Gametocyte Carriages of Plasmodium falciparum (pfs25) and Plasmodium vivax (pvs25) during Mass Screening and Treatment in West Timor, Indonesia a Longitudinal Prospective Study

Background: Three rounds of mass screening and treatment (MST) demonstrated no effect on Plasmodium falciparum and P. vivax incidence in West Timor, Indonesia. This study nested within that trial evaluated the effect of MST on gametocyte carriage. Methods: Microscopy and PCR diagnostics were applied to study subjects through 3 months of MST involving dihydroartemisinin-piperaquine (DHP) and primaquine based on infecting Plasmodium species. RT-qPCR targeting the pfs25 and pvs25 sequences was conducted to detect and quantify gametocytes in blood samples of P. falciparum and P. vivax- infected subjects. Data from the baseline and endpoint were compared (p<=0.05 as the signicance threshold).

spreader individuals that carry gametocytes and may contribute substantially to transmission in an area -pointing to the importance of treatment of the individual [7][8][9][10].
Gametocytes originate from the asexual cycle in humans. In an infected individual, a small proportion of the total parasites differentiate into gametocytes [6]. Plasmodium falciparum immature gametocytes sequester in the capillaries of inner organ until they mature, typically taking 10-14 days [6]. Since blood schizonticides do not kill mature gametocytes -they may be present 2-4 weeks post treatment [11][12][13].
Conversely, P. vivax development occurs concurrent to that of asexual parasites with treatment rapidly removing them from circulation [6,14].
Though frequently reported to be less infective compared to higher density symptomatic infections [20][21][22], these asymptomatic infections may contribute substantially to transmission due to their high prevalence in communities [23]. It has been estimated that asymptomatic individuals are the source of up to 50% of transmission [15], thus, transmission stemming from asymptomatic infections may represent an important problem in control and elimination of malaria [24].
Mass drug administration (MDA) and mass screen and treat (MST) strategies have been developed and implemented as means of attacking that asymptomatic reservoir [2][3][4][5]. In MDA, treatment is given to all residents of a prescribed area without regard to infection status, whereas in MST treatment is only given to residents who test positive for infection.
We reported that three rounds of MST administered in cluster randomized fashion at sites in endemic West Timor in eastern Indonesia failed to diminish the incidence of malaria [5]. In order to better understand that nding, we speci cally examined gametocyte carriage among residents living in those clusters.

Study area
This study was conducted in Wewiku subdistrict, Belu district, East Nusa Tenggara province, in eastern Indonesia (June-September 2013). The study area was reported to have the highest malaria endemicity in the province. The annual parasite index (API) per 1,000 population was 72 and 124 in 2011 and 2012 respectively (Belu district health o ce, personal communication), whereas in the entire East Nusa Tenggara province it was 25.8 and 21.1 during the same period [25]. Anopheles barbirostris was reported as the major malaria vector [5]. Other less dominant vectors included An. subpictus and An. vagus. A more detailed description on the study site has previously been reported [5].

Sample Collection
The original cluster-randomized trial evaluated the impact of MST conducted a) twice, and b) three times, both within 3 months [5]. Positivity for malaria parasites by microscopy was 8% in both MST at baseline, and 9% each at the last round of MST [5]. MST exerted no impact on measured incidence of diagnosed infections in both arms. In this analysis, round-1 of MST from both treatment groups were pooled and considered the 'baseline', while the last time-point from both groups was also pooled and considered the 'endpoint'.
Blood samples (n = 1551) were collected into EDTA tubes from 866 study subjects. All microscopypositive subjects were treated with dihydroartemisinin-piperaquine as a blood schizonticide, and primaquine (P. falciparum: 0.75 mg/kgBW single dose, P. vivax: 0.25 mg/kgBW/day for 14 days). For RNA extractions, 50 µL of blood was mixed with 250 µL of RNAProtect (Qiagen) within 4 hours of collection, and stored at -80 o C until processing.

Molecular Analysis
Microscopic and PCR screening results have been reported in the parent study [5]. DNA quanti cation was performed by species-speci c qPCR as described previously with some modi cation [26]. Details on the laboratory procedures are described in the Additional le 1. All P. falciparum and a set of randomly selected P. vivax positive samples underwent RNA extraction ( Fig. 1) using the Quick RNA Mini-prep kit (Zymo Research, USA) according to manufacturer's instruction. P. falciparum and P. vivax gametocytes were quanti ed by two-step RT-qPCR of the gametocyte markers pfs25 and pvs25 [27]. Transcriptor First Strand cDNA kit (Roche) was used to generate cDNAs for each sample in triplicates. During this rst step, 4 µL RNA was mixed with 2 µL random hexamer, 1 µL anchored oligo (dT) primers, 4 µL RT buffer, 2 µL dNTP, 0.5 µL RNAse inhibitor, and 0.5 µL Reverse Transcriptase enzyme for a nal volume of 20 µL. The incubation temperature was 10 min at 25 o C, followed by 30 min at 55 o C, then 5 min at 85 o C. The tube was then immediately put on ice. The presence of mRNA transcript was veri ed using RT-qPCR targeting the 18S rRNA [28]. Only samples positive for 18S were analyzed for pfs25 and pvs25. The pfs25 and pvs25 qPCR was conducted in 12 µL total volume containing 6 µL FastStart Essential DNA SYBR Green master (Roche), 4 µL cDNA, and 0.417 µM each of previously published primers [29]. The cycle conditions were as follows: 10 min at 95 o C, followed by 45 cycles of 15 min at 95 o C and 1 min at 58 o C. T M for P. falciparum was 74-75 o C, and 79-80 o C for P. vivax (Additional le 2). For quanti cation, series of plasmid harboring target sequence with concentration of 10 5 , 10 4 , 10 3 , 10 2 , 10, 5, and 1 copy per reaction were run in triplicate and a standard curve generated for each run. Negative (no template) controls were included in triplicates. The limit of detection (LOD) as assessed by running serial dilution of the plasmid in quintuples and determined as 10 copies/µL for pfs25 and pvs25. Performance of the assay is described in Additional le 3. All triplicates of the cDNA were run and recorded as positive if a minimum of two of the three demonstrated a positive result. Quantities of the transcript were reported as the average transcript numbers of the replicates to the nearest CT values.
All laboratory analyses were performed blindly to subject identi cation (ID). Upon completion of all laboratory work, the results were linked to the subject ID, MST time point, and treatment status. All laboratory analyses were conducted at the Indonesian Medical Education and Research Institute (IMERI), Faculty of Medicine, University of Indonesia, Jakarta.

Data analysis
Categorical variables were analyzed using chi square test. Numerical variables were analyzed using Student's t-test when the distribution was normal or Mann-Whitney when it was not normal. 18S rRNA gene copies and pfs25/pvs25 transcripts were log transformed for these statistical analyses. Linear regression was conducted to investigate the correlation between 18S and pfs25/pvs25 numbers. A pvalue ≤0.05 was considered as statistically signi cant. All analyses were performed using SPSS version 23 (IBM, Armonk, New York).

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. 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   Every parasitemic subject and gametocyte carrier was justi ed 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 eld 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).

Discussion
The current ndings corroborate and help explain the observed failure of MST to impact malaria incidence. Despite delivery of effective therapy of patent malaria against all parasite stages, including gametocytes, insu cient asymptomatic reservoir clearance by this intervention resulted in new parasitemic and gametocyte carriers that appeared to sustain untrammeled transmission within MST clusters. [5].
Towards understanding the gaps in protection with the MST intervention, it is particularly important to understand the origin of new parasitemic subjects and gametocyte carriers. There are two possibilities for the source of these infections. First, since a high proportion of them were negative at baseline, these infections may stem from newly infected individuals (Fig. 3). Being negative at baseline, most of these subjects (parasitemic and gametocytemic) were thus not given drugs at baseline. In addition, many infections were at too low densities to be detected by light microscopy and thus were also not treated.
Several studies have demonstrated that the numbers of parasites and gametocytes may uctuate in the blood [15,29] presenting the second possibility that a proportion of carriers, may have escaped a positive diagnosis with levels below the detection limit, and remained not given drugs at baseline. This was partly due to the sequestering parasites which resulted in a negative diagnosis -but were present and detectable at the endpoint [30]. In addition, it is possible that gametocytes in peripheral blood may constitute brief transience prior to more sustained sequestration in the shallow microvasclature of skin accessible to mosquitoes [31,32]. We did not explore that possible anatomic compartment in the current study. Lastly, in P. vivax there may be a considerable proportion of the population that are bloodstage negative but may have dormant infections in their livers [33].
The actual impact MST has on transmission dynamics raises questions on its role in eliminating malaria. In this context, MDA complying with appropriate safety procedures may be considered over MST. The MDA approach would counter the gaps in protection seen with MST -here diagnostic limits of detection resulted in sustained transmission with a stable infectious reservoir of gametocytes. MDA studies have demonstrated that this approach may reduce incidence in low endemic malaria areas [2,34]. However, combining this approach with vector control was shown to have bigger impact to lower the incidence [2].
The limitation of this study is the design which is nested within a community trial. The lack of randomization in the design of this study may possess confounding factors -even though multiple criteria, such as age and gender, were comparable between groups (Table 1).
Nevertheless, this study demonstrated the presence of a substantial number of new parasitemic subjects and gametocyte carriers at the end of the MST trial -pointing to a limitation associated with the persistence of malaria transmission. Programs that consider MST as an intervention need to consider its mechanistic impact on malaria transmission, while also taking MDA into consideration in the drive towards malaria elimination.