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Whether the number of concurrent clones in asymptomatic Plasmodium falciparum infections reflects the degree of host protection was investigated in children living in areas with different levels of transmission on the coast of Kenya. The number of concurrent clones was determined on the basis of polymorphism in msp2, which encodes the vaccine candidate antigen merozoite surface protein 2. In a low-transmission area, most children had monoclonal infections, and diversity did not predict a risk of clinical malaria. In an area of moderate transmission, asymptomatic infections with 2 clones were, compared with 1 clone, associated with an increased risk of subsequent malaria. In a comparative assessment in a high-transmission area in Tanzania, multiclonal infections conferred a reduced risk. The different nonlinear associations between the number of clones and malaria morbidity suggest that levels of tolerance to multiclonal infections are transmission dependent as a result of cumulative exposure to antigenically diverse P. falciparum infections.
The burden of Plasmodium falciparum malaria falls predominantly on sub-Saharan Africa, with >1 million children dying of the disease each year. The risk of encountering malaria varies widely within the African continent, however. In areas of high transmission, young children are at the highest risk of severe disease and death before gradually acquiring protective immunity after repeated exposure. The number of clinical episodes of malaria decreases with age, and older children and adults often harbor parasites without experiencing clinical symptoms. In areas of limited and unstable transmission, the population is more rarely exposed, and individuals of all ages are at risk of disease. Variations in malaria transmission and disease manifestations may be found even within restricted geographic areas .
Acquisition of protective immunity to malaria is considered to be developed in an antigen-specific manner. The level of antigenic diversity of P. falciparum populations in an area is, therefore, likely to affect acquisition of immunity. Several studies have shown that the antigenic and genetic repertoires of P. falciparum populations are wider in high-transmission areas [2-6], generated by more frequent recombination events in the mosquito . Characterization of P. falciparum populations (ie, clones) by typing polymorphic surface antigen markers has proven to be a useful tool to elucidate the molecular epidemiology of malaria and may contribute to a better understanding of the mechanisms of antimalarial immunity.
New parasite variants detected at the time of clinical episodes [8-10] and a lower number of concurrent clones in adults than in children [11, 12] have given support to the concept of strain-specific immunity. In high-transmission settings, the highest diversity is, however, found in school-age children, who are relatively protected against clinical malaria; this suggests that patterns of immunity are more complex [4, 12]. Whether the number of concurrent clones in asymptomatic P. falciparum infections reflects the degree of protection is not fully established. Among continuously exposed individuals, those who harbor few clones may be expected to have the most efficient immunity. In contrast, several studies have shown that multiclonal infections are associated with a reduced risk of malaria [13-16], whereas other studies have come to the opposite conclusion [17-19]. These conflicting findings may partly be attributed to age and transmission. Comparisons between studies are, however, restricted by differences in design and in molecular and data analyses.
With a view toward establishing the effect that transmission has on the diversity of P. falciparum infections and the degree of host immunity, we studied the relationship between number of clones and malaria morbidity in 2 groups of children living in areas with different levels of transmission on the coast of Kenya . The number of clones—determined by genotyping the highly polymorphic domain of msp2, which encodes the vaccine candidate antigen merozoite surface protein 2 (MSP2) —was investigated in relation to the risk of malaria in a cross-sectional survey with longitudinal follow-up. The study design allowed comparisons with a recent assessment conducted in a setting of high endemicity in Tanzania, in which an association between the number of clones and susceptibility to disease was found .
The study sites, Ngerenya and Chonyi, are located 20 km north and south of the town of Kilifi on the coast of Kenya. Malaria transmission is stable, with a long rainy period from May through July and a short period in November. Ngerenya has low transmission, with 10 infective bites/person/year , and Chonyi has moderate transmission, with 22-53 infective bites/person/year . Two cohorts of individuals have been followed since 1998 in the respective areas. The project was approved by the Kenya Medical Research Institute National Ethical Review Committee. Written informed consent was obtained from the guardians of all participants.
In October 2000, venous blood was collected from 1523 individuals, of whom 779 were <11 years old. Samples were available for DNA extraction from 748 children, 368 from Chonyi and 380 from Ngerenya.
Clinical episodes of malaria were continuously recorded by active and passive surveillance. All children were visited weekly by a fieldworker. In addition, the participants were encouraged to seek treatment whenever they fell ill. In the case of an axillary temperature ≥37.5°C or a history of fever, a blood smear was analyzed by microscopy. Children with fever and P. falciparum parasites were treated, free of charge, with sulphadoxine-pyrimethamine, according to the national policies. Children with signs of severe malaria were admitted to Kilifi District Hospital.
For the purpose of the study, clinical malaria was determined using 2 separate definitions: (1) fever (temperature ≥37.5°C or reported hot body) and the detection of P. falciparum parasites and (2) fever and the detection of >2500 P. falciparum parasites/μL (a more specific definition for malaria in this setting) . The observation period was 18 May 1999 to 26 August 2001.
To clearly define children who were asymptomatic at the time of blood sampling, individuals with fever (with or without parasites) at the time of or within 1 week after the survey and afebrile children with >10,000 parasites/μL were excluded. Because of the prophylactic effect of sulphadoxine-pyrimethamine , children treated within 4 weeks before the survey were excluded. This was considered a conservative approach, because the number of clones may be affected by ongoing disease [19, 25] and recent antimalarial treatment.
The presence of Plasmodium parasites was investigated by light microscopy of Giemsa-stained thick and thin smears. Parasite densities were enumerated against 200 leukocytes, estimating 8000 leukocytes/μL, or, if available, against measured leukocyte counts; in high-density samples, parasite densities were enumerated against 500 erythrocytes, estimating 5 × 106 erythrocytes/μL.
Genomic DNA was extracted from packed erythrocytes (500 μL) by means of a QiaAmp Blood Mini kit (Qiagen), adding 100 μL of elution buffer. The polymerase chain reaction (PCR) method included initial amplification of the outer msp2 domain, followed by 2 separate nested reactions with primers targeting the 2 allelic variants, FC27 and IC . PCR was performed in a total volume of 20 μL that included 1 μL of DNA, with 55 cycles in total and an annealing temperature of 58°C. The length of PCR products was determined by electrophoresis on 2% MetaPhor agarose gels (BMA), followed by ethidium bromide staining and UV transillumination. The number of msp2 alleles (FC27 plus IC alleles) determined the number of clones per infection.
Although in strict terms the term clone indicates parasites derived from a common ancestor, it is here used to describe a parasite population with the same genotype. The true genetic complexity is, therefore, likely to be underestimated, and figures show the minimum number of clones.
Analyses were performed by means of SPSS (version 14; SPSS). Data were categorized to avoid assumptions about linearity, as follows: for number of clones, 0, 1, 2, 3, and ≥4; for parasite density, microscopy negative and PCR negative, microscopy negative and PCR positive, and microscopy positive into quintiles of their distribution; for age, 0-1.99, 2.00-4.99, 5.00-7.99, and 8.00-10.99 years; and for number of malaria episodes, 0, 1-2, 3-4, 5-6, 7-9, and ≥10. Logistic regression was used to assess associations in the cohorts for parasite prevalence, parasite densities, gametocytes, number of clones, and episodes. Odds ratios (ORs) were adjusted for age and sex. Association between number of clones and parasite density was analyzed by linear regression.
Association between the number of clones and malaria risk was investigated in strictly asymptomatic individuals. Cox regression was used to assess the time to the nearest episode before and after the survey, respectively. Hazard ratios (HRs) were adjusted for age, sex, and parasite density. Additional analyses included adjustments for time to previous episode and stratification of children into ≤5 and >5 years old. The risk of malaria was further analyzed by Poisson regression assessing the number of episodes during 6-month period before and after the survey, respectively. Incidence rate ratios (IRRs) were adjusted for age and sex. Association between the number of clones and the total number of episodes during the observation period was analyzed by multinomial logistic regression, using 1-2 episodes for comparison.
The present study included 748 children (0-10 years old) who participated in a cross-sectional survey in October 2000; 368 were from Chonyi, which has moderate transmission, and 380 were from Ngerenya, which has low transmission (table 1). At the time of the survey, 25 children had an ongoing episode of fever and detection of P. falciparum, 7 children developed a clinical episode during the following week, 161 children had an episode of fever and detection of P. falciparum with antimalarial treatment within 4 weeks before the survey, and 15 children had parasite densities >10,000 parasites/μL. Three children had missing data. In total, 264 children from Chonyi and 273 from Ngerenya fulfilled the criteria of being asymptomatic.
The parasite prevalence by microscopy was 37.5% in Chonyi and 24.7% in Ngerenya. All samples were analyzed by PCR of the msp2 domain, resulting in detection of P. falciparum in an additional 62 children. In 30 microscopy-positive samples, the PCR assay failed to detect parasites despite duplicated analyses. Together, the 2 methods established a parasite prevalence of 49.0% and 31.6% in the Chonyi and Ngerenya groups, respectively. Among the asymptomatic children, 36.7% were positive by PCR in Chonyi, and 16.8% were positive by PCR in Ngerenya.
Parasite densities were higher among asymptomatic children in Chonyi (geometric mean± 2 standard deviations [SDs], 1274± 2457 parasites/μL) than in Ngerenya (geometric mean± 2 SDs, 558± 1612 parasites/μL), with more densities in the second to fifth quintiles of the distribution (ie, >240 parasites/μL) in Chonyi (P = .001). Gametocytes were detected in 43 children (5.7%), of whom 29 were asymptomatic; the frequency of gametocyte detection did not differ between the groups. Other Plasmodium species were distinguished in 29 children (3.9%), 18 in Chonyi and 11 in Ngerenya; 28 were P. malariae infections and 8 were P. ovale infections, of which 16 were mixed with P. falciparum.
Multiple msp2 genotypes (ie, clones) were detected in 70.1% of the asymptomatic children with parasitemia in Chonyi, compared with 41.3% in Ngerenya (figure 1). The mean number of clones was 3.01 (95% confidence interval [CI], 2.62-3.39; range, 1-9) in Chonyi and 1.70 (95% CI, 1.36-2.03; range, 1-6) in Ngerenya. Because the number of clones was not normally distributed, frequencies were used for comparisons (table 2). Infections with ≥3 clones were more prevalent in Chonyi (49.5%) than in Ngerenya (13.0%). The number of clones was positively associated with parasite density (regression coefficient β, 0.173 [95% CI, 0.05-0.30]; P = .013). Also, after adjustment for parasite density, the number of clones was higher in all age groups in Chonyi (P = .049).
The highest number of clones was found in the 5.00-7.99-year-old group in both cohorts (figure 2). Among the 25 children with ongoing fever and detection of parasites, the mean number of clones was 2.83 (95% CI, 2.13-3.54) in Chonyi and 2.30 (95% CI, 1.34-3.26) in Ngerenya, which were significantly different from those among the asymptomatic children. Gametocyte prevalence was not associated with number of clones. Children infected with multiple species had a high number of P. falciparum clones (mean, 3.58), as has been previously noted . Notably, the only 2 children from Chonyi who were concurrently infected with 3 species (P. falciparum, P. ovale, and P. malariae) had 7 clones.
Of the 2 dimorphic types of msp2, IC alleles were detected in 88.7% of asymptomatic infections in Chonyi and in 86.9% in Ngerenya, and FC27 alleles were found in 69.1% and 41.3%, respectively (figure 2). Infections composed of both types were more prevalent in Chonyi (57.7%) than in Ngerenya (28.3%). In Ngerenya, children with ongoing fever and detection of parasites at the survey had more FC27 types (75.0%).
Malaria morbidity was higher in Ngerenya than in Chonyi (mean± SD, 2.89± 2.59 vs 1.66± 1.69 episodes per person-year; mean± SD, 2.30± 2.22 vs 1.19± 1.45 episodes of fever and >2500 parasites/μL per personyear). The number of episodes was higher for all age groups in Ngerenya except for the 0-1.99-year-old group, in which the frequency was higher in Chonyi (0.83 vs 1.01 episodes per year). In Ngerenya, 46.9% had ≥5 episodes of fever and detection of parasites over the study period, compared with 18.2% in Chonyi (age-adjusted P < .001), and 55.7% and 25.4% had ≥3 episodes of fever and >2500 parasites/μL, respectively (age-adjusted P < .001). The mean number of episodes for children who were excluded due to ongoing or recent malaria was 4.78 (SD, 3.27) in Ngerenya and 2.87 (SD, 2.22) in Chonyi. To allow comparison with a recent study in Tanzania , fever included temperature ≥37.5°C and reported hot body, explaining the higher morbidity found here than in previous reports in which only measured temperature was used .
In Chonyi, children infected with ≥3 clones had, compared with children infected with 1 clone, a longer time to a previous episode of fever and detection of parasites (adjusted HR, 0.40 [95% CI, 0.17-0.94]; P = .035). Time to a malaria episode with >2500 parasites/μL was longer in children infected with ≥4 clones (adjusted HR, 0.17 [95% CI, 0.07-0.39]; P < .001) (figure 3A). Diversity was not associated with the number of episodes 6 months before the survey (table 3). The presence of parasites at the survey was not associated with the time to or the number of episodes before the blood sampling.
In Ngerenya, P. falciparum-negative children had fewer episodes during the previous 6 months (IRR, 0.44 [95% CI, 0.30-0.65]; P < .001). Children infected with 2 clones were, compared with children infected with 1 clone, more likely to have a recent episode of fever and detection of parasites (adjusted HR, 2.55 [95% CI, 1.16-5.60]; P = .020) and more episodes within 6 months before the survey (adjusted IRR, 1.68 [95% CI, 1.07-2.80]; P = .048) (table 3), likely reflecting subgroups of children with different exposure. The short time to previous episodes among children with parasitemia may also suggest incomplete parasite clearance after treatment due to partial sulphadoxine-pyrimethamine resistance in the less-immune population.
The number of clones in the asymptomatic children at the survey was further assessed in relation to their prospective risk of malaria by both case definitions. The definition of fever and >2500 parasites/μL was considered most specific in the prospective risk analysis, because it minimizes the effect of asymptomatic background parasitemia.
In Chonyi, children infected with 2 clones had a shorter time to a subsequent episode with >2500 parasites/μL, compared with children infected with 1 clone (adjusted HR, 4.23 [95% CI, 1.69-10.54]; P = .002) (figure 3B), as well as a higher number of episodes during the subsequent 6 months (table 3). In an age-stratified analysis, children ≤5 years old had higher risk of subsequent malaria when infected with 2 clones (adjusted HR, 13.61 [95% CI, 1.25-147.73]; P = .032), compared with children >5 years old, who only had borderline significant risk (adjusted HR, 3.97 [95% CI, 0.94-16.79]; P = .061). No significant associations were found with a subsequent risk of malaria among children infected with ≥3 clones, even when stratifying for age. Assessment of risk as the total number of episodes during the entire observation period revealed that children infected with ≥4 genotypes were more likely to not have experienced any episodes of fever and have >2500 parasites/μL (adjusted OR, 5.38 [95% CI, 1.35-21.52]; P = .017). No other associations were found between the number of clones and the total number of episodes.
In Ngerenya, children who were negative for parasites at the survey had an increased prospective risk of malaria (table 3), especially those >5 years old (adjusted HR, 4.39 [95% CI, 1.38-14.03]; P = .012). No associations were found between the number of clones and the time to subsequent malaria, the number of episodes 6 months after the survey, or the number of episodes during the entire observation period.
When the data for Chonyi and Ngerenya were combined and adjusted for area, infection with 2 clones was again associated only with time to subsequent malaria episode (adjusted HR, 2.69 [95% CI, 1.36-5.31]; P = .004).
P. falciparum has extensive antigenic and genetic polymorphism and, infections are often composed of several distinct parasite populations. The ability to control multiclonal infections is, therefore, likely to be an important component of protective immunity. We have assessed how the diversity of P. falciparum infection relates to the host’s susceptibility to disease in areas of different transmission.
Characterization of P. falciparum infections by msp2 genotyping in children living in 2 closely located areas on the coast of Kenya revealed that the number of infecting clones was highest in the area of most-intense transmission, in line with previous reports [2-6]. In Chonyi, which has moderate transmission, 70% of infections were multiclonal and 50% were composed of ≥3 clones. The highest diversity was detected in children for whom a long time has elapsed since receiving previous antimalarial treatment, suggesting accumulation of infections with time in partially immune individuals. Also, multiple clones are more likely to be transmitted via single mosquito bites in areas of higher transmission . In contrast, in Ngerenya (the area of low transmission) most infections were monoclonal, and only 6 children had ≥3 clones, reflecting very limited diversity and accumulation. The 2 closely located populations have the same ethnic origin, customs, and housing. The only apparent difference was higher use of untreated bed nets in Ngerenya, resulting from a previous trial in the mid-1990s . However, even the use of insecticide-treated bed nets has not been associated with reduced P. falciparum diversity .
The transmission of malaria might have varied over time in the study areas, and unfortunately there was no assessment of the entomological inoculation rate during the current observation period. However, repeated entomological studies in Kilifi District have revealed consistently higher transmission south of the town of town Kilifi, where Chonyi is situated (C. Mbogo, personal communication). The diversity of P. falciparum infection found here supports the notion that transmission intensity differed in the 2 settings during this period.
The present study was performed using blood samples collected at a single time point. Although different clones might be detected on consecutive days , the number of clones has been shown to be relatively consistent within individuals over time . Considering within-host dynamics, the present assessment is likely to have underestimated the overall diversity as well as the differences between the 2 areas.
The P. falciparum prevalence, densities, and number of concurrent clones were all higher in Chonyi. Considering the lower malaria morbidity, this suggests better tolerance to both parasite loads and antigenically diverse infections. When the risk of malaria disease was assessed in relation to P. falciparum diversity, the lowest subsequent risk was found in children infected with 1 clone and an increased risk was found in children infected with 2 clones, especially in children ≤5 years old. Higher diversity conferred no additional risk. In contrast, children infected with ≥3 clones had lower morbidity during the entire observation period and a longer time to a previous episode. The prospective risk was not reduced, however, which implies that a temporal difference in the association between the number of clones and disease exists. Children may be able to handle a certain number of clones throughout the low-transmission season but when infected with additional clones at the time of increased transmission they succumb to disease . Children with diverse infections may also be subgroups of individuals with different levels of tolerance.
In Ngerenya, the absence of parasites at the cross-sectional survey conferred an increased risk, especially in children >5 years old, suggesting higher susceptibility due to low exposure. There was, however, no association between the number of clones and prospective malaria risk.
An identical study design and analyses, including the same genotyping method  and statistical approach (Cox regression with the same adjustments), were used in our previous study in a high-transmission area in Tanzania . There, children who harbored 2-3 clones or were parasite negative had a reduced risk of subsequent malaria, whereas children with 1 clone had the highest risk (figure 4), suggesting that multiclonal infections are a marker of protection. In both studies, associations between the number of clones and malaria risk were nonlinear and could not be revealed when clones were modeled as continuous data. Assumptions about linearity should indeed be avoided because host-parasite interactions may be level dependent, with regard to both pathogenesis and protection .
Moreover, only clearly asymptomatic children were included, to avoid the influence of ongoing clinical malaria or recent treatment [19, 25]. This may have underestimated the associations because the most vulnerable children are likely to be excluded, as reflected by their higher malaria morbidity. Inclusion of all children, however, did not reveal any associations between the number of clones and risk in either of the studies, demonstrating the need for distinct selection criteria in population-based assessments.
Transmission accounts for the main difference between the settings, suggesting that intensity and diversity of exposure lead to different levels of tolerance to multiclonal infections (figure 4). In the low-transmission area (Ngerenya), rarer exposure restricts acquisition of protective immunity, most infections lead to disease, and antigenic diversity does not appear to be of overall importance. In the moderate-transmission area (Chonyi), individuals are immune to 1 or a few clones, but additional clones lead to disease. Susceptibility may vary, however, because of individual differences in exposure. In high-transmission areas (Tanzania), continuous exposure to antigenically polymorphic parasites leads to tolerance to multiclonal infections, which maintain cross-protective immune responses.
The rate of acquiring and clearing P. falciparum infections is dependent on age . The reduced risk conferred by multiclonal infections in high-transmission settings have mainly been found in school-age children [13, 14, 29], whereas younger children and infants were at increased risk [17, 18, 29, 30], suggesting a gradual acquisition of tolerance to multiple clones with age. Here, in the moderate-transmission area, infections with 2 clones generated the highest risk in children ≤5 years. There was, however, no reduced risk in older children, suggesting that they do not reach the same level of tolerance as in high-transmission settings.
The diversity of P. falciparum infections reflects simultaneous acquisition of new infections and immunity. The persistence of antigenically diverse parasites is also likely to affect the diversity of antibody responses. Analysis of anti-MSP2 antibodies in this area showed a reduced malaria risk, especially for antibodies directed against the same allelic types as the concurrent parasites . Detailed assessments of antigen-specific antibody responses in relation to infecting clones may help us to further understand the importance of persistent parasites per se.
In conclusion, associations between the number of concurrent clones in asymptomatic P. falciparum infections and the prospective risk of malaria are dependent on transmission level. The same diversity is both a marker of risk and a marker of protection in different areas and is of no apparent importance in a low-transmission area. Tolerance to multiclonal infections is thus a result of the intensity and antigenic repertoire of exposure. Understanding how and when immunity to antigenically diverse P. falciparum is acquired under different transmission conditions is a prerequisite for the development of future control interventions, such as antimalarial vaccines or preventive treatment.
We thank the children and their guardians for their participation; the staff at the outpatient department; Sophie M. Uyoga, Moses Mosobo, Mohammed Shafi, and Spencer Polley for technical assistance; Edna Ogada for data management; and Margaret Mackinnon, Faith Osier, and Charles Mbogo for most valuable comments on the manuscript.
Financial support: Wellcome Trust (financing to the project); Swedish Research Council and Swedish Society of Medicine (support to A.F.)
Potential conflicts of interest: none reported.