Background
Malaria still poses a public health problem in Malaysia despite continuous efforts to control the spread of the disease. Four species of
Plasmodium were responsible for the spread of malaria in humans for a long time. However, currently the fifth species,
Plasmodium knowlesi, which is also life threatening [
1], has spread to many parts of Malaysia [
2‐
4]. Besides Malaysia, knowlesi malaria has also been reported in countries in Southeast Asia. These include Thailand [
5], Singapore [
6], Philippines [
7], Vietnam [
8], Myanmar [
9,
10], Indonesia [
11] and Cambodia [
12]. This disease has also been reported in Europe and America by travellers who visited endemic sites in Southeast Asia, particularly Malaysia [
13‐
17].
The first reported case of human knowlesi malaria in Malaysia was from Pahang. An American surveyor working in the jungles of Pahang contracted the disease [
18]. Work was then carried out to determine the epidemiology of the disease and its vectors [
19‐
23]. No further cases were detected in spite of screening more than 1,000 people in that area. No vectors were incriminated in that area during their study [
21]. The main aim of the investigation was to determine if malaria was a zoonosis. Studies were also conducted in other areas of peninsular Malaysia. New species of simian malaria in macaques were described [
24‐
26] and
Anopheles hackeri was incriminated as the vector of
P. knowlesi[
23]. However,
An. hackeri was found biting only macaques and was not attracted to humans. Thus, from the studies it was hypothesized that
P. knowlesi will not be easily transmitted to humans and that the first case was an extremely rare event.
In peninsular Malaysia, malaria, which is transmitted largely by
Anopheles maculatus is on the decline. However, cases of
P. knowlesi are occurring in areas which have been free of malaria [
3]. Early studies have incriminated the
Anopheles Leucosphyrus group of mosquitoes as the vectors of simian malaria [
21,
27]. The epidemiology of knowlesi malaria is thus strongly linked to the Leucosphyrus group of mosquitoes. Recently
Anopheles latens has been incriminated as a vector of
P. knowlesi in Kapit, Sarawak Malaysian Borneo [
28] and
Anopheles cracens as the vector in Kuala Lipis Pahang, peninsular Malaysia [
3]. The results of a longitudinal study to understand seasonal variation in two different ecotypes in Kuala Lipis is presented here.
Discussion
Knowlesi malaria is an emerging zoonosis of public health importance in Malaysia. Owing to Malaysia’s rapid development, tropical climate, presence of vectors and long-tailed macaques, timely and effective disease control is required to prevent the spread of the disease. Since the emergence of knowlesi malaria in the country in 2004 [
2], entomological investigations have been carried out to elucidate the vectors.
Anopheles latens and
An. cracens have been incriminated as the vectors in Kapit, Sarawak [
28] and Kuala Lipis, Pahang [
3] respectively. Both these species are members of the An. leucosphyrus subgroup of mosquitoes known to be natural vectors of simian malaria. Species belonging to this group are also important vectors for human malaria and are distributed in the South and Southeast Asia region [
36‐
38]
. Anopheles latens is also a predominant vector of human malaria in Sarawak and has widespread distribution there [
39,
40]. However,
An. cracens has limited distribution in the northern state of Perlis [
19], in the east coast state of Terengganu [
32,
41] and currently in Pahang. It was also reported from southern (peninsular) Thailand (Chumphon, Phangnga, Phattalung) [
42], and Indonesia (Aceh, Sabang, Sumatra) [
32].
Anopheles cracens, was found to be an important vector not only for human malaria, but was also positive for
P. inui and
P. cynomolgi[
19]
. This mosquito was found to be attracted to monkeys at canopy and humans at ground level [
43]. However, the current study has shown that
An.
cracens is biting macaques more at ground level than at canopy. The propensity of
An. cracens to bite monkeys at ground level or canopy, and humans, demonstrates the importance of this species in the transmission of knowlesi malaria. Due to changes in the ecosystem, the behaviour of the mosquito also seems to change. Although extensive vector studies have been carried out in Pahang in the 1980s and 1990s by researchers from the Institute for Medical Research, (Kuala Lumpur, Malaysia) [
29,
44,
45] there have been no previous reports of
An. cracens there. Earlier studies by Wharton
et al. in other parts of Malaysia also show no reports of this species [
22].
Studies carried out by Baimai [
42] in Thailand showed that
An. cracens was an anthropophilic species and peak biting activity was from 19:00 to 21:00 hours. Thus, it seems that
An. cracens has not changed its biting activity since similar times has been demonstrated in this study. In addition,
An. cracens was known to enter shelter but rarely rested on walls and readily exited after a bloodmeal.
Anopheles cracens was an important vector of human malaria because it was involved in the human
Plasmodium transmission in areas where houses were close to the jungle [
46]. After the 1960s very little is known about
An. cracens in Malaysia
. The distribution of
An. cracens in peninsular Malaysia remains unknown, although cases of knowlesi malaria are reported from all states in Malaysia.
Despite high prevalence of simian malaria in macaques in Kuala Lipis [
3], the infected mosquitoes obtained from the study were low. Thus, there is a possibility that other species besides
An. cracens are involved in the transmission of simian malaria at least among macaques. Transect studies through the forest will determine the other species involved in transmission. Studies carried out in Kapit, Sarawak where a large number of knowlesi malaria cases were reported from that area,
An. latens was incriminated as the vector and the monkey to human biting ratio was 1:1.3 [
47]. In comparison to that, the monkey to human biting ratio for
An. cracens was 1:2. From this study, it was observed that
An. cracens is attracted to both monkey and human. However, it prefers to bite humans compared to monkeys. This could possibly be the reason why there was fewer knowlesi malaria cases in peninsular Malaysia compared to Malaysian Borneo. The frequency of a man-monkey-mosquito natural cycle transmission is dependent upon the various hosts in the area of natural transmission.
Anopheles hackeri was incriminated as the natural vector of
P. knowlesi among the monkey population in peninsular Malaysia after the discovery of the first human case of
P. knowlesi in Pahang [
23]. However,
An. hackeri is not attracted to humans and is zoophagic. Thus, at that time it was concluded that
P. knowlesi would not be easily transmitted to humans due to the nature of that mosquito. Thus, it is evident that more studies on vectors are needed throughout Malaysia in order to understand the dynamics of simian malaria being transmitted to humans.
Laboratory studies have shown both
An. kochi and
An. maculatus are susceptible to simian malaria parasites especially to
P. cynomolgi[
21]. However, the numbers of
An. maculatus coming to the monkey-bait trap was very scarce and this could be a reason why
An. maculatus has not been found with natural infection of simian malaria. As for
An. kochi, it was found predominantly at ground level and not at canopy level and this could be one of the reasons why it was not positive for sporozoites. In nature the macaques roost on trees at night and thus mosquitoes biting at canopy level will be able to pick up infection.
Although knowlesi malaria has been reported from many countries in Southeast Asia, studies on vectors in relation to knowlesi malaria are lacking. Besides Malaysia, studies in Vietnam have incriminated
Anopheles dirus to be the vector of
P. knowlesi[
48,
49]. In Vietnam
An. dirus has been found positive with mixed infection of human and simian malaria sporozoites. However, cases of knowlesi malaria in Vietnam was cryptic and only a few cases have been reported [
8], while in Malaysia, mixed infection of human and simian malaria occurs [
2,
3] but no mixed infection was found in
An. cracens. This leads to further gaps in our knowledge with regard to human to human transmission or monkey to human transmission. In the case of Malaysia, deforestation has certainly disrupted the ecosystem and the forest mosquitoes that were found in the northern part only of peninsular Malaysia is now reported in the east coast of the peninsular and were found in the edge of the forest and in villages surrounding the forest. How much further they have spread remains unknown. It is also difficult to decipher why the numbers of
An. cracens were more in the fruit orchard compared to the forest edge. With deforestation this species of mosquito is still able to colonise the plantations and the forest edge. It seems similar to a report in Thailand where by
An. dirus a forest mosquito colonised plantations when deforestation took place [
50]. Since cases of knowlesi malaria have been reported from all states of Malaysia, vectors must be present everywhere. However, a species may play a primary role in an area and a secondary role elsewhere. Thus, vector control activities will be hampered unless vectors are elucidated throughout the country. In order to eliminate malaria in Malaysia it is important to study the vectors in various ecological zones so as to design an effective control programme. From this study it is also clear that current vector control tools being used in the malaria control programme will not be effective to reduce vector population as these mosquitoes are exophagic and early biters. Extensive studies should also be carried out on village population to determine the prevalence of knowlesi malaria in the country. Besides microscopy, molecular techniques should be used in order to determine the species of malaria. Only then can proper strategies be instituted to control malaria and work towards its elimination.
Molecular techniques are very useful in identifying the infection, in describing the epidemiology, and in characterizing mixed infections, which are otherwise under reported. Thus, with the improvement in molecular diagnostics methods, one is now able to distinguish between P. knowlesi and P. malariae. However, the levels of the problem strongly rely on the cohabitation of the monkeys, humans and the presence of the vectors, which are simio-anthropophagic and exophilic. The frequency of man-monkey-mosquito natural cycle transmission is dependent upon the various hosts in the area of natural transmission.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
IV conceived the study. AIJ, IV and FMY were responsible for the preparation of the manuscript. IV, AIJ, NAY, YY and AAH were responsible for field collection, supervision, identification and processing of mosquitoes. AIJ and IV were responsible for the molecular work. AIJ, FMY and IV analysed sequence data. All authors have read and approved the manuscript.