Epidemiology of forest malaria in central Vietnam: a large scale cross-sectional survey

Study area and population

The survey was carried out from November to December 2003 in Ninh Thuan province, located on the southern coast of Central Vietnam (Figure 1). Forty three villages corresponding to the forested and mountainous part (north-west) of the province and with the highest annual malaria morbidity and mortality figures according to the Provincial Malaria Station were selected. The population is distributed over 12 communes located in 4 districts (Bac Ai, Ninh Son, Ninh Phuoc and Ninh Hai) and is mainly inhabited by the Rag Lays, traditionally nomadic but recently settled in permanent villages. Most people are farmers, cultivating maize in forest fields or and rice around the villages, and collecting forest products. The dry season lasts from December to April, the rainy season from May to November with one of the lowest mean annual rainfalls in the country (<800 mm/y). The monthly mean temperature is 25–30°C, with an annual mean of 27.3°C. The mean relative humidity over the past 3 years has been 74.7%, ranging from 70 to 80% throughout the year. Malaria transmission is perennial with 2 peaks (May-June and October-November), the monthly incidence of malaria cases following closely the monthly rainfalls (Figure 2). The two main malaria vector species are An. dirus A and Anopheles minimus A [14].

Data collection

Households were numbered and a full census carried out. A sample size of 150 individuals per cluster of 1,000 inhabitants was calculated at 5% significance level assuming a 5% parasite rate with 3.5% precision. Households were randomly chosen and within each of them, an individual aged 10 to 60 years was randomly selected (the latter age range was estimated to represent the active population most at risk of malaria [5]). Socio-demographic data, risk factors for malaria infection as well as current prevention methods were collected on pre-coded standardized questionnaires. At clinical examination, axillary temperature and spleen size were measured. Blood samples for parasitaemia (thick film) and P. falciparum antibodies (filter paper) were collected. Suspected malaria cases were treated with a 7-day course of artesunate according to the national guidelines; other common diseases were treated accordingly.

Village coordinates were recorded using a Geographic Position System or GPS (eTrex Summit, GARMIN Corporation) and later reported on the land-cover digital map for the year 2000 provided by the Institute of Geography – Hanoi (adapted from land-cover map compiled by the Department of Forestry – FIPI). The original map with 22 land-cover classes was simplified into the 5 following categories:

Natural forests – including rich forest, mixed forest, medium forest, wood and bamboo mixed forests, bamboo forest and clear forest;

Plantations – including different types of plantations;

Dry forest deciduous;

Other – including rice fields, land tenure, habitat with garden, swamps or wetlands, and different types of bare land;

Water – including natural river & lake.

Elevation varied from 10 m (Phuoc Chien commune) to 323 m above the sea-level in Phuoc Binh.

The numbers presented on the map refer to the village unique identifier. Parasite rates and seroprevalences for each village (Table 1) were reported onto the map with different coloured symbols corresponding to the following categories: 0–10%; 10–30%; = 30% (Figure 1).

Laboratory methods

Thick films were stained with a 5% Giemsa solution for 30 minutes. Parasite densities were computed based on the number of asexual forms and gametocytes per 200 white blood cells (WBCs), assuming a mean WBC count of 8,000/μl. A slide was classified as negative if no Plasmodium asexual form was found after counting 1,000 WBCs. Filter papers (Whatman N°3, Kent, United Kingdom) were stored at -20°C, and Indirect Fluorescent Antibody Tests (IFAT) were carried out to determine the total immunoglobulin titres against P. falciparum as described previously [15]. P. falciparum antigen was prepared from in vitro cultures of an isolate originating from a patient in Southern Vietnam. Negative control serum was obtained by pooling the sera of five malaria-free individuals; positive control serum, by pooling the sera of 5 patients who had already suffered several malaria episodes. The serum dilutions ranged from 1:80 to 1:640. Both slide and IFAT reading were blinded and carried out at NIMPE (National Institute of Malariology, Parasitology and Entomology), Hanoi. Quality control was performed at ITM (Institute of Tropical Medicine), Antwerp, on all positive and 10% negative samples and all discrepant results were re-read and confirmed by a third technician.

Case definition

- Suspected malaria: patient with typical malaria symptoms without microscopic diagnosis;

- Malaria infection: positive slide with Plasmodium asexual forms, regardless of symptoms;

- Clinical malaria: fever (BT≥37.5°C) and/or history of fever in the past 48 hours with a positive blood slide;

- Positive IFAT: titre ≥ 1/80. The immune response to an active malaria infection is species and strain specific, and lasts 6–9 months without additional exposure [16,17]. Usually titres above 1:20 are considered positive. However, we chose a more specific screening titre of 1:80.

- Current & recent malaria infections: to perform the multivariate adjusted risk-factor analysis for malaria infections (Table 4) all cases with either a positive slide and/or a positive IFAT were combined.

Statistical analysis

Data were entered in Epi Info 6.04 (CDC, Atlanta; WHO, Geneva 1996) and analysed in STATA 8 software (Stata Corp.2003, College Station, TX). Malaria parasite rate and sero-prevalence were computed by village and a survey chi-square test ("svytab" command in STATA) was used to test for significant differences for these two variables between villages of each commune and between the communes themselves. A survey logistic regression ("svylogit" command in STATA) was carried out taking into account the survey characteristics, with villages as primary sampling units and communes as strata. Uni- and multivariate adjusted analysis for the risk of malaria infection during the rainy season was carried out. Therefore, current (positive slide) and recent (IFAT ≥ 1:80) malaria infections were combined in order to include most malaria infections during the previous six months up to the time of the survey. The primary exposure was forest work and this variable was coded as follows: 1) not working in forest (other job); 2) occasionally (less than weekly); 3) regularly (at least weekly), but not staying overnight; 4) regularly working in forest and sleeping there at various frequency. The category – no profession, i.e. children, students and retired people, was included in the category occasionally working in forest since they do so with their family.

The Ethical Committee of ITM, Antwerp and that of NIMPE, Hanoi approved the study (Ethical clearance registration n°: OG 018). Informed consent was received from all village leaders and People Committees after explaining the study objectives and methodology. Selected individuals were informed and invited to be part of the survey some days in advance but were free to refuse. Other people not chosen but being sick at the time of the survey were examined and treated accordingly but were not included in the survey database.

The study population (Table 2)

4,306 (92%) of the 4,679 selected individuals participated to the survey with a sex ratio of 1.3 in favour of females. The Rag Lay ethnic group represented the vast majority of the study population (86%), and educational level was generally low since almost half of the population had never attended school. However, communication was easy since most people (91.7%) could speak Vietnamese. Bed-nets (usually double nets) were widely used (98% of households) with a median of 2.5 persons/net. Most of the nets had been treated with insecticide the previous year, but no indoor spraying had been done. The socio-economic status was very low since half of the population was living in houses made of thatched bamboo and more than 60% owned none of the three items asked for (radio, television, or motorbike). About 80% of the study population was defined as forest workers, i.e. their main source of income was from forest activities (farming, hunting, logging, etc...).

Forest work characteristics (Table 2)

Farming represented the main forest activity (99.5%), mostly maize and manioc, rice being generally cultivated around the villages. During the month prior to the survey, forest workers spent a median of 27 days in the forest. Most of them usually went to the forest with other family members, including children and elderly people (categorized in our study as "no profession"). About 30% of the forest workers stayed sometimes overnight in the forest (or forest fields), with a median of 12 nights during the month prior to survey. Important seasonal variations exist: April (preparation of fields before rains), July-August (first harvest) and for some villages November (second harvest) are the periods of highest forest activity. Few forest workers reported using hammocks (because they could not afford them) and hammock-nets were even rarer. Other preventive measures, such as standby and/or preventive malaria treatment, were rarely used (2.3%).

Malariometric indices (Table 3)

Six hundred sixty eight (15.5%) individuals had fever at the time of the survey, 125 (18.7%) of them being attributable to malaria. The spleen rate was 7% (all but two individuals had a Hackett Class 1) and the median age among people with enlarged spleen was 30 years (range [11–60]). The mean parasite rate (PR) was 13.3%, ranging from 0 to 42.3% across the 43 villages, with a large majority of asymptomatic infections (74.1%). P. falciparum and Plasmodium vivax were equally represented, though the mean parasite density was significantly lower for the latter, and only three patients were found positive with Plasmodium malariae. About 5% (31) of the infections were mixed, mostly P. falciparum with P.vivax (28), one patient had the three species, two had Pf+Pm and one P.v +P.m.

P. falciparum seroprevalence (SPV) was generally high, 25.5%, ranging from 2.1 up to 75.6% across villages.

Mapping of PR and SPV (Figure 1 & Table 1)

All 43 villages were located in places classified as 'dry forests deciduous' or 'others', situated at various distance from the natural forest. Within each commune the respective distances to the different types of vegetations are similar between the villages, since the latter are usually close to each other, i.e.12 km (except in Phuoc Binh commune where the 6 villages stretch over almost 10 km and in Phuoc Tan, over 5 km).

The mapping of PR and SPV shows an extremely patchy distribution without geographic clustering or trends per commune, per direction or elevation. Significant differences for both PR and SPV were found between the 12 communes, and between villages in almost all (10/12) communes (F (design based) p < 0.05)).

Four villages (n°1-3-23-25) had both low PR and SPV, below 10%. These villages are situated in the plain, near the main district road leading to Dalat or to the district town in Phuoc Dai commune. Nui Ray was the only village among the 43 with a PR at zero. At the other extreme, four villages had a SPV above 50% (n°14-30-33-43): all of them were remote with difficult access and near to the forest. In these villages, the PR was relatively low (ranging from 6.7% in Ma Lam to 36.6% in Cha Dung), reflecting higher transmission rates during the past six months, at the time of higher forest activity. Similarly, in Phuoc Ha and in Phuoc Dai communes (villages n° 9–12 and 26, 27), all six villages had a PR<10% but a SPV>10% (from 15.2 to 42.7%). However, remoteness was not the only determining factor for high PR and SPV since some remote villages such as Tanoi (n°8) in Manoi commune and Bac Ray1 (n°18) in Phuoc Binh commune, did not have higher PR or SPV than the other villages within the same commune. Overall, more than half of the villages (24) had either a PR>10% and/or a sero-prevalence>25%. Usually the SPV was higher than the PR, though some villages had a SPV lower than the PR. This could be explained either by the higher current exposure or by the higher prevalence of P. vivax (as in Bo Lang or Da Ban (data not shown)).

Risk factors for malaria infection (Table 4)

All current and recent malaria infections were combined to perform a risk-factor analysis. A total of 1,434 infections (33.3%) were included. Among risk factors identified by uni-variate analysis, Rag Lay ethnicity, education, socio-economic status and regular forest work had the strongest effect on the odds of malaria infection. After adjusting for the effect of potential confounders, regularly working and sleeping in the forest was a strong risk-factor with the odds almost three times higher than that of people not working in the forest. Even occasional forest work was a significant risk-factor for malaria (Adjusted OR = 1.48; 95%CI [1.01; 2.17]). The malaria risk was significantly lower in people living in houses made of dried mud, while wooden or brick houses were no more protective, after adjusting for socio-economic status. There was a decreasing trend for the odds of malaria infection with increasing socio-economic status, the highest category had about 50% lower malaria risk compared to the poorest one.