Prevalence and Population Dynamics of 14 MSP-119 Haplotypes
Of the 2,309 samples collected from the study participants during the three transmission seasons, 1,375 were parasite-positive (by PCR). Of the samples positive for parasites by microscopy, 96% were PCR positive, and of microscopy-negative samples, 40% were PCR positive. Of the 1,375 parasite-positive samples, 1,369 gave successful genotyping results. A haplotype-estimating algorithm was used to determine the presence of 14 haplotypes in samples that yielded allele frequency data. The algorithm was able to resolve haplotype frequencies for 1,363 of the 1,369 genotyped samples. The overall prevalence of each haplotype in the 1,363 samples is shown in . The QKSNGL and EKSNGL haplotypes (corresponding to the FVO and FUP strains, respectively) had the highest prevalence in the cohort with prevalences of 46% (95% confidence interval [CI] 44%–49%) and 36% (95% CI 34%–39%), respectively. The ETSSRL haplotype (corresponding to the 3D7 strain) had a lower overall prevalence of 16% (95% CI 14%–18%). Ten of the 14 haplotypes had prevalences below 10%, and eight haplotypes had prevalences below 5%.
Prevalences of 14 MSP-119 Haplotypes in a Cohort from Bandiagara, Mali
shows the prevalence of MSP-119 haplotypes over time in three age groups. As seen in the figure, the frequency of individual haplotypes varied considerably over time. However, despite this variability, the QKSNGL (FVO) and EKSNGL (FUP) haplotypes were most prevalent during all three years and in all three age groups. In addition, it appeared that the difference in prevalence between the two most common types and the remaining types increased with age. The same patterns were observed when the analysis was limited to microscopy-positive samples (unpublished data).
Prevalence of MSP-119 Haplotypes over Time in Three Age Groups
Multiplicity of Infection
shows the average MOI in three age groups during the three years of the incidence study. Average MOI fluctuated over time, but overall tended to decrease over the course of the transmission season (Mantel-Haenszel Chi-square test for trend p = 0.074, p < 0.001, p = 0.027, for 1999, 2000, and 2001, respectively, for all age groups combined). In addition, MOI was higher in the oldest age group (≥11 y) than in the two younger age groups. In a multivariable regression model including month, study year, age, and parasite density and taking into account repeated measurements from the same individual, persons 11 years of age or older had a 2.7 times greater odds of having a mixed infection (defined as having more than one MSP-119 haplotype) compared to children 5 years of age or younger (odds ratio [OR] 2.71, 95% CI 1.76–4.15, p < 0.001). The odds of having a mixed infection was not significantly different between the two younger age groups (OR 1.22, 95% CI 0.84–1.78, p = 0.24). The odds of a mixed infection was also greater in infections with moderate parasitemia (500–5,000 parasites/μl) compared to low-parasitemia infections (<500 parasites/μl) (OR 1.66, 95% CI 1.20–2.31, p = 0.0022). The odds of a mixed infection was not significantly different between higher-parasitemia infections and low-parasitemia infections (OR 1.11, 95% CI 0.80–1.55, p = 0.53 for 5,000–25,000 parasites/μl, and OR 1.07, 95% CI 0.75–1.53, p = 0.70 for >25,000 parasites/μl).
MSP-119 Haplotype Frequencies in Symptomatic and Asymptomatic Infections
shows the frequency of symptomatic and asymptomatic infections in the cohort for all three study years combined. As seen in A, the incidence of symptomatic infections peaked in October and decreased to almost zero by January, and was lower in individuals aged 11 years or older. Asymptomatic infection was less common in individuals aged 5 years or younger, and even in the absence of clinical malaria, asymptomatic parasitemia persisted, with a prevalence as high as 60% in the oldest individuals (B).
Frequency of Symptomatic and Asymptomatic Infections during the Malaria Transmission Season in Three Age Groups
When the frequency of MSP-119 haplotypes was compared between symptomatic and asymptomatic infections, three haplotypes (ETSSRL [3D7], QKNNGL, and EKSNGF) had a significantly lower prevalence among symptomatic infections compared to asymptomatic infections (). The same results were observed in a multivariable model adjusting for time and age, while taking into account longitudinal measurements. With QKNSGL (FVO) as the reference group, the ETSSRL (3D7), QKNNGL, and EKSNGF haplotypes had decreased odds of being present in a symptomatic infection (ETSSRL [3D7]: OR 0.67, 95% CI 0.49–0.92, p = 0.012; QKNNGL: OR 0.48, 95% CI 0.35–0.68, p < 0.001; and EKSNGF: OR 0.29, 95% CI 0.09–1.02, p = 0.053). When parasite density was added to the model, these associations were no longer significant, suggesting that the associations between these three haplotypes and asymptomatic infection may be accounted for by differences in parasitemia. To test this hypothesis, GEE was used to model the association between each of the three haplotypes and parasite density, adjusting for time, age, and MOI. Indeed, both ETSSRL (3D7) and QKNNGL were more prevalent in the lowest-parasitemia infections (<500 parasites/μl) compared to the highest-parasitemia infections (>25,000 parasites/μl) (ETSSRL [3D7]: OR 2.12, 95% CI 1.41–3.20, p < 0.001; QKNNGL: OR 3.65, 95% CI 2.32–5.72, p < 0.001). QKNNGL was also more prevalent in moderate-parasitemia infections (500–5,000 parasites/μl) than in the highest-parasitemia infections (OR 2.44, 95% CI 1.38–4.31, p = 0.0021). There were no statistically significant associations between the EKSNGF haplotype and parasite density, although as one of the more rare haplotypes, the data may have been too few for meaningful multivariable analysis. The raw data indicate a frequency of 2.8% in the lowest parasitemia infections compared to a frequency of 0.9% in the highest frequency infections.
Prevalence of MSP-119 Haplotypes among Symptomatic and Asymptomatic Infections
Within-Host Dynamics of MSP-119 Polymorphisms
On average, individuals aged 5 years or less were parasite positive at 14 time points during the 3-y study period, seven of which corresponded to clinical episodes. Individuals aged 6–10 y were positive at 16 time points (six clinical episodes), and individuals 11 y and older were positive at 12 time points (three clinical episodes). shows cumulative hazard functions generated from Cox proportional hazards models of the time to next clinical episode in individuals' consecutive clinical infections as a function of change in predominant amino acid at each of the six polymorphic residues in MSP-119, year, and age group. The hazard of a subsequent clinical infection when there was a change in overall haplotype was not significantly different from the hazard of a subsequent clinical infection when there was not a change in haplotype (hazard ratio [HR] 1.17, 95% CI 0.87–1.58, p = 0.30). However, as seen in graphs for amino acid positions 1691, 1700, and 1701 in , when a change in predominant amino acid at each of the polymorphic residues was considered, the hazard of a subsequent clinical episode was greater when there was a change in predominant amino acid at these positions than when there was no change at these positions (1691: HR 1.48, 95% CI 1.13–1.94, p = 0.0044; 1700: HR 1.48, 95%CI: 1.13–1.93, p = 0.0039; 1701: HR 1.38, 95% CI 1.05–1.80, p = 0.021). The hazard of a subsequent clinical episode when there was a change at the other three polymorphic positions (1644, 1699, and 1701) was not significantly different from when there was no change (1644: HR 1.05, 95% CI 0.82–1.34, p = 0.72; 1699; HR 1.17, 95% CI 0.86–1.60, p = 0.32; 1716: HR 1.07, 95% CI 0.78–1.48, p = 0.67). These associations were not changed when mixed infections were excluded (i.e., going from a predominant to mixed infection was not considered a change) or when multiple events from the same individual were taken into account (unpublished data). In addition, the hazard of a subsequent clinical episode in 2000 and 2001 was less than the hazard in 1999 (2000: HR 0.53, 95% CI 0.38–0.74, p < 0.001; 2001: HR 0.56, 95% CI 0.41–1.76, p < 0.001), and as expected, was also less for individuals aged 11 y or more compared to children aged 5 y or less (HR 0.64, 95% CI 0.45–0.92, p = 0.016).
Cumulative Hazard of a Subsequent Clinical Infection according to Change in Predominant Amino Acid
shows point estimates and confidence intervals comparing the odds of a change in predominant haplotype or predominant amino at each polymorphic residue in intervals including an asymptomatic infection followed by a symptomatic infection to intervals including consecutive asymptomatic infections, adjusting for year, age, and time between infections. The odds of a change in predominant amino acid at residues 1691, 1700, and 1701 were significantly greater in intervals ending with a clinical infection compared to intervals containing consecutive asymptomatic infections (1691: OR 2.01, 95% CI 1.08–3.73, p = 0.028; 1700: OR 1.91, 95% CI 1.04–3.48, p = 0.036; 1701: OR 1.92, 95% CI 1.04–3.52, p = 0.036). For changes of any type, the longer the amount of time between infections, the greater the odds of a change. The patterns were the same when intervals including mixed infections were excluded and when GEE was used to take into account multiple intervals from the same individual; point estimates were similar, but confidence intervals were wider, resulting in some associations no longer being significant at α = 0.05 (unpublished data).
Association between Change in Predominant Haplotype or Amino Acid and Development of Clinical Symptoms