In this study we found that HbAS was associated with a delayed time to first malaria episode, an association that remained statistically significant in multivariate Cox regression analysis. Numerous case-control [7
] and longitudinal [12
] studies have established that HbAS decreases the risk of malaria, as measured by odds and incidence rate ratios, respectively. However, the effect of HbAS on the time to first malaria episode had not been established using time to event analysis. Aidoo et al. showed by time to event analysis that HbAS is associated with a reduction in all-cause mortality, but malaria-specific outcomes, including severe malarial anemia and high-density parasitemia, were reported as incidence rates [21
]. In Gabon, investigators found no association between HbAS and time to malaria episodes [19
], but the results of this study are difficult to interpret for several reasons: 1) individuals were initially enrolled in a hospital-based, case-control study of severe malaria, 2) age and other covariates were not taken into account when HbAS and non-HbAS individuals were compared longitudinally, and 3) statistical methods were chosen that were inappropriate for the analysis of correlated sojourn times between consecutive malaria episodes of the same subject. This approach is further complicated by the problem of defining when a malaria episode ends, and when an individual becomes susceptible to subsequent episodes after treatment.
Several features of our study made it well-suited to assess the impact of RBC polymorphisms on the time to first malaria episode. The study population was an age-stratified random sample representing 15% of all individuals living in a rural, well-circumscribed, non-migratory community where anti-malarial drugs were provided exclusively by the study investigators. Enrollment occurred over a short period of time approximately 1 month prior to the abrupt onset of malaria transmission (). Ninety-three percent of individuals had negative blood smears for P. falciparum
at the time of enrollment, obviating the need for a treatment-reinfection study design [22
], an approach that may alter the subsequent risk of malaria [23
]. Moreover, other aspects of our study favored an unbiased detection of malaria episodes: 1) follow-up at regular cross-sectional visits was >99% among 2–10 year-old children, indicating a high degree of study awareness and participation, 2) the average distance of individuals’ homes to the study clinic was 382 meters, minimizing geographic and logistic barriers to study participation, and 3) a study physician was available at all times at the only easily accessible health care facility in the area.
The use of time to first malaria episode as a study endpoint is increasingly common in clinical trials of malaria vaccines [24
] and anti-malarial drugs [2
], and in observational studies of malaria immunity [3
]. Indeed, the WHO recently recommended that the time to first malaria episode serve as the primary endpoint in phase III clinical trials of candidate malaria vaccines [1
]. An imbalance in the distribution of HbAS between comparator groups, whether in randomized trials or observational studies, may occur by chance and lead to invalid conclusions. The clear impact of HbAS on this endpoint, and the possibility of a biological interaction between HbAS and certain interventions (e.g. candidate blood stage vaccines), provides a rationale for routine Hb typing in such studies that are often conducted in areas where the prevalence of HbAS exceeds 25% [6
]. To date, Hb typing has not been consistently reported in such studies [5
]. Hb typing not only ensures a balanced distribution of HbAS in randomized trials, but incorporating the information into data analyses can increase the statistical power for detecting the effect of an intervention and hence reduce the required sample size. This is particularly important in malaria vaccine trials in which current vaccine candidates are anticipated to have relatively low efficacy (e.g.≤50%) [26
Several mechanisms have been proposed to explain how HbAS confers protection against malaria, including decreased RBC invasion or poor growth under low-oxygen tension [27
], enhanced removal of parasitized RBC [29
], accelerated acquisition of antibodies specific for PfEMP-1 and other variant surface antigens [31
], and more recently, reduced cytoadherence of parasitized RBC [33
]. Anti-PfEMP-1 antibodies could thus work cooperatively with HbS to further reduce cytoadherence [33
], which may explain the apparent effects of age-associated acquired immunity on malaria protection by sickle trait [34
]. It is conceivable that any one or a combination of these processes underlies the association between HbAS and the delayed time to first malaria episode observed in this study, either by prolonging the time it takes to achieve symptomatic parasite densities or increasing the probability that asymptomatic infections will be aborted. Since we did not actively survey for asymptomatic parasitemia we could not distinguish between these two possibilities. However, the former is supported by the results of a study in Senegal in which weekly blood smears after quinine treatment showed a delay in the reappearance of asymptomatic parasitemia in HbAS individuals [22
]. However, the relationship between asymptomatic parasitemia and the subsequent risk of malaria is complex and not fully understood [35
It is unlikely that differential mosquito exposure confounded the association between HbAS and the time to first malaria episode, since HbAS individuals appeared to be randomly distributed within the small, well-circumscribed village that lacks a dominant body of water (). Moreover, the frequency of bednet use was evenly distributed between HbAS and non-HbAS individuals. It is also unlikely that access to the study clinic played a significant role, since distance to the clinic from individuals’ homes did not differ by hemoglobin type.
*A-, −α/αα, and blood group O have been associated with protection from severe malaria [9
], but their role in protection against uncomplicated malaria is less clear. Consistent with this, we did not observe an association between these polymorphisms and the time to first malaria episode, or malaria incidence, although more subtle effects may have been detected with a larger sample size. Notably, we did not detect an increase in the incidence of uncomplicated malaria among G6PD
*A heterozygous females that has been observed by others [19
]. Consistent with a recent study [20
], we observed negative epistasis between HbAS and −α/αα. However, the frequency of −α/−α in this cohort was too low to confirm the negative epistatic interaction with HbAS reported previously [39
Interestingly, asymptomatic parasitemia at enrollment (end of the dry season) was associated with a decreased risk of subsequent malaria in multivariate analysis. This finding is consistent with those of a recent longitudinal study in Senegal where malaria is also highly seasonal [36
]. In Uganda, however, where malaria is meso-endemic, asymptomatic parasitemia was reported as a risk factor for malaria [35
], suggesting that the clinical outcome of asymptomatic parasitemia might vary with the epidemiological setting. In neither of these studies, however, were HbAS and other malaria-protective polymorphisms investigated as potentially contributing factors. In-depth studies of both host and parasite factors may reveal important mechanisms by which host immune responses and parasite immune evasion are balanced.
The delayed presentation of malaria associated with HbAS in this study adds to the measures by which the protective effect of this remarkable polymorphism has been documented. Clinical studies of malaria that use the time to first malaria episode as an endpoint should incorporate routine Hb typing in the study design to account for its effect. It appears that other common RBC polymorphisms do not significantly impact this endpoint, though this may vary in different epidemiological settings. Although this study was designed to investigate the acquisition and maintenance of malaria immunity, the observed effects of HbAS validate this study’s ability to detect factors that influence malaria morbidity.