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

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

Spatial Effects on the Multiplicity of Plasmodium falciparum Infections

As malaria is being pushed back on many frontiers and global case numbers are declining, accurate measurement and prediction of transmission becomes increasingly difficult. Low transmission settings are characterised by high levels of spatial heterogeneity, which stands in stark contrast to the widely used assumption of spatially homogeneous transmission used in mathematical transmission models for malaria. In the present study an individual-based mathematical malaria transmission model that incorporates multiple parasite clones, variable human exposure and duration of infection, limited mosquito flight distance and most importantly geographically heterogeneous human and mosquito population densities was used to illustrate the differences between homogeneous and heterogeneous transmission assumptions when aiming to predict surrogate indicators of transmission intensity such as population parasite prevalence or multiplicity of infection (MOI). In traditionally highly malaria endemic regions where most of the population harbours malaria parasites, humans are often infected with multiple parasite clones. However, studies have shown also in areas with low overall parasite prevalence, infection with multiple parasite clones is a common occurrence. Mathematical models assuming homogeneous transmission between humans and mosquitoes cannot explain these observations. Heterogeneity of transmission can arise from many factors including acquired immunity, body size and occupational exposure. In this study, we show that spatial heterogeneity has a profound effect on predictions of MOI and parasite prevalence. We illustrate, that models assuming homogeneous transmission underestimate average MOI in low transmission settings when compared to field data and that spatially heterogeneous models predict stable transmission at much lower overall parasite prevalence. Therefore it is very important that models used to guide malaria surveillance and control strategies in low transmission and elimination settings take into account the spatial features of the specific target area, including human and mosquito vector distribution

A prospective study of Plasmodium falciparum multiplicity of infection and morbidity in Tanzanian children

Several studies suggest that in individuals with substantial previous exposure to malaria, co-infection with multiple clones of Plasmodium falciparum can protect against subsequent clinical malaria attacks. Other studies, mainly of individuals with little previous exposure, found the converse relationship. To test whether acquisition of such cross-protection tracks the acquisition of clinical immunity in general, 610 Tanzanian children aged 0-6 years were enrolled in a nine-month prospective study of the risk of morbidity in relation to parasitological status and merozoite surface protein 2 genotypes on enrolment. Prevalence of parasitaemia and multiplicity of infection increased with age. In the first year of life, the incidence of clinical malaria was almost three times higher in children with parasites at baseline than in those without. In older children, baseline P. falciparum infections appeared to protect against both parasitaemic and non-parasitaemic fever episodes. In children aged less than three years, baseline multiple infection tended to be associated with higher prospective risk of clinical malaria than single infection while in children aged more than three years the converse was found, but these effects were not statistically significant. These results provide further evidence that relationships between asymptomatic malaria infections and clinical malaria change with cumulative exposur

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

9. Effect of insecticide-treated bed nets on haemoglobin values, prevalence and multiplicity of infection with Plasmodium falciparum in a randomized controlled trial in Tanzania

A randomized controlled trial of insecticide-treated bed nets (ITNs) was conducted in an area of high malaria transmission in Tanzania in order to assess the effects of ITNs on infection and anaemia. One hundred and twenty-two children, aged 5 to 24 months, were randomly allocated to 2 groups, one of which received ITNs. Outcome measures were assessed in 6 consecutive months with monthly cross-sectional surveys. These measures were haemoglobin values, Plasmodium falciparum prevalence and density, and multiplicity of infection determined by polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) of the msp2 locus. There was a significant increase in mean heamoglobin values and a significant decrease of 16·4% in microscopically determined P. falciparum prevalence in children in the ITN group six months after the start of the trial. Both effects were more pronounced in younger children. However, no significant difference was observed in parasite density or multiplicity of infection among infected children. Comparison with PCR results indicated that microscopically subpatent parasitaemia was more frequently found in children in the ITN group. This, together with the observed similar multiplicity in the 2 groups, suggests that infections are maintained despite ITN use, owing to the chronicity of infections. This study shows that ITNs reduce the risk of anaemia in highly exposed young children. The virtually unchanged multiplicity of infection indicates that the potentially protective concomitant immunity is not compromise