Forest malaria in Cambodia: the occupational and spatial clustering of Plasmodium vivax and Plasmodium falciparum infection risk in a cross-sectional survey in Mondulkiri province, Cambodia

Seventeen villages were selected in the Kaev Seima district, Mondulkiri province, in the Kingdom of Cambodia. The rainy season in Cambodia normally runs from June to October with the high transmission period from June through December [1, 4, 24]. Approx. two-thirds of the population in Mondulkiri is comprised of national ethnic minorities, with the Phnong ethnic group comprising the largest proportion [25, 26]. In November–December 2017, a census was conducted by visiting each household in the 17 villages and collecting basic demographics of household participants such as age and gender. A person’s household was defined as location of main residence in the village according to adult members of the household or the village head. GPS coordinates were collected using Garmin® GPSMAP® 64s devices. When no adult household member could be found, demographic information was obtained from the village head’s registry book.

Based on the census, a random selection of households was drawn oversampling small villages to ensure sufficient coverage (Additional file 1: Table ST1). Selected households were visited from mid-December 2017 until mid-April 2018 and all household members aged 2–80 years who had resided in the study area for at least 3 months were invited to participate in the survey. Upon informed consent, a questionnaire on household variables was administered to the head of household or another adult household member by trained interviewers. A questionnaire on individual-level variables was administered to each consenting household member. Children were interviewed assisted by a parent (or rarely another caregiver in the absence of the parents). Data were collected on tablets and run through automated data quality checks within days after the interview. In case of missing data or discrepancies, the field team was informed for immediate resolution if possible. Finger-prick blood samples were collected as thick and thin film slides and in K⁺EDTA-microtainers. Participants were screened for symptoms, i.e. feeling sick or feverish on the day of interview, having felt feverish over the preceding two days, or having an axillary body temperature of at least 37.5 °C. Upon any indication, the participant was administered a standard malaria RDT (Malaria Ag P.f/P.v, Standard Diagnostics Inc., South Korea) and referred to a local health care provider for treatment if positive.

The microtainer blood samples collected at the interview site were stored in 4 °C ice boxes. At a field laboratory, they were separated into plasma and cell pellet and frozen at −20 °C. Following transport to the main laboratory at Institut Pasteur of Cambodia in Phnom Penh, cell pellets were stored at −20 °C and plasma at −80 °C. Infections with any of the four human Plasmodium parasites were determined by real-time PCR [27]. In case of a positive genus-specific result, qPCR specific for P. falciparum, P. vivax, Plasmodium malariae, and Plasmodium ovale followed. All positive and a random selection of 10% negative samples were assessed by independent double LM readings of asexual and sexual stages and parasite densities calculated. No discrepancy in parasite densities of above 30% occurred. Parasites were counted per approximately 500 leukocytes and densities were inferred assuming 8000 leukocytes per microlitre blood.

Villages were classified based on the forest cover in a 750 m radius around the households, computed from the land cover analysis in [28] (Additional file 1: Figure S1). Villages with ≥ 50% of households with ≥ 10% forest cover in their vicinity were considered “inside the forest”, with ≥ 30% of households with ≥ 5% forest cover as “at the forest fringe”, or “outside the forest” otherwise. Because of very low sample sizes, the two small, neighbouring villages Beng (11 individuals) and Gaty (95 individuals) were analysed as one. Population prevalence was estimated based on post-sampling weights assigned to each participant according to their representation by village, gender, and 10-year age bins compared to the census population (raw numbers of positive survey samples accompany the estimates in brackets). Categorical covariates were compared using the chi-squared test or the Fisher’s exact test when low strata sizes required it.

The association of covariates with infection by P. vivax, P. falciparum, or all four species was assessed by mixed-effects logistic regression with random intercepts per household and village. Those covariates that were statistically significantly associated with Plasmodium infection at two-tailed α = 5% in univariate regressions were included in the multivariate model. The villages’ proximity to the forest, work-unrelated overnight travels (incl. to forest sites), and work in the deep forest were considered part of the multivariate model a priori in order to assess the association of forest exposure with risk of infection. Having slept outdoors or under a bed net last night were kept as part of the model based on causal reasoning or prior knowledge due to their association with infection risk in previous publications, e.g. [11]. Gender and age were retained in the model as proxies for risk-related behaviour and thus potential confounders. The other covariates comprised potential proxies for socioeconomic status or further exposure variables and their subset significantly associated with infection risk was assessed by backwards variable selection. Akaike Information Criterion (AIC) was used to assess model fit. If collinearities and interactions were identified, covariates were retained comparing fit of the respective models by AIC. Statistical significance in the multivariate regressions was calculated per covariate by likelihood ratio tests. Spatial hotspots were identified by a purely spatial scan statistic via a discrete Poisson model with maximally a third of the population in a scanning window, adjusted for all covariates of the final multivariate model except for the villages’ forest proximity [29].

All questionnaires were applied on tablets using the REDCap (Research Electronic Data Capture) software hosted at Institut Pasteur in Paris [30, 31]. Data quality control as well as all descriptive and analytical statistics were performed in R 3.6.3 [32]. The SaTScan™ software version 9.6 was used for the spatial scan statistic [33].

From among the 10,053 individuals in 2351 households identified in the census, the survey recruited 4200 participants from 1147 households and oversampled smaller villages on average (Additional file 1: Table ST1). A map of the survey households and the categorization of villages by proximity to the forest is shown in Fig. 1a. Mean age was 26 years in both the census and the survey and with 51% (5135/10,053) and 53% (2231/4200) women, respectively, women were slightly oversampled in the survey. In men, ages of 16 to 40 years were mildly underrepresented (Fig. 1b). The main income source for the survey households was by large majority farming (89.8%, 1030/1147). In terms of mobility, three-quarters (75.9%, 3188/4200) of the survey participants reported work-unrelated trips to forest or field sites in the last month (8.5%, 358/4200) or any work trip in the last two months (75.8%, 3184/4200). By vast majority, these trips were short and frequent: In only 4.4% (140/3188) of the instances, stays for longer than a week were reported and among those who reported any work trip, 89.4% (2847/3184) went for work at least once a week.

Infection by Plasmodium parasite was detected by PCR in 8.3% (349/4200) of participants. The proportion of samples positive for P. vivax was 6.4% (268/4200) compared to 3.0% (125/4200) for P. falciparum (p < 0.001, Table 1). Plasmodium vivax was found in 77% (268/349) of all infections compared to 36% (125/349) for P. falciparum. Four samples were positive for P. malariae mono-infections and none for P. ovale. Speciation by PCR was unsuccessful in 4 samples which were analysed as negative. Extrapolation to the entire Kaev Seima population yielded an estimated prevalence of 8.9% for Plasmodium infection, 6.8% for P. vivax, and 3.3% for P. falciparum. Estimated prevalence was highly heterogeneous across the villages, ranging from 0.6% (3/594) to 36.3% (54/152) and from 0% (0/594) to 25.1% (27/106) for P. vivax and P. falciparum, respectively (both p < 0.001, Table 1). Village-level prevalence of P. vivax infection was on average higher in villages inside the forest compared with those at the forest fringe and outside the forest (medians 23.2, 7.2, 5.6% respectively, Fig. 2; similarly for P. falciparum in Additional file 1: Fig. S2).

Of all PCR-detected infections, 83.2% (223/268) were sub-microscopic for P. vivax compared to 74.4% (93/125) for P. falciparum (p≈0.056, Table 2). Plasmodium vivax infections coincided less often with reported or measured symptoms at the interview than those by P. falciparum, with 6.7% (18/268) and 16.8% (21/125), respectively (p < 0.01). Only 2.2% (6/268) of all P. vivax infections were symptomatic and also positive by RDT or LM (i.e. detectable through the Cambodian health care system), less often than for P. falciparum (7.2%, 9/125, p < 0.05). Asexual parasite stages were detected in all LM⁺ samples for P. vivax (37/37) and in most (81.5%, 22/27) for P. falciparum (Table 3). In LM⁺ samples, the geometric mean parasite densities were 149.7 parasites/μL and 431.3 parasites/μL (t-test: p≈0.07), respectively. While gametocytes were found in only one (2.7%, 1/37) P. vivax LM⁺ sample, more than a third (37.0%, 10/27) of LM⁺ samples for P. falciparum were gametocyte-positive with a geometric mean of 51.1 gametocytes/μL.

Estimated prevalence was more than twice as high in men as in women, i.e. 10.4% (188/1969) vs. 3.6% (80/2231) for P. vivax (p < 0.001), 4.8% (83/1969) vs. 1.9% (42/2231) for P. falciparum (p < 0.001), and 13.3% (239/1969) vs. 5.0% (110/2231) regardless of species (p < 0.001). The patterns across age were similarly heterogeneous for both species (Additional file 1: Figure S3). While there was no difference in genus-wide prevalence across age in women, men showed an elevated risk at working age (p < 0.001), regardless of the proximity of the village to the forest (Fig. 3). A fitted interaction term of gender and age was significant in the strata of villages outside the forest (p < 0.001), but not across those villages at the forest fringe or inside the forest.

Risk of infection was associated with individual covariates for both P. vivax and P. falciparum in a highly similar fashion (Table 4 for behavioural variables, full list of variables with odds ratios in Additional file 1: Table ST2). For both species, prevalence was associated with work-unrelated overnight travels and highest if those occurred to forest sites (22.4%, 60/268, and 14.9%, 40/268, for P. vivax and P. falciparum, respectively) and lowest for urban destinations (1.7%, 1/59, and 0%, 0/59, for both p < 0.001). Infections of both species were also more prevalent among those who reported work trips to sites in the nearby and deep forest (assessed separately). In particular, a significantly higher prevalence was observed in participants who reported work trips into the deep forest (25.5%, 37/145, and 18.6%, 27/145) compared to those who did not (5.7%, 231/4055, and 2.4%, 98/4055, for P. vivax and P. falciparum, respectively, p < 0.001). Once clustering at household and village-level was taken into account in univariate mixed-effects logistic regression, work in the nearby forest was no longer significantly associated with risk of infection though (Additional file 1: Table ST2). There were fewer infections among those who reported the use of standard protection measures such as sleeping under a bed net (6.1%, 236/3880, and 2.7%, 104/3880, vs. 10.0%, 32/320, and 6.6%, 21/320, if no net was used, p < 0.01 and p < 0.001 for P. vivax and P. falciparum, respectively). A higher risk in men, at working age, in villages at the forest fringe or inside the forest, and with indicators of lower socio-economic status was also found similarly for both species (Additional file 1: Table ST2).

Behavioural covariates related to activities in the forest were statistically significant in the multivariate model of Plasmodium infection (Table 5). Having travelled to forest sites (nearby or deep forest) or having worked in the deep forest independently increased the odds of infection two to three-fold (adjusted odds ratio, aOR, 2.17, p < 0.01 and aOR 2.88, p < 0.001, respectively). Risk of infection was not significantly attributed to the other behavioural covariates that were included in the model a priori, namely having slept outdoors (aOR 1.99, p≈0.08) and under a bed net (aOR 0.99, p≈0.96). Risk of infection was increased in males (aOR 3.06, p < 0.001) and working age (aOR 7.84 in 21–25 years old compared to children, p < 0.001). A fitted interaction term of gender and age improved the model (AIC 1786 vs. 1802 without interaction, p < 0.001, Additional file 1: Table ST3). Other covariates linked higher socio-economic status with lower odds of infection, such as living in a house with a roof built of relatively high-quality material (aOR 0.50, p < 0.01). The covariate on recent information on malaria via TV (aOR 0.37, p < 0.01) most likely also acts as a proxy for higher socio-economic status, i.e. being able to afford a TV in the first place.

Work-unrelated overnight travels to forest sites was a predominantly male domain (reported by 10.7%, 211/1969, of the men vs. 2.6%, 57/2231, of the women, p < 0.001) and particularly frequent in male adolescents (with 24.3%, 70/289, highest in 21–30 years old males, p < 0.001). Forest work was independently assessed for sites of the nearby and deep forest. Only for the latter, a statistically significant association with risk was observed. As for travels, they were reported more frequently by men than by women (6.9%, 135/1969, vs. 0.4%, 10/2231, p < 0.001) and most often by 21–30 years old males (17.6%, 51/289, p < 0.001). However, working in the deep forest occurred in both men aged between 8 and 60 years and in 8–43 years old women. Forest travels were reported in 3–62 years old men and 3–63 years old women. Both kind of trips occur usually frequently and of short duration. In 81% (217/268) of the times, the travels to forest sites lasted less than a week. Work trips into the deep forest occurred in 75% (108/145) of the times at least weekly. When going for work several times a month or once a month, the reported duration was also most often less than a week (83%, 15/18, and 53%, 8/15, of the times, respectively). A net was used during overnight work trips into the forest in only 25% (28/113) of the instances. While equally few participants from villages outside the forest, at the forest fringe, and inside the forest reported work in the deep forest (3.4%, 105/3060, 2.9%, 18/625, and 4.3%, 22/515, respectively, p≈0.44), travelling to forest sites was reported more often in forest fringe and forest villages (8.6%, 54/625, and 11.5%, 59/515, respectively, compared to 5.1%, 155/3060, outside the forest, p < 0.001).

Living in a village inside the forest remained associated with risk of infection when adjusting for the significant demographic, socio-economic, and behavioural covariates (aOR 12.47, p < 0.001, Table 5). Households from all four villages inside the forest also formed hotspots of Plasmodium infection, i.e. purely spatial clusters of elevated risk of infection that cannot be explained by the other covariates (Fig. 4, Additional file 1: Tables ST4–ST5).