Characteristics of the cohort
The birth cohort included 1855 infants and the second of 36 twin births were excluded to prevent clustering effects during analyses (Figure 1). Table 1 summarizes characteristics of the birth cohort data examined. The 1819 infants included in the analyses had in total 19231 scheduled monthly (active) visits, corresponding to a compliance rate (observed visits/expected visits × 100) of 88% (Table 1). For infants who reported illness between any two successive active visits, 74% (1341/1819) had a corresponding passive visit record.
Malaria microscopy test was performed for 92% (1674/1819) of infants at their active or passive visits (Table 1 and Figure 1). The infants had a mean follow up time of 6 (SD: ± 3) visits.
Malaria parasites were detected among 58% (971/1674) of infants at least once during the follow up period. In total, 55% (919/1674) of infants had malaria parasites and with corresponding temperature test results and/or history of fever, and these were used to define symptomatic or asymptomatic outcomes.
Malaria parasites, symptomatic malaria and asymptomatic infections in the cohort
Out of a total of 12926 malaria microcopy tests performed during the follow up for 1674 infants, 19% (2394/12926 from 971 infants) were positive for malaria parasites. Majority of the infections were P. falciparum (96%; 2293/2394), followed by P. malariae (2%; 41/2394) and ‘other species’ (P. ovale, or P. vivax: 1%; 21/2394). Mixed species infections were rare (1%; 27/2394) and mainly P. falciparum and P. malariae co-infections (70%; 19/27). Gametocytes were detected in 1.6% (39/2394) of all infections. P. falciparum contributed 85% (33/39), P. malariae 5% (2/39) and other species 10% (4/39) of all gametocytes.
Of 2394 malaria parasite infections, 92% (2194/2394 - among 919 infants) had corresponding temperature tests and/or history of fever and were classified as asymptomatic or symptomatic. On both active and passive visits, proportion of parasite negative tests decreased while the proportion of asymptomatic infections and symptomatic malaria increased (Figures 2a and 2b). Of all infections classified, 61% were accompanied with symptoms (1340/2194); and 68% (913/1340) of the symptomatic malaria were detected at passive ill visits while 32% ( 427/1340) were detected at active visits.
Prevalence of infections (symptomatic or asymptomatic) increased from birth to twelve months of age and was highest (20%, 261 infants infected/1314 total number of infants) at seven months of age (Figure 2c). From birth to five months of age, infections were predominantly asymptomatic and from six to twelve months of age both symptomatic malaria and asymptomatic infections became more frequent among the infants (Figure 2c). This trend was also reflected in the cumulative incidence of symptomatic malaria which was similar to asymptomatic infections until five months of age, after which most infections were symptomatic (Figure 2d).
For both symptomatic malaria episodes and asymptomatic infections, the median P. falciparum parasite density was significantly lower from birth to five months of age compared to six months of age and above (Supplementary Figure 2).
Profiles of longitudinal symptomatic malaria and asymptomatic infections
For detailed description of infection dynamics throughout the first year of life, infants (70%, 1264/1819) who had microscopy test results and recorded temperature and/or fever results on eight or more active visits and any passive visits were analyzed (Supplementary Figure 1a and 1b), to identify the temporal occurrence of asymptomatic infections and symptomatic malaria.
Overall temporal profiles
Four main groups were identified from the longitudinal profiling of asymptomatic infections and symptomatic malaria among the 1264 infants: i) “parasite negative” [36% (459/1264)], if an infant did not have malaria parasitemia during all follow up visits, ii) “Always symptomatic” [35% (444/1264)], if an infant had symptomatic malaria on all occasions parasites were detected. iii) “Always asymptomatic” [7% (87/1264)], if an infant had asymptomatic infection on all occasions parasites were detected, and iv) “Alternating” [22% (274/1264)], if both asymptomatic infection and symptomatic malaria were detected over the first year of life.
In infants with malaria parasites, infections occurred intermittently throughout the year on both passive visits (where microscopy was performed on the same day and infants were treated) and active visits (where infants were ‘healthy’, and microscopy was performed at a later date and no efforts were made to treat) (Figure 3). Infections among infants in the always asymptomatic group were identified during active visits only, unlike the other groups whose infections were identified during both active and passive visits (Figure 3).
Infection profiles among groups of infants
Malaria parasites were detected among 64% (805/1264) of infants in this sub-cohort at least once during the active and passive visits. A proportion [37% (299/805)] of infants belonging to the always symptomatic and always asymptomatic groups had only one detectable infection over the twelve months of follow up (Figure 4). While 55% (246/444) of infants within the always symptomatic group had only one symptomatic malaria episode, 61% (53/87) of infants in the always asymptomatic group had only one asymptomatic infection.
Among the parasite positive infants, 63% (506/805) had two or more separate infections over the one year period (Table 2). Of these, 54% (274/506) had both symptomatic and asymptomatic infections i.e. alternating group, 39% (198/506) were in the always symptomatic group and 7% (34/506) in the always asymptomatic group (Table 2).
The cumulative incidence of infections from birth to twelve months of age was highest in the alternating group, followed by always asymptomatic group – where it was similar to the always symptomatic group from eight months of age onwards (Figure 5a).
The mean number of infections were similar in the always symptomatic [1.75 infections/child (779/444)] and always asymptomatic groups [1.85 infections/child (161/87)] (P = 0.715). Within the alternating group, the mean number of symptomatic malaria episodes was 2.17 infections/child (595/274) and asymptomatic infections was 1.86 infections/child (509/274). Per the default definition, single infections were not determined for infants in alternating group (Figure 4).
When comparing infants with two or more infections, the alternating group had the highest mean number of multiple infections [4.03 infections/child (1104/274)] compared to the always symptomatic [2.69 infections/child (533/198)] and always asymptomatic [3.18 infections/child (108/34)] groups (P = 0.001).
Analysis of the number as well as the sequence of infections in the alternating group showed 87 different combinations of asymptomatic infections and symptomatic malaria (Table 3). Half (57%; 50/87) of these combinations of alternating asymptomatic infections and symptomatic malaria were unique to individuals (Table 3). Assessing the number of times asymptomatic infections and symptomatic malaria alternated in any of the 87 combinations showed three alternating infections [observed in six different sequences among 25% (68/274) of infants] were frequent, while ten alternations at the most occurred (Table 3). The most common alternating sequence was to first have asymptomatic infection(s) followed by a symptomatic episode(s) (46%; 125/274). The second most common sequence was first symptomatic episode(s) followed by asymptomatic infection(s) (19%; 51/274) (Table 3).
The time span between any two infections peaked between 27 and 38 days for all groups. Infections which were six months (180 days) or more apart were rarely observed in all groups (Supplementary Figure 3).
Age at infections among groups of infants
First infections were detected from one to twelve months of age [median = 6 months, (IQR: 5 – 8)]. Age at first infection was lowest in the alternating group [median = 5 months (IQR: 4-7)] compared to the always asymptomatic group [median = 7 months (IQR: 4-9)] or always symptomatic group [median = 7 months (IQR: 5-9)], all P < 0.001.
In the alternating group, first infections which were asymptomatic [61% (167/274) of infants] were more frequent than first infections which were symptomatic [39% (107/274) of infants] (P = 0.003).
Whereas the first symptomatic malaria episodes begun from two to twelve months of age for infants in the always symptomatic group (Figure 5b), first infections were detected from one to eleven months of age for infants in the alternating group (Figure 5c). For infants in the always asymptomatic group the first infections occurred between one and twelve months of age (Figure 5d).
Although infections were detected at all ages, only few infants (5%; 36/805) had infections at two months of age, and they were mainly from the alternating group [69% (25/36)]. For all groups with malaria parasites, the number of times infants were infected (on separate occasions) increased as they aged (Figure 5b, 5c and 5d), but the proportion of infants infected decreased with age (Table 2).
The median age of infection was seven months (IQR: 6 – 10) for the alternating group and eight months for both always asymptomatic (IQR: 5 – 9) and always symptomatic groups (IQR: 6 – 10).
Parasite densities among groups of infants
The range of P. falciparum asexual parasite densities overall was 2 – 974759 parasite/ul. Parasite densities increased with age in all groups (Figure 6), and the median parasite density at the first infection was highest in the always symptomatic group, followed by the alternating group and lowest in the always asymptomatic group (P < 0.001) (Table 4). Compared to other groups, the always symptomatic group had the highest median parasite density from one to six months of age (P < 0.001) as well as from seven to twelve months of age (P < 0.001) (Table 4).
Within the alternating group, the median parasite density [14812 parasite/ul (IQR: 2847 – 77843)] was higher when infants were symptomatic compared to when they were asymptomatic [2848 parasite/ul (IQR: 858 – 8228)] (P < 0.001).
In all groups, the parasite densities were lower the first six months of age compared to seven months of age and above (all P < 0.001).