Evaluating the technical feasibility of serological testing and treatment for Plasmodium vivax in mobile at-risk of malaria Cambodian populations

Summary

Background

Cambodia is targeting malaria elimination by 2025, aligning with the WHO's Mekong Malaria Elimination program. While elimination of Plasmodium falciparum is nearly achieved, Plasmodium vivax elimination presents challenges inherent to this species due to the occurrence of dormant parasite stages, known as hypnozoites. A new approach has been proposed to serologically identify individuals likely carrying hypnozoites that should receive appropriate antimalarial treatment: P. vivax serological testing and treatment (PvSeroTAT). This study aims to determine the technical feasibility of a PvSeroTAT approach in malaria endemic communities with highly mobile populations in Eastern Cambodia.

Methods

From October 24th 2021 to February 26th 2023, two successive rounds of PvSeroTAT were conducted in adult and adolescent males in three villages of Mondolkiri, Eastern Cambodia. At each round, capillary blood samples were collected from consenting participants to be used for P. vivax serology and G6PD activity determination. Seropositive participants, who were G6PD normal, were then recontacted to be provided an anti-hypnozoite primaquine regimen following Cambodian treatment guidelines (0.25 mg/kg for 14 days). Cross-sectional surveys to evaluate P. vivax prevalence were conducted before, during and after the PvSeroTAT interventions in the same three villages and in three additional neighboring control villages where interventions were not implemented.

Findings

Participation was high, with 96% (456/477) of eligible individuals enrolled in at least one round of PvSeroTAT. However, only 63% of participants enrolled in the first PvSeroTAT round agreed to participate in the second round. In the first and second round of PvSeroTAT, 31% (101/327) and 30% (98/334) of enrolled participants, respectively, were seropositive and among those, 82% (163/199) were eligible for primaquine treatment. All 163 seropositive eligible individuals could be recontacted and offered a primaquine treatment, this occurred within 10 days for 96% of individuals (157/163). P. vivax prevalence decreased in all villages, including the control ones, after the first round of PvSeroTAT from 7.7% to 2.7% overall.

Interpretation

The participation rates and overall technical feasibility of PvSeroTAT in highly mobile individuals living within communities in malaria endemic areas of Cambodia were very promising. PvSeroTAT with a lab-based assay is feasible in Cambodia even if it is logistically more challenging than using point-of-care assays. Further studies to understand community perspectives about test and treat approaches in the absence of clinical symptoms will be important for the development of tailored community education and awareness material to improve participation in multiple rounds of test and treat interventions.

Funding

The PvSeroTAT interventions received funding from the Global Fund RAI3 initiative. Cross-sectional surveys were funded by the NIH International Centers of Excellence for Malaria Research (ICEMR) Asia–Pacific (U19AI129392).

Research in context.

Evidence before this study

We searched PubMed using the terms “Plasmodium vivax” [Title/Abstract] AND “serology” [Title/Abstract]. Among the 59 articles retrieved, most reported epidemiological studies to determine areas of transmission or using seroprevalence as surveillance tools for malaria transmission. Few investigated etiologies of febrile illness. No article reported using serology to drive radical cure treatment for P. vivax infections.

Added value of this study

This study is the first to report the technical feasibility of using serology to identify individuals at risk of P. vivax relapses and drive anti-hypnozoite treatment in hard-to-reach mobile individuals living in malaria endemic areas. This is of particular interest for countries approaching malaria elimination that require dedicated strategies for P. vivax.

Implications of all the available evidence

We report here that despite a reduction in malaria incidence over the past years, there is still a significant burden of asymptomatic P. vivax carriage in malaria endemic communities in Eastern Cambodia. We demonstrate excellent feasibility of PvSeroTAT among the most at risk of malaria and mobile individuals in communities. Studies investigating the impact of PvSeroTAT on P. vivax prevalence and transmission are needed.

Introduction

Malaria elimination is a major public health priority in South-East Asian countries, with a commitment to achieving this goal by 2030. Cambodia in particular has set an ambitious target, aiming for malaria elimination by 2025.¹ While tremendous progress has been made concerning Plasmodium falciparum, now nearly eliminated from the country with a few dozen cases reported in 2023, the situation is drastically different for Plasmodium vivax, with several thousand clinical infections reported during the same period.² Two main reasons explain this difference in the effectiveness of control efforts between the two Plasmodium species. First, P. vivax establishes dormant, clinically silent infections in hepatocytes of infected individuals through parasite forms named hypnozoites. These hypnozoites can reactivate weeks to months (or even years) after mosquito inoculation and initial blood-stage infection, causing subsequent relapses.³ Currently, there is no direct diagnostic test for hypnozoite presence. Only 8-aminoquinolines such as primaquine or tafenoquine can kill P. vivax hypnozoites but the use of these drugs is challenged by serious safety issues if administered to glucose-6-phosphate-dehydrogenase (G6PD) deficient individuals.⁴ Consequently, G6PD activity must be assessed to safely administer these anti-hypnozoite drugs, a procedure not routinely performed in most remote malaria-endemic areas. The second major issue related to P. vivax is the high proportion of asymptomatic infections among individuals living in endemic areas. These asymptomatic cases comprise infections caused by hypnozoite reactivation as well as active infections by blood-stage parasites for which immunity is rapidly acquired. In addition, there is increasing evidence of a significant cryptic biomass of P. vivax in the spleen and bone marrow of infected individuals.^5,6 Several studies have shown that asymptomatic infections can represent up to 90% of all P. vivax cases.⁷ By definition, these individuals do not seek treatment and therefore fuel ongoing transmission in communities. Despite their typically low parasite densities, they can readily transmit the infection to mosquitoes.^8,9 Because of P. vivax resilience to control measures, dedicated strategies tailored to P. vivax specific biology are urgently needed.¹⁰

A new approach has been suggested to address the issue of asymptomatic hypnozoite carriers, extending beyond the conventional focus on clinical case detection and targeting individuals who may not exhibit symptoms. This approach relies on detecting the presence of antibodies targeting a panel of 8 P. vivax blood-stage antigens.¹¹ By analyzing serological profiles of individuals enrolled in longitudinal cohorts conducted in Thailand, Brazil and the Solomon Islands, a panel of 8 antigens was selected allowing the identification of individuals recently (within the prior 9 months) infected by P. vivax with 80% sensitivity and 80% specificity.¹¹ We recently further validated this panel by analyzing a longitudinal cohort of Cambodian individuals living in malaria-endemic areas followed monthly for 12 months.¹² Additionally, we have shown that these serological markers are predictive of subsequent P. vivax infections indicating that such a serology test can accurately identify individuals at risk of recurrent P. vivax parasitaemia.^11,12 Leveraging these results, a strategy tailored to P. vivax elimination has been proposed based on serological testing and treatment (PvSeroTAT): residents of a given area are screened for the detection of serological signatures of recent exposure to P. vivax, and seropositive individuals are administered anti-hypnozoite drugs adapted to their G6PD activity.^11,13,14 Mathematical modeling has shown that two rounds of PvSeroTAT six months apart could decrease P. vivax prevalence by up to 70% and could be of great utility in countries such as Cambodia that are approaching malaria elimination.^14,15

In Cambodia as well as in most countries in the Greater Mekong Subregion, malaria transmission primarily occurs in forests where the primary Anopheles vectors thrive.^7,16, 17, 18, 19, 20 Consequently, the most at risk of malaria individuals are those engaging in forest activities and spending substantial time (several days) there (referred to as forest-goers). Multiple studies have shown that the forest-goers are typically adolescent and adult males making this group the most at risk of malaria infections within endemic communities.^7,12,21 There are some challenges inherent to targeting this high risk group for malaria elimination based on their remote location but also on their mobile behavior. Two main approaches targeting forest goers have been proposed and evaluated to determine impact on malaria transmission, the first being usage of topical insect repellent and the second using chemoprophylaxis to prevent malaria. Vector control measures using topical repellent showed some impact on P. falciparum but had no significant effect on P. vivax infections.²² Offering chemoprophylaxis to forest goers with a schizonticidal drug (such as artemether-lumefantrine or artesunate-mefloquine) had significant impact on P. falciparum but more limited impact on P. vivax.^21,23 However, because these chemoprophylaxis did not include a hypnozoitocidal drug such as primaquine or tafenoquine, it is expected that despite a reduction in P. vivax prevalence, these strategies would not be sufficient to eliminate P. vivax. There is, however, reluctance by Malaria Control Programs to implement large-scale, mass treatment or chemoprophylaxis using 8-aminoquinolines because of the risk of hemolytic adverse events in G6PD deficient individuals. In such approaches, these drugs would be over-administered to a high number of individuals that do not need any treatment as they are not infected by P. vivax, thus outweighing the benefits of administration of 8-aminoquinolines. Since only individuals that present a signature of recent exposure to P. vivax are targeted in a PvSeroTAT strategy, this approach would provide the benefits of 8-aminiquinoline administration to a maximum number of individuals likely requiring such treatment and minimize over-administration of such drugs to individuals not requiring them, making PvSeroTAT an appealing alternative to mass strategies.

Here, we present the results of a study conducted to evaluate the technical feasibility of a PvSeroTAT approach in forest-going, highly mobile and hard-to-reach adult and adolescent males residing in communities in endemic areas of Cambodia. Specifically, this study was done to investigate if the most difficult to reach community members (i.e., highly mobile adults and adolescent males), that are the key high-risk group for continued malaria transmission, could be reached with PvSeroTAT.

Methods

Study sites

The PvSeroTAT intervention was conducted in three villages of Kaev Seima district, Mondolkiri province, Eastern Cambodia: Sraektum, Sraempilkroam and Aukaupreas. Three additional villages of the same area were selected as controls where no intervention was rolled out: Poucha, Sraempillieu and Sraepreas (Fig. 1). These six villages are located outside or at the fringe of forests where malaria transmission occurs. In these communities, adult and adolescent males face the highest risk of contracting malaria due to occupational activities conducted within the forests such as wood-logging and hunting.^7,12

Fig. 1.

Location of the study site in Mondolkiri province, Eastern Cambodia. The three intervention villages (red landmarks) are Aukaupreas (A), Sraempilkroam (B) and Sraektum (C) and the three control villages (green landmarks) are Poucha (D), Sraempilieu (E) and Sraepreas (F). The white bar indicates 5 km distance.

Study design and participants

Two successive rounds of PvSeroTAT were conducted in the three intervention villages with the first round taking place from July 6th to July 18th 2022 and the second round performed between December 6th and December 19th 2022 (Fig. 2). Participants included in the study were males aged more than 15 years old. Community awareness was facilitated through face-to-face community meetings, where the study team trained community workers for questionnaire and sample collection. These workers were instrumental in promoting word-of-mouth communication through community leaders to disseminate study information within the village and foster engagement and knowledge among the population. At each intervention, participants received a detailed explanation of the study. After providing written informed consent, they completed a brief questionnaire. A capillary blood sample was collected by finger-prick using 1.6 mm depth safety lancets into two EDTA microtainer tubes (each with a capacity of up to 500 μl) and kept at 4 °C in sealed plastic bags in cooler boxes filled with ice-cold water. While the target volume for each microtainer was 500 μL, the actual volume of blood collected varied between participants, ensuring it was sufficient for spectrophotometric G6PD activity testing (300 μL), as well as for PCR and serological assays (60 μL). The collection was performed in two tubes to minimize manipulation of the sample used for G6PD assessment. A single finger-prick was enough for most participants, but for some, two different pricks were needed. Samples were transported to the central laboratory of the Institut Pasteur du Cambodge in Phnom Penh within 12 h of collection and processed upon arrival, with an average transport time of approximately 6 h. In the laboratory, the blood samples were used for serological analysis, G6PD activity measurement and subsequently DNA extraction for retrospective PCR diagnostic of Plasmodium infection. Briefly, an aliquot of whole blood was used to determine G6PD activity by spectrophotometry. The second aliquot was separated into plasma and cell pellet, with plasma used for serological testing and cell pellet for DNA extraction and retrospective PCR detection of Plasmodium. Once serology and G6PD activity results were obtained (within 48 h of sample reception in the lab) they were communicated to the study team in Mondolkiri who conducted a follow-up visit with seropositive G6PD normal participants to provide them with their serology and G6PD testing results and with a supply of 14-days primaquine (0.25 mg/kg/day). Primaquine treatment was self-administered by participants with daily phone call from the study nurse to verify compliance and monitor any self-reported adverse events.

Fig. 2.

Schematic representation of the study. PvSeroTAT was conducted in 3 intervention villages in two successive rounds. Seropositive consenting participants with G6PD normal activity were offered a 14-day primaquine therapy. Cross-sectional surveys to evaluate P. vivax prevalence by qPCR were conducted before, during and after the interventions in these 3 villages as well as in the 3 control villages where no intervention was implemented.

Cross-sectional surveys

Cross-sectional surveys (CSS) were conducted in the three intervention and in the three control villages, covering the same study population of males aged 15 years and older. All eligible individuals were included in the CSS. The CSS were performed before the first round of intervention (October 24th to November 23rd 2021), between the two intervention rounds (October 4th to October 22nd 2022) and after the second round (February 7th to February 26th 2023). At each CSS, a capillary blood sample was collected by finger-prick in EDTA microtainer tubes and used for DNA extraction and retrospective PCR diagnostic of Plasmodium infection.

Laboratory analyses

G6PD activity was determined by spectrophotometry from 300 μl of blood using an ABX Pentra C400 instrument and Randox G6PD chemistry kits. Serological analysis was performed on 1 μl of plasma following established protocols.²⁴ IgG antibody responses to 8 P. vivax proteins (listed in Appendix Table S1)¹¹ were assessed using antigen-conjugated magnetic fluorescent beads in a multiplexed 96-well plate assay as described elsewhere.^24,25 Briefly, each antigen was conjugated to Luminex Magplex magnetic beads, mixed with a plasma sample diluted at 1/100 and allowed to bind. After washing, the antibody-protein-coated microspheres were incubated with a PE-conjugated anti-human IgG secondary antibody. The mean fluorescence intensity (MFI) detected on the Luminex platform (Bio-Plex 200) served as an indicator of the level of antibodies binding to the antigens present on the beads. The MFI values were transformed to an arbitrary relative antibody unit (RAU) value, calculated using a 5-parameter logistic model coded in R version 4.3.1.^24,25 This was based on the positive control standard curve: pooled plasma from Papua New Guinea in serial dilution (1/50 to 1/25,600) added on each plate to generate a standard curve for adjusting plate–plate variation.^24,25 Quantitative antibody levels obtained were then fed into a published Random Forest classification algorithm to categorize individuals as seropositive (recently exposed to P. vivax antigens) or seronegative (unexposed) using an RShiny app accessible at https://gitlab.pasteur.fr/tobadia/pvserotat-rshiny-app as previously described.²⁵ The Random Forest classification algorithm was trained on IgG antibody data from individuals in Thailand, Brazil and the Solomon Islands.¹¹

DNA extraction was performed from 10 μl of blood using InstaGene matrix and Plasmodium infections were determined by real-time PCR targeting cytB following established protocols.²⁶ Plasmodium positive samples were then tested using species-specific cytB real-time PCR to identify P. falciparum, P. vivax, Plasmodium malariae and Plasmodium ovale infections.

Statistical analyses

Proportions were compared using Chi-squared or Fisher’s exact tests while means were compared using t-tests or Mann–Whitney tests as appropriate. All analyses were performed using Graphpad Prism version 10.2.0.

Ethics approval

The study was approved by the National Ethics Committee for Health Research (NECHR) of Cambodia on May 10th 2021 (#080NECHR). The cross-sectional surveys were approved by the Walter and Eliza Hall Institute HREC (21/31).

Role of the funding source

Funders had no role in the study's design, data collection, analysis, interpretation, or report writing.

Results

Participation rates in at least one round of PvSeroTAT

We performed two successive rounds of PvSeroTAT in the same three villages. The population size of eligible individuals (males aged over 15 years) varied across the villages, ranging from 73 in Sraempilkroam to 261 in Aukaunpreas (Table 1). It is important to note that these population sizes are based on estimates provided by the head of each village. For the first round conducted in July 2022, the vast majority (94%, 446/477) of eligible individuals were approached and offered to participate in the study for all three villages (92% in Aukaunpreas, 94% in Sraektum, and 96% in Sraempilkroam). Of the individuals that could not be approached (n = 31, 6%), the majority (17/31, 55%) were engaged in forest activities at the time of the study. Among those offered to participate, 69% (166/241), 74% (100/135) and 87% (61/70) agreed to participate in Aukaunpreas, Sraektum, and Sraempilkroam, respectively, resulting in an overall enrollment rate of 73% (327/446).

Table 1.

Participants enrolled in the two rounds of PvSeroTAT.

Village	Pop sizea	1st round (July 2022)					2nd round (December 2022)					p-valued	Participated in
		Unreachable		Reachable	Of the approached, those who:		Unreachable		Reachable	Of the approached, those who:			Both roundse	At least one roundf
		In forestb	No infoc	Approached	Accepted	Refused	In forestb	No infoc	Approached	Accepted	Refused
Aukaunpreas	261	5% (13)	3% (7)	92% (241)	69% (166)	31% (75)	1% (2)	3% (7)	97% (252)	79% (198)	21% (54)	0.0144	54% (89)	105% (275)
Sraektum	143	3% (4)	3% (4)	94% (135)	74% (100)	26% (35)	4% (6)	3% (4)	93% (133)	64% (85)	36% (48)	0.0720	71% (71)	80% (114)
Sraempilkroam	73	0	4% (3)	96% (70)	87% (61)	13% (9)	5% (4)	4% (3)	89% (65)	78% (51)	22% (14)	0.1801	74% (45)	92% (67)
Total	477	4% (17)	3% (14)	94% (446)	73% (327)	27% (119)	3% (12)	3% (14)	94% (450)	74% (334)	26% (116)	0.7584	63% (205)	96% (456)

^aEligible population size as reported by heads of villages.

^bEligible participants unreachable and reported by relatives to have engaged in forest activities at the time of the study.

^cUnreached eligible participants for whom no information on their location was available.

^dComparison of individuals accepting to participate among those approached between each round of PvSeroTAT (Chi² test).

^eProportion of participants enrolled in the first round also enrolled in the second round.

^fProportion of individuals enrolled in at least one round of PvSeroTAT among the reported total eligible population size.

Participation rates were similar between the two rounds of PvSeroTAT, with 74% of approached individuals agreeing to participate in the second round (p = 0.7584). In detail, recruitment did not significantly differ between the two rounds in two villages (Sraektum: 64%, 85/133, p = 0.0720; Sraempilkroam 78%, 51/65, p = 0.1801) while in Aukaunpreas, enrollment significantly increased in the second round (79%, 198/252, p = 0.0144). Overall, only 63% (205/327) of individuals enrolled in the first round could be retrieved and agreed to participate in the second intervention. Specifically, 54% (89/166), 71% (71/100) and 74% (45/61) of participants enrolled in the first round agreed to participate in the second round in Aukunpreas, Sraektum and Sraempilkroam, respectively. Considering both rounds, 96% (456/477) of the estimated total number of eligible individuals agreed to participate in at least one of the two rounds of PvSeroTAT.

Primaquine treatment in seropositive eligible participants

1st round of PvSeroTAT

Among the participants enrolled in the first PvSeroTAT round, 31% (101/327) tested seropositive for recent exposure to P. vivax blood-stage parasites by serology. Of these, 22% (22/101) were not eligible for primaquine treatment. The reasons for ineligibility included G6PD deficiency, defined as enzyme activity below 4 U/g Hb as per Cambodian Treatment Guidelines (13%, 13/101), hemoglobin levels below the threshold for primaquine administration (7 g/dL) as per Cambodian Treatment Guidelines (1%, 1/101), or blood sample clotting, which prevented reliable G6PD analysis (8%, 8/101).

Of the 79 seropositive individuals eligible for primaquine treatment, all were successfully followed-up by the study team and offered primaquine treatment. The study team provided instructions relating to primaquine administration. Instructions included (i) to take primaquine with food intake to mitigate gastrointestinal adverse events, (ii) to continue the primaquine regimen according to the initial schedule in case a dose is forgotten, (iii) to notify the study team and stop primaquine treatment in case of occurrence of any of these symptoms: dark urine, pallor or yellow eye color, shortness of breath, back pain or increased heart rate. All but one participant (99%, 78/79) agreed to receive primaquine. Among them, 87% (68/78) self-reported compliance with the full 14-day regimen while 13% (10/78) reported interrupting the treatment before completion. The reasons for interrupting primaquine treatment were because participants forgot to take a dose and then stopped the entire treatment despite instructed otherwise (6%, 5/79) or self-reported adverse events (6%, 5/79). Non-compliance mainly occurred due to participants traveling to the forest or other locations for several days and forgetting to bring their medication. One individual interrupted the treatment on Day 4 and reported headaches and muscle pain, three participants reported diarrhea, vomiting and headache on Day 6 and Day 9 and one participant reported difficulty breathing in addition to diarrhea and vomiting on Day 5. Whether these adverse events were caused by primaquine is unknown but the study nurse, who followed all participants receiving primaquine daily with phone calls, advised treatment interruption in case of any signs of adverse events and to seek medical attention in local health center facilities.

2nd round of PvSeroTAT

During the second round of PvSeroTAT, 30% (98/334) of individuals were seropositive for recent exposure to P. vivax by serology, a proportion similar to that of the first round (p = 0.6649). Of these, 14% (14/98) were not eligible for primaquine treatment, all due to G6PD activity below the Cambodian threshold for primaquine administration. No invalid samples were found at this round of PvSeroTAT. All the 84 seropositive eligible individuals were successfully followed-up by the study team, provided serology and G6PD deficiency results and offered a primaquine regimen, and all agreed the treatment. The vast majority (90%, 76/84) of participants self-reported completing the 14-day course of primaquine, while 5% (4/84) interrupted primaquine treatment because they forgot to take it and 5% (4/84) due to self-reported adverse events. Adverse events included headache (4 participants), diarrhea (2 participants), vomiting (3 participants) and muscle pain (1 participant) with some participants experiencing multiple symptoms. These occurred on Day 5 (1 participant), Day 7 (2 participants) and Day 10 (1 participant) of primaquine treatment.

Of the 205 individuals who participated in both rounds of PvSeroTAT, 61% (125/205) were seronegative at both rounds, 19% (39/205) were seropositive at both rounds, 14% (29/205) were seronegative in the first round and seropositive in the second, and 6% (12/205) were seropositive in the first round and negative in the second (Fig. 3).

Fig. 3.

Percentage and number of individuals with each seropositivity status among the 205 participants enrolled in the two successive rounds of PvSeroTAT. The legend displays the serology status in the first round followed by the status in the second round.

Among the 39 individuals seropositive at both rounds, 11 (28.2%) did not receive primaquine in the first round due to G6PD deficiency (5 individuals) or low hemoglobin levels (1 participant) or due to clotted blood samples (5 individuals). Of these, the 6 G6PD normal individuals received a primaquine treatment in the second round. For the 12 participants seropositive in the first round and seronegative in the second, 10 received primaquine in the first round, while two did not as one individual was G6PD deficient and the other had an invalid blood sample preventing G6PD activity testing.

Delay between enrollment and treatment

The median time between enrolling participants and providing primaquine to those eligible (seropositive G6PD normal) at both PvSeroTAT rounds was 6 days (IQR: 5–8.3) and 97% (157/162) of participants were offered primaquine within 10 days or less from enrollment (Fig. 4). This time to treatment was significantly longer for the second round of PvSeroTAT conducted in December 2022 (median: 8 days, IQR: 6–9 days, p < 0.0001) as compared to the first round performed in July 2022 (median 5 days, IQR: 4–5 days). Two participants were followed-up 19 and 20 days after enrollment, respectively, and both reported being in the forest at the time.

Fig. 4.

Time between enrollment and treatment for seropositive eligible individuals during the first and second PvSeroTAT round.

Serology results in PCR detected active P. vivax blood-stage infections

We retrospectively examined whether participants had an active blood-stage infection by PCR and found that 4.0% (13/327) and 2.7% (9/334) of participants were positive for blood-stage P. vivax in the first and in the second PvSeroTAT round, respectively. Among seropositive participants, 8% (16/199) were PCR positive and 92% (183/199) were PCR negative. Conversely, 73% (16/22) of PCR positive individuals (11/13 and 5/9 in the first and in the second round, respectively) were also seropositive. Among the 16 individuals who were both PCR-positive and seropositive, three were ineligible for primaquine treatment due to G6PD activity below Cambodian thresholds for primaquine administration, while the others were offered the treatment and self-reported completing the 14-day course.

P. vivax prevalence over the course of the study

Cross-sectional surveys (CSS) were conducted in all 6 villages (intervention and control) before the first round of PvSeroTAT, between the two rounds, and after the second round. The overall prevalence of asymptomatic malaria among males aged over 15 years old in November 2021, before the first PvSeroTAT round, was 7.7% (45/586) as determined by PCR. In the intervention villages, the prevalence was 6.9% (26/375) and ranged from 3.6% to 11.1% (Fig. 5). In the three control villages, the prevalence was 9.0% (19/211) and ranged from 6.3% to 12.3%. Among Plasmodium infections, the majority were caused by P. vivax (91.1%, 41/45) followed by a single mixed P. falciparum/P. vivax infection (2.2%, 1/45). Plasmodium species could not be determined for three infections (6.7%, 3/45). This prevalence did not differ between control and intervention villages (p = 0.4194).

Fig. 5.

Plasmodium and P. vivax prevalence (with 95% CI) measured by PCR in intervention and control villages before the first PvSeroTAT round (November 2021), between the two rounds (October 2022), and after the second round (February 2023).

During the second CSS, performed in October 2022, between the two rounds of PvSeroTAT, the overall malaria prevalence significantly decreased to 2.7% (18/655) compared to the first CSS (p < 0.0001). This decrease in prevalence was observed in all villages, whether intervention (2.6%, 11/425, p = 0.0039) or control (3.0%, 7/230, p = 0.0086) with no significant difference between them (p = 0.8036). Again, most infections (77.8%, 14/18) were caused by P. vivax with four undetermined species (22.2%, 4/18). In February 2023, during the last CSS conducted after the second round of PvSeroTAT, the overall prevalence was 2.2% (13/603), which was not significantly different from the prevalence measured in October 2022 (p = 0.5864). Similar prevalence rates were observed in both intervention (2.3%, 8/348) and control villages (2.0%, 5/255, p > 0.9999). Of all infections, the majority were caused by P. vivax (76.9%, 10/13) followed by two P. falciparum infections (15.4%, 2/13) and one infection of undetermined species (7.7%, 1/13).

Discussion

We present here the first PvSeroTAT study conducted in highly mobile, forest-going adult and adolescent males in malaria-endemic communities. Our primary objective was to assess the technical feasibility of this approach, specifically whether PvSeroTAT could achieve its intended objectives within the constraints of the study area and limited resources, and whether it could be effectively implemented in the targeted high-risk population.

Overall, we achieved high enrollment rates, despite potential concerns that could have arisen about the laboratory-based serological assay applied and the proposed screen, re-contact and treat intervention. The participation rates we obtained here are lower than those achieved in a previous study evaluating chemoprophylaxis to reduce malaria prevalence in forest goers with 1242 individuals completing the study protocol out of 1613 assessed for eligibility (77%) and 1480 enrolled (84%).²³ However, the study population and inclusion criteria in the mentioned study were different than ours as it recruited forest-goers only and over a 9-month period, making direct comparison challenging. We nevertheless show that PvSeroTAT with a lab-based assay is feasible in Cambodia even if it is logistically more challenging than using point-of-care (POC) assays. While such POC tests are under development but not yet available for serological testing of markers of recent exposure to P. vivax, there are now POC quantitative devices for G6PD activity determination.²⁷ Using such a device would have avoided the issues we encountered during the first round with invalid samples preventing spectrophotometric G6PD activity determination, although following refresher training of the field staff, this issue was prevented in the second round.

Out of the 199 seropositive individuals from both rounds combined, 82% (163) were retrieved and offered primaquine treatment. The 36 individuals who did not receive primaquine were mostly ineligible because they were G6PD deficient (28/36) or had hemoglobin levels below the thresholds for primaquine administration. We used a G6PD activity threshold of 4 U/g Hb as per Cambodian Treatment Guidelines of Cambodia to provide primaquine treatment. This threshold corresponds to 46% of the adjusted male median (AMM) G6PD activity measured in our study (8.7 U/g Hb). Primaquine could be safely provided to male individuals with a G6PD activity as low as 30% of the AMM.²⁸ Using 4 U/g Hb instead of 30% AMM (2.6 U/g Hb) led here to misclassification of 4% (1/28) of individuals as G6PD deficient. Nevertheless, our data illustrate the major challenge of G6PD deficiency, and that adapted therapeutic options such as weekly primaquine administration are needed to achieve elimination. A weekly primaquine regimen (0.75 mg/kg per week for 8 weeks) is currently being operationally evaluated by the Cambodian Malaria Control Program in selected pilot areas of the country. Few studies have shown that this regimen provides adequate anti-relapse efficacy and is relatively well tolerated in G6PD deficient individuals despite a drop in hemoglobin after the first dose.²⁹

Of note, our estimates of enrollment rates were based on population size information provided by the head of villages, and our results indicate that these estimates were inaccurate, as the total number of people we enrolled in one village exceeded the reported total provided by the head of the village. Nevertheless, despite this imperfection and the resulting uncertainty over the denominator of overall participation, our results show that nearly all eligible individuals agreed to participate in at least one round of PvSeroTAT. This high overall participation can be attributed in part to the strong push for malaria elimination by Cambodian health authorities, with communities well aware of the disease burden. Even though P. falciparum malaria has drastically decreased in recent years, communities’ awareness of P. vivax specificities and the need for the implementation of tailored elimination strategies is likely increasing. However, it is important to recognize that these high rates occurred within the context of a study where incentives may have influenced participation, rather than in a programmatic intervention. Additional studies are needed to determine how such PvSeroTAT approach could be translated into real world programmatic interventions. In addition, in this work, we focused on the most at-risk individuals and enrolled only male aged more than 15 years old. Future studies should also be conducted to determine feasibility of PvSeroTAT in women and other age groups to fully capture all community members residing in malaria endemic areas.

Although there were some differences between villages, overall enrollment rates remained very similar between both rounds (73–74%). Our work demonstrated that the willingness of individuals to participate in the study remained high even six months after a first intervention as well as their adherence to the intervention, and high treatment completion rates. However, the number of participants who attended both rounds was comparatively relatively low. While this is likely to reflect the highly mobile nature of our target population, in-depth qualitative acceptability studies will be important to design specific approaches to increase the number of people that participate in at least two PvSeroTAT rounds. This is particularly important as mathematical modeling has shown that multiple rounds of PvSeroTAT would likely be necessary to interrupt transmission within communities.¹⁴ More than two PvSeroTAT rounds may thus be required in these mobile, high risk populations to achieve maximum impact.

A key factor enabling the success of such decentralized interventions, where analyses are performed away from the field sites where the participants are enrolled, is the duration between enrollment and recontacting participants to provide treatment. Our results show an efficient turnaround time, with most individuals (96%) recontacted within 10 days or less. This rapid response time is crucial for enabling retention of participants, especially for these mobile adult males. Our results indicate that despite focusing on the most mobile individuals, we achieved very satisfying feasibility. Interestingly, the two individuals retrieved by the study team the latest (19 and 20 days, respectively), were not found earlier because they had returned to the forest.

Our study was designed to evaluate the technical feasibility of PvSeroTAT in highly mobile forest going populations and was not intended or powered to assess efficacy on reducing P. vivax prevalence or transmission. Indeed, we focused on the most hard-to-reach individuals rather than the entire communities, leaving potential hypnozoite carriers unenrolled and untreated. Although we included three neighboring control villages where interventions were not carried out to estimate P. vivax prevalence, no buffer zones were established between the intervention and control villages, allowing for spillover from one area to the other. Nevertheless, we conducted three cross-sectional surveys (CSS) before, during and after the interventions to determine P. vivax prevalence. Our results underscore the significant asymptomatic burden of P. vivax in communities living in endemic areas, even in villages located outside transmission zones (forests), with an overall prevalence of 7.7% of P. vivax carriage before the start of the study and up to nearly 14% in some villages. Following the first round of PvSeroTAT, P. vivax prevalence decreased in all villages, whether intervention or control. However, further reduction in prevalence was not observed during the CSS conducted after the second round of PvSeroTAT. We speculate that the reduction in prevalence resulted from the first round of PvSeroTAT, which eliminated a significant number of hypnozoites from the asymptomatic reservoir. Because malaria infections are acquired in forests, and individuals living in both control and intervention villages are infected in shared forest locations, the reduction of carriers in intervention villages might have also impacted the neighboring control villages. Randomised controlled trials evaluating the efficacy of PvSeroTAT on P. vivax prevalence in different epidemiological settings are being undertaken and will enable this hypothesis to be tested.

Our study has demonstrated that it is feasible to target residual, clinically silent P. vivax infection in mobile high-risk populations in Cambodia with PvSeroTAT, even in the absence of a rapid, POC serology test. Sending samples to a reference laboratory for sero-diagnosis did add significant logistical complexity and delays to treatment, but the very high re-contact and treatment adherence rates are encouraging and demonstrate that these challenges can be overcome. While two rounds of PvSeroTAT reached almost all high-risk individuals at least once, further PvSeroTAT rounds may be needed to achieve maximum impact. Additional studies are required to understand community members’ perceptions about PvSeroTAT, particularly taking treatment in the absence of symptoms. This will inform the development of tailored and effective education and awareness material for future implementation of PvSeroTAT.

Contributors

BW and JP conceived the study. CT, ST, ME, NK, AO were responsible for data collection. CT, DL and JP oversaw the study. CT, RJL, MW, LJR, IM and JP did the data analysis. CT and JP wrote the first draft. All authors had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Data sharing statement

De-identified patient data will be made available upon reasonable request.

Editor note

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Declaration of interests

We declare no competing interests. RJL, MW and IM are inventors on patent PCT/US17/67926 on a system, method, apparatus and diagnostic test for Plasmodium vivax.