 2 (msp2) gene were studied in 120 children aged 5 months to 2·5 years, in a randomized controlled trial of insecticide-treated bed nets (ITNs) in Kiberege village, Tanzania. Analysis of longitudinal changes in positivity for individual alleles in samples collected at intervals of one month indicated that the average duration of infections, allowing for undetected parasite genotypes, was 73 d in those aged <18 months and 160 d in children aged ≥18 months, consistent with a shift from acute to chronic infection with age. Overall, 51% of genotypes infecting the host were estimated to be detected by polymerase chain reaction-restriction fragment length polymorphism analysis in any one sample of 0·5 μL of packed peripheral blood cells. In children less than 18 months old this sensitivity was 61% (SE = 6%) compared with 41% (SE = 6%) in older children. Conversely, the rate of appearance of new parasite genotypes was higher in children <18 months of age than in older children, but this partly reflected the difference in sensitivity. The overall incidence of new infections was estimated to be reduced by 17% in ITN users. There was no statistically significant difference between users and non-users in observed infection multiplicity, sensitivity, recovery rate, or estimated infection rates for individual alleles. This suggests that, in areas of high P. falciparum endemicity, ITNs have little effect on the establishment of chronic malaria infectio

7. Dynamics of multiple Plasmodium falciparum infections in infants in a highly endemic area of Tanzania

The force of infection and recovery rate for malaria in infants in a highly endemic area of Tanzania were analysed using polymerase chain reaction-restriction fragment length polymorphism genotyping of the Plasmodium falciparum msp2 locus in 99 paired blood samples. Overall, new genotypes were acquired at a rate of 0·064 per day, and the average duration of infections was estimated to be 23 d. The highest recovery rates were in children under 4 months of age. The higher susceptibility of infants to clinical malaria in comparison with older children, in areas of very high transmission, may be largely a consequence of the short duration of infections which precludes the establishment of concomitant immunity. The high turnover of infections also implies that infection prevalence and multiplicity approach an equilibrium even in very young children, and calls into question the use of infant conversion rates as a measure of transmission intensit

PCR-correction strategies for malaria drug trials: Updates and Clarifications

Malaria drug trials conducted in endemic areas face a major challenge in their analysis because it is difficult to establish whether parasitaemia in blood samples collected after treatment indicate drug failure or a new infection acquired after treatment. It is therefore vital to reliably distinguish drug failures from new infections in order to obtain accurate estimates of drug failure rates. This distinction can be achieved for Plasmodium falciparum by comparing parasite genotypes obtained at the time of treatment (the baseline) and on the day of recurring parasitaemia. Such PCR correction is required to obtain accurate failure rates, even for new effective drugs. Despite the routine use of PCR correction in surveillance of drug resistance and in clinical drug trials, limitations inherent to the molecular genotyping methods have led some researchers to question the validity of current PCR correction strategies. Here we describe and discuss recent developments in these genotyping approaches, with a particular focus on method validation and limitations of the genotyping strategies. Our aim is to update scientists from public and private bodies who are working on the development, deployment, and surveillance of new malaria drugs. We aim to promote discussion around these issues and argue for the adoption of improved standardised PCR correction methodologies

A point mutation in codon 76 of pfcrt of P. falciparum is positively selected for by chloroquine treatment in Tanzania

This study was undertaken to validate the relevance of Chloroquine (CQ) resistance markers pfert76 and pfmdrl86 in an endemic area in Tanzania. After treatment with CQ, recrudescence was distinguished from new infection by msp2 genotyping, and the number of concurrent infection was also determined. The rate of children with recrudescent parasites at day 7 and/or day 14 amounted to a parasitological failure rate of 22.4% using PCR. The mean multiplicity of infection at day 0 was 3.2 (n = 71). The allelic frequencies of the mutated pfert76 and pfmdrl86 were estimated to be 92 and 77%, respectively. Both values exceeded by far the observed frequency of 14% of recrudescent parasites as calculated on the whole analysed parasite population taking multiple infections into account. Although neither mutant allele is of predictive value for parasitological resistance, there is evidence for a role of pfert76 in CQ resistance in the natural parasite population. All wild-type pfert76 alleles were eliminated before day 3, after the onset of CQ treatment and no recrudescent parasite with the wild-type allele was observed at later time points. The discrepancy between the rate of resistant parasites (14%) and the frequency of the mutant pfert76 allele (92%), however, indicates that other polymorphisms and other factors must be involved in CQ resistance. No selective elimination of the pfmdrl86 wild-type allele was observed. © 2002 Elsevier Science B.V. All rights reserved

Improving methods for analysing anti-malarial drug efficacy trials: molecular correction based on length-polymorphic markers msp-1, msp-2 and glurp.

BACKGROUND:Drug efficacy trials monitor the continued efficacy of front-line drugs against falciparum malaria. Over-estimates of efficacy result in a country retaining a failing drug as first-line treatment with associated increases in morbidity and mortality, while under-estimating drug effectiveness leads to removal of an effective treatment with substantial practical and economic implications. Trials are challenging: they require long durations of follow-up to detect drug failures, and patients are frequently re-infected during that period. Molecular correction based on parasite genotypes distinguishes reinfections from drug failures to ensure the accuracy of failure rate estimates. Several molecular correction "algorithms" are proposed, but which is most accurate and/or robust remains unknown. METHODS:We used pharmacological modelling to simulate parasite dynamics and genetic signals that occur in patients enrolled in malaria drug clinical trials. We compared estimates of treatment failure obtained from a selection of proposed molecular correction algorithms against the known "true" failure rate in the model. FINDINGS:(i) Molecular correction is essential to avoid substantial over-estimates of drug failure rates. (ii) The current WHO-recommended algorithm consistently under-estimates the true failure rate. (iii) Newly-proposed algorithms produce more accurate failure rate estimates; the most accurate algorithm depends on the choice of drug, trial follow-up length, and transmission intensity. (iv) Long durations of patient follow-up may be counterproductive; large numbers of new infections accumulate and may be misclassified, over-estimating drug failure rate. (v) Our model was highly consistent with existing in vivo data. INTERPRETATION:The current WHO-recommended method for molecular correction and analysis of clinical trials should be re-evaluated and updated

6. Multiple Plasmodium falciparum infections in Tanzanian infants

Paired blood samples from 99 Tanzanian infants were analysed to examine the infection dynamics of Plasmodium falciparum during the first year of life. Infecting parasites were genotyped by polymerase chain reaction amplification of the polymorphic gene for the merozoite surface protein 2 and subsequent analysis according to the resulting restriction fragment length polymorphism pattern. The same samples served as controls in a parallel case-control study for which an additional blood sample was taken from each child during a fever episode. The relationship of the number of concurrent infections (multiplicity) with age and morbidity was analysed and results were compared to those of a similar study on older children between 2 and 7 years of age, carried out in the same village at the same time. The mean of 2 infecting genotypes per positive blood sample from community surveys was low compared to that in older children, and there was no significant age-dependency of multiplicity within the first year of life. Multiplicity of infection in fever cases was also independent of age. In infants, multiplicity was positively associated with parasite density and risk of clinical malaria, in contrast to the situation in older children (>2 years). The findings help in the understanding of infection dynamics, premunition, and development of semi-immunity in malari

8. Effect of iron supplementation and malaria prophylaxis in infants on Plasmodium falciparum genotypes and multiplicity of infection

During a randomized placebo-controlled trial of chemoprophylaxis against Plasmodium falciparum malaria and iron supplementation, in infants living under conditions of intense transmission, all samples of P. falciparum obtained from children aged 5 and 8 months were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis for the msp2 locus. One hundred and six blood samples were analysed for the number of concurrent infections (multiplicity), and the allelic family of each msp2 genotype was determined. Mean multiplicity of infection was, overall, 2·76 infections/child, and it was significantly reduced in infants receiving chemoprophylaxis. This finding might help to explain the rebound effect in morbidity observed after prophylaxis was ended. Iron supplementation did not affect multiplicity of infection. In infants receiving placebo only, or placebo and iron supplementation, a significant positive association was observed between the number of infections and parasite densities (Spearman's ϱ = 0·25, P − 0·047). This association was lost in the group receiving chemoprophylaxis alone, or in combination with iron. This study showed a significant association of FC27-like msp2 alleles with prospective risk of clinical malaria in children (relative risk = 1·487, P = 0·013). Such an association was also found for the present risk of clinical malaria in infants receiving prophylaxis (odds ratio = 3·84, P = 0·026), which might imply that chemoprophylaxis may impair the development of premunitio