Ethics Statement

This study has been approved by Medical Ethics Committee, UMMC. Ref No. 817.18 and National Institutes of Health (NMRR-09-881-4764). Permission to review malaria cases was obtained from the Ministry of Health, and Selangor Department of Health (JKNS/KA/V/734/03-05J1D.5). Participants provided verbal informed consent. Ethics approval was given on the basis that blood taken from participants were meant for diagnostic purposes of two circumstances; the first, patient was admitted to hospital and blood was drawn to diagnose (covered by Medical Ethics Committee, UMMC. Ref No. 817.18) and the second, in areas of an outbreak or of which cases of malaria had been reported, blood samples were collected by the District Health officers after verbal informed consent had been given (covered by National Institutes of Health, NMRR-09-881-4764).

Verbal informed consent of patients in the University of Malaya Medical Centre was documented in a diagnostic form which receives final verification by the Head of the Department of Parasitology, University of Malaya (Medical Ethics Committee, UMMC. Ref No. 817.18). In areas with outbreaks or reported potential malaria cases, verbal informed consent was documented in a form which had to be filled up by the District Health Officers that contains name, age, ethnicity, gender and address of each participant. The completed forms are kept in the database of Klang Vector Laboratory (National Institutes of Health, NMRR-09-881-4764).

This consent procedure was approved by Medical Ethics Committee, UMMC. Ref No. 817.18 and National Institutes of Health, NMRR-09-881-4764 ethics committees.

Malaria Cases and Treatment

Malaria transmission in Selangor is sporadic and varies from one area to another. During this research project, malaria cases were detected by the health clinics of respective districts in Selangor. Blood smears were obtained and sent to the laboratory. When malaria diagnosis was confirmed, the case was registered to the source of infection (locality of infection) accordingly. The cases were confirmed by the Klang Vector Laboratory. Thereafter, the Malaria Surveillance Unit of the Klang Vector Laboratory initiates an epidemiological investigation to determine origin of the cases (whether local or imported) and conduct mosquito collections to implicate the vector.

Treatment consisting of a single course of chloroquine was prescribed to patients with uncomplicated infections of P. falciparum, P. malariae and P. knowlesi. For patients infected with P. vivax and P.ovale, a course of chloroquine and primaquine was given. In cases of severe malaria, intravenous artesunate was used in combination with another drug such as doxycycline or chloroquine, regardless of the causative species. For vivax malaria patients, post treatment follow up of one year was performed whereas for patients infected with other species of malaria parasites (including P. knowlesi infection), a six-month-medical follow up was given.

Study Area and Entomological Mapping

The state of Selangor, Peninsula Malaysia was the target site of this study. Malaria cases from all districts were reported to the Klang Vector Laboratory. Entomological sampling was done on localities with reported malaria cases between year 2006 and 2012 whereby initial investigation revealed possibility of local malarial infection. The exact coordinates of potential vectors collected were obtained using the Garmin Geographic Positioning System (GPS) and directly plotted into GIS software system [17] as in Figure 1. Larvae obtained from the surrounding vicinity were brought and raised to adults in the laboratory. Subsequently, species identification of these adult mosquitoes was performed.

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Figure 1. Map of the study area showing the distribution of Anopheles species mosquitoes, macaque monkeys, and P.knowlesi malaria cases.

Most of Selangor districts are exposed to Anopheles species malaria vectors and Macaca fascicularis. Only Hulu Selangor has reported P. knowlesi cases according to vector lab records.

https://doi.org/10.1371/journal.pone.0077924.g001

Entomological Sampling and Mosquito Identification

Sampling was done following reports of any malaria cases. Larvae were collected from water bodies adjacent (less than 1000m radius) to the vicinity where malaria cases were reported. Adult Anopheles mosquitoes were collected using bare leg landing method and double-bed net method for three consecutive days at 41 locations reported to have malaria cases between 2006 to 2012 (Table 1). Collection was carried out from 1800 to 0000 hours.

Collected mosquitoes were identified taxonomically using Anopheles mosquito identification keys of Reid and Jeffery [18,19]. The dissected mosquito was examined with a Leica ICC50 microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Leica DC 300F colour video camera (Leica Microsystems, Cambridge, UK), which was connected to a computer. Photographs were analysed with Leica IM500 software (Leica Microsystems, Wetzler Germany). Several organs of each mosquito specimen were investigated: midgut to check for oocysts and salivary glands for sporozoites.

Long tailed macaque distribution

Since the long-tailed macaque monkey (Macaca fascicularis) is a known natural host of P. knowlesi [20] and is native to Peninsula Malaysia, its distribution was also taken into account in this study. Data on the macaque distribution was obtained from the Department of Wildlife and National Parks (DWNP/ PERHILITAN).

Data analysis

Raw data of malaria patients from the Klang Vector Laboratory were archived using Microsoft Excel 2010. Data analysis was done using Sigma Plot for Windows Version 12.0 Build 12.2.0.45 and Microsoft Excel 2010 (Version 14.0.6129.500 - 32-bit). Based on the Garmin GPS Device, the coordinates of sampling points within study sites that were captured were then digitally plotted and mapped using ArcGIS software[17]. All photographs were taken with an Olympus camera (OM-D E-M5).

Malaria cases

A total of 1623 laboratory confirmed malaria cases were registered in the 7-year-period between 2006 and 2012. The malaria cases (both local and imported cases) were found positive by microscopic examination for Plasmodium spp. and have been reported from all the nine districts of Selangor (Figure 2). Records of confirmed positive malaria cases for the year 2006 to 2012 of each district were analysed, and percentages of malaria cases in Selangor subsequently determined, and were represented with maps accordingly.

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Figure 2. Maps of confirmed reported malaria cases by district, 2006 to 2012.

Malaria endemicity showing the distribution of malaria cases, as indicated by district-level estimates based on available survey data from the Klang Vector Laboratory, Selangor, Department of Health. The districts were generated into shaded maps, in which the values for the cases are represented in shades from light (low cases) to dark (high cases). (A) Map showing cases that originated from outside the state of Selangor (Imported cases) (B). Map showing malaria cases that originated locally within the State of Selangor (Local cases).

https://doi.org/10.1371/journal.pone.0077924.g002

Based on microscopic findings, of the total 1623 samples, malaria parasite with the highest prevalence was P. vivax, with 1239 cases (76.3%). This was followed by P. falciparum, with 211 cases (13.0%), P. knowlesi, with 75 cases (4.6%), P. malariae, with 71 cases (4.4%), 26 cases (1.6%) of mixed infections of P. vivax and P. falciparum, and 1 (0.06%) P. ovale infection (Figure 3).

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Figure 3. Malaria incidences caused by the different species of Plasmodium in Selangor (2006- 2012).

Five different species of Plasmodium (excluding mixed cases) were identified as causes of malaria in Selangor (2006-2012). Based on the graph, from 2006 to 2012, the most common agent of malaria is P. vivax, followed by P. falciparum (with the exception of 2012, where P. knowlesi cases are second most common). More P. knowlesi cases are reported from 2008 onwards. The least common agent is P. ovale, with only one case in 2010. Mixed cases of P. vivax and P. falciparum are also noted in this graph.

https://doi.org/10.1371/journal.pone.0077924.g003

The age-specific infection prevalence and species distribution of all reported malaria cases are shown in Figure 4. From this study, the incidence of malaria ranged between the age of 1 and 55 year-old. The highest prevalence was found in the age group of 25- 29 year-old (peak age: 25 year-old).

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Figure 4. Malaria incidence in Selangor (2006-2012) based on age group.

Malaria cases are seen in in all age groups, with most cases occurring in a specific group (20-39).

https://doi.org/10.1371/journal.pone.0077924.g004

Based on the distribution of imported and local malaria cases in Selangor (Figure 5), imported cases showed a sharp increase in 2007 and thereafter dropped to a level that was higher than that of year 2006. This level was generally maintained throughout the study. However, the number of local malaria cases increased. In 2012, the number of local and imported cases had almost equalised (114 local cases as compared to 139 imported cases). This is in contrast with the 5 local cases and 83 imported cases reported in 2006.

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Figure 5. Malaria case classification in Selangor (2006-2012).

Malaria cases were classified as either local, imported, relapsed cases or unclassified/unknown. Classification was based on patient history. Cases of patients residing in Selangor for 2 weeks or less would be classified as imported. As shown above, there is an increase of local cases from 2006 to 2012 (except 2010), with the highest number of local cases (116) being reported in 2011. Imported cases, though decreasing from 2007, still remain higher than local cases with the highest number of imported cases (300) being reported in 2007. By 2012, there is an almost equal percentage of imported and local cases. Relapsed cases were also reported in all years except 2007 and 2012, with 17 cases being the highest in 2010. In all relapsed cases, Plasmodium vivax was the infective agent. Some cases were denoted as unclassified due to lack of data.

https://doi.org/10.1371/journal.pone.0077924.g005

Malaria and mosquito correlation

To determine the correlation between the causative agent of malaria (Plasmodium) and the vector (Anopheles mosquito), we tried to find out the potential vectors of malaria in the districts of Selangor with reported human cases (Figure 1; Table 1). The mosquitoes collected were taxonomically identified. Sixteen species were found, namely, the An. hyrcanus species group, An. aitkenii species group, An. leucosphyrus species group, An. vagus, An. umbrosus, An. baezai, An. karwari, An. collessi, An. kochi, An. brevipalpis, An. barbirostris, An. tessellatus, An. maculatus, An. sinensis, An. philippinensis and An. separatus.

In this investigation, the Anopheles mosquitoes were found in a total of 41 localities. The established malarial vector of Peninsula Malaysia, An. maculatus was relatively frequently collected in places with reported malaria cases. It was found in 18 out of the 41 locations with Anopheles mosquitoes. Based on examinations, none were found positive for malarial parasites. Moreover, at least 2 areas had Macaca fascicularis present coinciding with that of reported malaria cases and Anopheles mosquito prevalence (Figure 1). Both human malaria cases were that of P. knowlesi infections.

Malaria Epidemiology and Strategy in Selangor

This study is among the few which aims to investigate the correlation between malaria cases, macaque distribution and Anopheles mosquitoes in Selangor, Peninsula Malaysia. The National Strategic Plan (NSP) for Malaria Elimination 2010-2020 has been initiated by the Ministry of Health Malaysia. One of the aims of this national plan is to reduce locally acquired malaria incidence rate in Peninsula Malaysia by less than 1 per 1000 population by 2015 and in East Malaysia (Sabah and Sarawak) by 2017 and eventually, to ensure that Malaysia is free from local malaria transmission (except P.knowlesi transmission) by 2020. However, Anopheles mosquitoes would still be present in Malaysia, and possibilities of outbreaks occurring in such locations still exists if malaria cases were imported from other countries. Protection in malarious areas are provided through the use of residual spraying, impregnated bed net and anti-larvicidal control methods.

One of the main obstacles faced by NSP is the mobility of infected workers. A locality that has the source of infection (human cases) is termed vulnerable areas whereas an area that has malaria vectors is categorized as receptive areas. Receptive and vulnerable areas indicate areas that have both local malaria cases being reported as well as vector mosquito being found within that malarious area simultaneously. In the event where two or more human malaria cases occur in a certain area, an “outbreak” is said to have occurred in that area. In 2012, only two out of nine districts in Selangor were classified in this category, namely Hulu Langat, and Gombak. In order to prevent outbreaks in ‘receptive and vulnerable areas’ as well as in other areas, time-tested malaria prevention and control measures of the Malaria Elimination Programme must be implemented continuously.

Transmission of malaria in Selangor

In this study, we show that malaria transmission is still being reported in the State of Selangor. From the GIS data (Figure 2), local and imported malaria cases were still being reported from all districts of this state. It is interesting to note that although no malaria was reported within Kuala Lumpur, imported malaria transmission was reported from all the districts within the State of Selangor, which surround the Federal Territory strongly suggesting that foreigners and travellers from areas infected with malaria are crucial in the introduction of malaria to non-malarious areas [6].

On the other hand, local malaria transmission was only reported from three districts (Gombak, Hulu Langat, Hulu Selangor) that adjoins sub-urban areas and the Titiwangsa mountain range. We suggest that such distribution pattern is mainly attributed to the clearing of forests for development in these districts. Similar correlation of deforestation and malaria transmission was observed in the State of Perak, Peninsula Malaysia in 1997 [11].

Factors contributing to a potential risk of malaria transmission in Selangor

Macaque distribution

The overall data on macaque distribution in Peninsula Malaysia was obtained from the Department of Wildlife and National Parks, and detailed data on malaria cases were obtained from Health Departments of the Petaling District and Kuala Kubu Bharu District (highest imported and local cases respectively), the Klang Vector Laboratory and the University of Malaya Medical Centre (UMMC), Kuala Lumpur. P. knowlesi cases were seen in areas with Macaca fascicularis activities and mosquitoes belonging to the Anopheles (Cellia) leucosphyrus group (the established vector of P. knowlesi) had been collected from Kuala Kubu Bharu.

Other factors

Detection and control of malaria remains a public health concern, especially among the Orang Asli who stay in remote areas and make forays into the jungle in search of jungle produce, and thus exposing themselves to Anopheline mosquitoes infected with malaria parasites. The most prevalent malaria parasite in this study was P. vivax, followed by P. falciparum. This was similar to a previous study conducted in Pos Piah, Perak by Norhayatiet al., 2001 [26]. Other studies among Orang Asli population in Selangor reported that the most prevalent species was P. falciparum, followed by P. vivax [5].

Human activities such as hydroelectric dam construction, paddy field activities and irrigation canals which ecologically alter the jungle environment and encourage re-invasion of the typically efficient Anopheles mosquito which may play the role as vectors [11]. When a forested area is disturbed, streams and seepage water that were originally shaded are now exposed to sunlight. These clean, sun-lit water bodies are the breeding sites of choice for An. maculatus, the predominant mosquito vector of Peninsula Malaysia [23,25,27]. The ability of some soil types to trap water, such as those of granite origin, further contributes to this problem. Moreover, the replanting activities in agricultural industry such as those of rubber tree plantations and oil palm plantations further encourage the prevalence of the Anopheles mosquitoes [18,25,27]. It is possible that the construction of the dam in Kampung Pertak, Hulu Selangor district has allowed exposure of clean water bodies to sunlight, forming the breeding sites for Anopheles mosquitoes, such as, An. maculatus.

Plasmodium knowlesi infection in Selangor

Over the 7-year-period, the first report of P. knowlesi infections (2 cases) were reported to the Klang Vector Laboratory in 2008. P. knowlesi cases reported in this study were mainly from jungle fringes. There were no cases reported from urban areas. In 2012, two districts reported P. knowlesi cases; Hulu Selangor (32 cases, 11.8%) and Gombak (5 cases, 1.8%). Both these districts had sightings of Macaca fascicularis, which were also verified by distribution data given by the DWNP. Prior to 2008, in this study there was no malaria reported caused by P. knowlesi. The discovery of a large cohort of P. knowlesi cases thought to have been P. malariae by Singh et al. [28] has led to awareness of the presence of human knowlesi malaria in the region. Laboratory personnel and clinicians have thus become more aware of this parasite and since then, P. knowlesi infections have been reported in neighbouring countries such as Thailand [29], Myanmar [30], Vietnam [31], Singapore [32], the Philippines[33], and China [34]. Besides, there were reports of P. knowlesi infection acquired by travellers visiting Peninsula Malaysia [35]. P. knowlesi infection is potentially fatal due to its rapid development (24 hour-erythrocytic cycle) and great potential of achieving high parasitemia (> 5000/μl blood) [36,37].

Limitations

Malaria parasites species in this study were detected using light microscopy alone and thus could have led to premature analysis of results as early trophozoites that appear as ring forms resembling those of P. falciparum while late trophozoites “band forms” appear similar to P. malariae [28]. Furthermore, the malaria results provided for this study did not include infections that were not reported or those that did not go for treatment. Therefore, it may not reflect the actual malaria transmission in the state.

Another potential limitation in this study was that vegetation cover was not represented in the GIS map to show human activities such as deforestation, as well as encroachment in the sampling sites in the various districts into the potential Anopheles inhabited environments. However, this correlation was partially verified by our cursory field observations and findings.

Frequent immediate spraying by health authorities subsequent to a malaria case report resulted in low number of mosquitoes caught and might have led to the negative infection findings on the captured mosquitoes. Furthermore, we were limited by our capacity to enter into the depths of the jungle and had to restrict our collection close to human habitation.