- Open Access
Infectious reservoir of Plasmodium infection in Mae Hong Son Province, north-west Thailand
© Pethleart et al; licensee BioMed Central Ltd. 2004
Received: 26 May 2004
Accepted: 22 September 2004
Published: 22 September 2004
It was unknown whether the main reservoir of Plasmodium falciparum and Plasmodium vivax, which infects mosquitoes in Thailand, was (a) in people feeling sufficiently ill with malaria to come to a clinic or (b) in people who had remained in their home villages with some fever symptoms or with none.
Mass surveys were carried out in Thai villages to identify people with Plasmodium infections and with fever. Malaria patients were also located at a clinic which served these villages. Adults from both sources whose blood slides registered positive for Plasmodium spp. were requested to allow laboratory-bred Anopheles minimus to feed on them. Seven to nine days after the blood feeds the mosquitoes were dissected and checked for presence of oocysts.
Results and Discussion
There were higher rates of Plasmodium infection among people in the villages with fever than without fever and much higher rates of infection among clinic patients than among people who had remained in the villages. People with malarial infections identified via the clinic and the village surveys could infect mosquitoes, especially, but not only, if their blood slides showed visible gametocytes. Because only a very small minority of the village populations were visiting the clinic on any one day, assessment indicated that the main reservoir of infection was not primarily among clinic patients but among those in the villages, especially those feeling feverish.
Efficient use of an anti-gametocyte drug to suppress the parasite reservoir in a population requires that it be given, not just to clinic patients, but to infected people located by mass surveys of the villages, especially those feeling feverish.
An estimate of the infectiousness of the Plasmodium reservoir to mosquitoes is of interest in understanding the epidemiology of malaria and its changes after application of certain types of control measures. Different approaches have been used to investigate this, including direct feeding of mosquitoes through the skin of human subjects [1–5] or feeding through an artificial membrane [6–13]. The present study was designed to determine whether the reservoir of infection of vectors was mainly in people ill enough to go to a clinic or whether it was mainly in those with slight or no malarial symptoms who have remained in their villages.
Patients and Methods
This study was approved by the Thai Ethics Committee and the Ethics Committee of London School of Hygiene and Tropical Medicine. Subjects were recruited from mass blood surveys in villages and from a clinic in Muang District, Mae Hong Son Province, which is in north-western Thailand. Anti-malarial drug use is tightly controlled in Thailand and these drugs are generally available on prescription. Self-medication is, therefore, unusual in this area. Mass blood surveys were conducted twice a year. After slides were examined for parasites, the people whose slides were positive and were more than 14 years of age were invited to take part in this study. The Thai ethical committee did not allow direct feeding of mosquitoes on children aged less than 15 years. Adults positive for malaria parasites were informed about the purpose of the study and, if they agreed, a consent form was signed and patients were interviewed.
Human subjects for the study were also enrolled from the Vector-borne Disease Control Unit No. 8 (VBDU: described as "Clinic" throughout this paper). In this Clinic, blood smears were taken by the clinic staff and stained with Giemsa to check for malaria. Patients who had presented with clinical malaria and/or parasitaemia and were more than 14 years of age were informed about the purpose of the study and asked to participate in it. If they agreed and signed the consent form, then the mosquito feeding was performed.
Mosquito feeding and dissection
Before feeding, patients' arms were cleaned with 70% alcohol. Then, 50 laboratory-bred female Anopheles minimus species A (aged 4–6 days), which had been starved for 9–12 hr, were allowed to feed on their arms for 30 minutes. After feeding was completed, anti-malarial drugs were given to all the human subjects by a malaria worker. Two hours after feeding the unengorged mosquitoes were removed from the cups using a sucking tube and destroyed, leaving only fully engorged mosquitoes in the cup. The mosquitoes were brought back to the Chiang Mai insectarium and maintained at 25 to 27 C and 70–80% relative humidity with permanent access to sucrose solution and without any further blood meals. Mosquitoes at 7–9 days post-feed were anaesthetized and the wings and legs were removed. Midguts were dissected on glass slides in a drop of 0.85% NaCl and examined at a 40× magnification. The number of oocysts present on the midgut to each mosquito was counted and recorded individually.
Quality control of blood slide data
Asexual parasitaemia and gametocytaemia were quantified as the number of aseuxal forms/200 white blood cells on a thick film. Throughout the period of the study, 20% of the negative blood smears and all positive slides which had been examined by the microscopist of the Clinic were chosen randomly and re-examined by a team from the Office of Vector-borne Disease Control No.2, Chiang Mai. All cases of positive slides without visible gametocytes and which led to oocyst production were re-examined by an expert team from the London School of Hygiene and Tropical Medicine to ascertain whether a few gametocytes might have been present but were initially missed.
The database from mosquito feeding included 1) densities of gametocytes and trophozoites, 2) number of infected mosquitoes, and 3) mean number of oocysts per infected mosquito. The chi-square test or Fisher's exact test was used to examine the significance of differences in the proportion of people in various categories who transmitted malaria to the mosquitoes (i.e. yielded at least 1 oocyst). Regressions on the natural log of gametocyte density were also computed. Stata statistical software (version 6) was used for analysis.
Data from village surveys and the clinic on reported fever and malaria infection.
Fever not reported
Fisher's exact tests of association of fever with infection
Village surveys, age <15 yrs
P. falciparum +ve
P = 0.082
P. vivax +ve
P = 0.000007
Village surveys, age >14 yrs
P. falciparum +ve
P = 0.00006
P. vivax +ve
P = 0.000003
Clinic (age >14 yrs)
P. falciparum +ve
χ2 = 0.39, P = 0.53
P. vivax +ve
χ2 = 0.14, P = 0.51
Results of feeding mosquitoes on human blood
No. people from whose blood some mosquitoes developed oocysts
No. people from whose blood no mosquitoes developed oocysts
Significance of differences
Fisher not sig.
Fisher P = 0.06
No observable gametocytes
Fisher P = 0.32
Fever not reported
χ2 = 0.41, P = 0.52
χ2 = 7.3, P = 0.007
No observable gametocytes
Fisher P = 0.56
Fever not reported
Parasite density ( P. falciparum and P. vivax )
χ2 = 1.30, P = 0.25
Considering each species separately, the probability of infectiousness to mosquitoes was significantly related to being observably gametocyte positive in P. vivax (P= 0.007), but the relationship was only of borderline significance for P. falciparum (Fisher's exact test, P = 0.06). Approximately 50% (7/13) of the P. falciparum cases with observable gametocytes failed to infect mosquitoes and 30% (14/45) of the P. vivax cases with observable gametocytes failed to infect. However, the difference was not significant (Fisher's exact test, P= 0.19). Approximately 21% (13/62) with no observable gametocyte of either species could infect mosquitoes.
58 of the infections with either species had observable gametocytes (11 cases from the village survey and 47 from the Clinic). Feeds on 37 of these 58 gametocyte carriers led to oocyst production. The regression of percent of mosquitoes infected on the natural log of gametocyte density was not significant (t = 0.87, df= 36, P= 0.39). Similarly, there was not a significant association of mean oocyst load per infected mosquito on the natural log of gametocyte density (t = 0.87, df = 36, P= 0.38).
The results from the experiments with mosquitoes showed the infectiousness of subjects from the village surveys as well as from the Clinic. These indicated that some symptomatic and asymptomatic infections of each species could infect mosquitoes. The differences in percentages of infection in different studies [2, 4, 14] might be explained by several possible factors. First, the method of feeding: a recent study comparing the infectivity of gametocyte carriers to mosquitoes, using membrane and direct feeding, found significantly higher proportions of mosquitoes infected and higher oocyst burdens in mosquitoes fed directly on the skin . Conversely, Vanderberg  reported that infectivity in mosquitoes fed through a membrane usually equaled or exceeded infections by direct methods. However, most studies gave better results for direct feeding than membrane feeding. Thus studies such as the present one using direct feeding may provide a more reliable estimate of the infectious reservoir. Second, recruited subjects: in several studies mosquitoes were fed on individuals selected randomly and not on the basis of gametocytaemia [5, 10, 14]. But in other studies mosquitoes were fed on selected gametocyte carriers [4, 17] or parasitaemic cases with or without gametocytes (as in the present study).
Another relevant factor is variability in mosquito populations [18, 19], such as mosquito size [20–22]. The number of blood meals may also affect the infection rate [8, 23]. A careful comparison of Anopheles dirus, An. minimus and Anopheles maculatus infectivity in relation to size and blood-feeding behaviour would be of interest. Moreover, variability in susceptibility between different mosquito colonies is possible [4, 24].
The results in the present study showed that some cases with undetectable gametocytes could infect mosquitoes. This apparent anomaly is presumably at least partly due to larger volumes of blood in mosquito bloodmeals than are observed on slides, so that sufficient gametocytes to infect a mosquito may have been below the level of detection on blood slides. An attempt was made to see if gametocyte densities are higher in bloodmeals than finger pricks. However, it was found that gametocyte density was not significantly higher (and actually appeared to be lower) in the blood taken up by mosquitoes than in the blood from finger pricks. In some cases high densities of gametocytes were not infectious and similar results have been reported in most studies assessing human malarial infectivity to mosquitoes [3, 4, 11, 24, 25]. It has been suggested that the prevalence of gametocyte carriers is not a good indication of the infectiousness of a population to mosquitoes [10, 25–27].
It is clear that, as the Ethical committee only authorized us to request people over 14 years of age to take part in our experiments, this excluded a sector of the reservoir population, the children, from the study. It is also clear that the occurrence of transmission-blocking immunity and prior histories of the subjects taking anti-malarial drugs might have influenced whether they infected mosquitoes. No data on these factors was collected and so no comment can be made on their possible influence on infectivity of people's blood to mosquitoes. However, the fact that availability of anti-malarial drugs is much more tightly controlled in Thailand than in most malarious countries should be emphasized.
We would like to thank the staff from the Vector-borne Disease Control Unit No.8, Mae Hong Son Province for blood collection. We wish to acknowledge the help and co-operation of Mrs Suparp Chatchatreechan, Mr Pongnarin Dee-in, Mr Somkid Sumonmard and the staff in the Entomological Department of the Office of Vector-borne Disease Control No.2, Chiang Mai (VBDO) for their help in looking after and dissecting mosquitoes. We would also like to extend our thanks to staff in the Investigation Department of VBDO and to John Williams and his team from the London School of Hygiene and Tropical Medicine, UK, for re-checking the slides. We are also thankful to Dr Apinun Aramratana, Dr Walter RJ Taylor and Dr Ilona Carneiro for their suggestions on collection, analysis and presentation of data. This work was supported by the Royal Thai Government and World Health Organization (TDR).
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