Characteristic of the study subjects
A total of 811 samples were collected at baseline and 740 at endpoint. These subjects represented 78% of the total parent study samples. Of the 102 P. falciparum positive samples (baseline = 50, endpoint = 52), 83 were available for RNA extraction (Fig. 1). Of the 334 P. vivax positive samples (baseline = 192, endpoint = 142), 231 samples were available for RNA extraction (Fig. 1). Comparable demographic characteristics were observed among study subjects between baseline and endpoint (Table 1). Furthermore, 685 (84%) subjects were sampled at both time points.
Table 1
Characteristic of the study subjects
| Baseline | Endpoint |
Total subjects | 811 | 740 |
Age | | |
< 5 y5-15 > 15 y Unknown | 90 (11.1%) 177 (21.8%) 543 (67.0%) 1 (0.1%) | 88 (11.9%) 160 (21.6%) 490 (66.2%) 2 (0.3%) |
Male (%) | 387 (47.7%) | 337 (45.5%) |
pfs25 / pvs25 RT qPCR assay
A single peak was demonstrated in the melting analyses of both pfs25 and pvs25 RT qPCR. PCR efficiency for pfs25 and pvs25 were 94.7% and 88.1%, respectively. Both assays demonstrated r2 of more than 0.98 (Pf: 0.986, Pv: 0.988) and LOD of 10 copies/µL (Supplementary file S3 Assay performance).
For both species, gametocyte transcript numbers were positively correlated with 18S gene copy numbers, with a stronger correlation observed for P. vivax than P. falciparum (Pf: r = 0.321, p < 0.001; Pv: r = 0.778, p < 0.001, Fig. 2A and B).
Parasitemia (microscopy and/or PCR) and gametocyte carriage between baseline and endpoint
Plasmodium falciparum: Between baseline and endpoint, no significant difference in prevalence was observed (6%=52/811 versus 7%=50/740, p = 0.838) (Table 2, Fig. 1). Furthermore, no significant difference was detected for the proportion of gametocyte positive infections (43%=19/44 versus 59%=23/39, p = 0.189) (Table 2, Fig. 1). The geometric mean pfs25 transcript numbers did not show any significant difference either (baseline: 132.5 transcripts/uL, CI95 30.2-580.5, endpoint: 210.9 transcripts/uL, CI95 56.8-782.7, t-test, p = 0.625) (Table 2).
A higher number of pfs25 transcripts were observed among the subjects taking drug compared to those without drug (with drugs: 182.8, CI95 29.1-1150.5, without drugs: 39.6, CI95 3.3-472.1, t-test, p = 0.39). Among gametocyte carriers, all children < 5 year of age (n = 4), 80% (4/5) of school-aged children (5–15 y), and 70% (7/10) adults (> 15 y) were positive by microscopy and thus given drugs.
Plasmodium vivax
Although the proportion of parasitemic subjects was significantly lower at the endpoint compared to baseline (23.7%=192/811 versus 19.2%=142/740, p = 0.035) (Table 2, Fig. 1), no significant difference was seen in the proportion of gametocyte positive infections (39%=49/125 versus 37%=39/106, p = 0.786) (Table 2, Fig. 1) as well as the geometric mean of transcript numbers (56.4, CI95 23.8–134.0 transcripts/uL and 65.6, CI95 26.5-162.2 transcripts/uL, p = 0.810) (Table 2).
Subjects identified for treatment harbored approximately 37-fold higher transcripts than the untreated carriers (geometric mean & 95%CI: 356.6, 97.2-1309.8 versus 9.6, 5.1–18.2, t-test, p < 0.001).
Table 2
Parasitemia and gametocyte carriages at baseline and endpoint by species
| Baseline | Endpoint | p value | OR (95%CI) |
Total subjects | 811 | 740 | | |
P. falciparum | | | | |
Positive by LM and/or PCR | 52 (6%) | 50 (7%) | 0.838 | 1.06 (0.69–1.61) |
No. screened for pfs25a | 44 | 39 | | |
Gametocyte carriage (%) | 19 (43%) | 23 (59%) | 0.189 | 1.89 (0.72–4.97) |
Transcript numbers | 132.5 (30.2-580.5) | 210.9 (56.8-782.7) | 0.625 | 0.6 (0.1–4.2)c |
P. vivax | | | | |
Positive by LM and/or PCR | 192 (24%) | 142 (19%) | 0.035* | 0.77 (0.59–0.98)* |
No. screened for pvs25b | 125 | 106 | | |
Gametocyte carriage (%) | 49 (39%) | 39 (37%) | 0.786 | 0.90 (0.51–1.59) |
Transcript numbers | 56.4 (23.8–134.0) | 65.6 (26.5-162.2) | 0.810 | 0.9 (0.2-3.0)c |
aBased on RNA availability |
bBased on random selection and RNA availability |
cMean ratio (95%CI) |
Abbreviation, LM: light microscopy |
Dynamic of parasitemic subjects and gametocyte carriers at baseline and final time point
Every parasitemic subject and gametocyte carrier was justified individually at each timepoint, and their status was followed or traced backwards. Furthermore, parasitemic/gametocyte status was linked to whether or not they took antimalarial drugs. The drugs were given under direct observation of the field study team.
Plasmodium falciparum
At baseline, 28 of 52 P. falciparum were microscopic positive and thus given drugs (Table 3). Of these subjects, 22 were followed at the endpoint and six subjects were lost to follow up. Of those 22, 20 were negative at endpoint while 2 remained positive (Fig. 3A). Among the 28 drug treated subjects, 15 were gametocyte carriers (Table 3). Ten of those 15 subjects were followed, and all were negative at the endpoint.
At the endpoint, on the other hand, there were 50 parasitemic subjects (Table 2). Of these subjects, 40 were negative at baseline, and 6 were not previously seen (Fig. 3A). The other four parasitemic consisted of two subjects with a previous drug treatment (originally from the 22 subjects taken drugs above), and two subjects without treatment (originally from 24 subjects without drugs above) (Fig. 3A, Table 3). Among the 50 parasitemic subjects, 23 were gametocyte carriers (Table 2). Of these carriers, 20 were negative at baseline, while the other 3 consisted of one carrier did not receive drugs and two subjects not seen at baseline (Fig. 3A).
Plasmodium vivax
At baseline, 54 of 192 parasitemic subjects were microscopic positive and thus given drugs (Table 3). Of these subjects, 50 were followed and included in the endpoint sample while four were lost to follow up. Of these 50, 44 were negative at endpoint while 6 were positive (Fig. 3B). Among the 54 subjects with drugs at baseline, 26 were gametocyte carriers (Table 3, Fig. 3B). Of these carriers, 23 were followed at endpoint and 22 were negative (Fig. 3B).
At endpoint, there were 142 parasitemic subjects (Table 2). Of these, 85 were negative at baseline, eight were newly diagnosed subjects, six were given drugs at baseline, and 43 were infected but not treated at baseline (diagnosed positive by PCR) (Fig. 3B). Among the 142 parasitemic subjects, 39 were gametocyte carriers (Table 2). Among these 39 carriers, 30 were negative at baseline, seven were newly diagnosed subjects, one was given drugs at baseline, one was without drug treatment (Fig. 3B).
Table 3
Subjects with and without drug treatment at baseline
Description | |
Plasmodium falciparum | 52 |
Positive by LM (drug treated) | 28 |
Positive by PCR (not treated) | 24 |
Gametocyte carriage (pfs25) | 19 |
Drug treated | 15 |
Not treated | 4 |
Plasmodium vivax | 192 |
Positive by LM (drug treated) | 54 |
Positive by PCR (not treated) | 138 |
Gametocyte carriage (pvs25) | 49 |
Drug treated | 26 |
Not treated | 23 |