Human blood samples
Blood samples or Giemsa stained blood films were sent to the Parasitology Unit of the Institute from hospitals and health centres that wanted a confirmation of Plasmodium malariae or in some cases to rule out P. knowlesi.
DNA extraction from whole blood
DNA was extracted from whole blood using the Qiagen D Neasy Blood Tissue Kit (Hilden, Germany), following the manufacturer's recommendations.
DNA extraction from blood films
In some cases only Giemsa stained blood films were provided. Before the extraction of the DNA, the slides were first cleaned with chloroform to remove oil. Fifty microliters of TE buffer was then pipetted on to the thin blood film. Two discs were punched out from a Whatman 1 filter paper (Whatman USA) using a pre flamed paper puncher. The discs were placed on the slide to soak up the buffer. Using a clean, flamed forceps, at least half of the smear was completely wiped off the slide with the filter paper and was transferred to 1.5 ml centrifuge tubes (Axygen). The DNA was extracted using the Qiagen D Neasy Blood Tissue Kit (Hilden, Germany).
Nested Polymerase Chain Reaction
Nested PCR assays [3, 20], based on the Plasmodium DNA sequence of the small subunit ribosomal RNA (SSUrRNA) genes, were used to detect and identify the species of malaria parasites found in the blood. The maximum number of samples processed at any one time was not more than 10. Positive controls for P. falciparum, P. knowlesi, P. malariae and P. vivax were included for all nested PCR species assays. A negative control was also included for each batch of assays. Nest 1 reaction was carried out in a 50 μl reaction mixture containing 1× reaction buffer (5× Green Go Taq Flexi Buffer, Promega Madison USA) 3 mM MgCl2(Promega), 200 mM of each deoxynucleoside triphosphate (Promega), 300 nM of each primers and 1.25 U of Go Taq DNA polymerase (Promega) and 5 μl of DNA template was used for each reaction.
Nest 2 PCR amplification was done in a 20 μl reaction mixture containing 1× reaction buffer (5× Green Go Taq Flexi Buffer Promega) 2 mM MgCl2(Promega,), 200 mM of each deoxynucleoside triphosphate (Promega,), 300 nM of each primers and 0.5 U Go Taq DNA polymerase (Promega,) and 2 μl of the nest 1 PCR products were used as DNA templates. All PCR reactions were carried out using thermal cycler (Techne TC 152 -Barloworld Sci Ltd UK). Ten microliters of the nest 2 amplicons were analyzed by agarose gel.
Sequencing of the Plasmodium csp genes
The csp genes of malaria parasites from all human isolates positive for P. knowlesi were amplified with primers, PKCSP-F and PKCSP-R . However, for the mosquito and monkey isolates, primers PKCSPF2 (5' TACAAGAACAAGATGARGAAC 3') and PKCSPR2 (5' TCAGCTACTTAATTGAATAATGC 3') were used since many non- specific bands were obtained with PKCSP-F and PKCSP-R.
PCR was carried out in a 20 μl reaction volume containing 1× Phusion HF buffer (Finnzymes), 200 mM/l of each dNTP (Finnzymes Finland) and 250 nM/l of each primer, and 0.02 U of Phusion DNA Polymerase (Finnzymes Finland). The PCR was carried out using a thermal cycler (Techne TC 152 -Barloworld Sci Ltd UK). The PCR conditions were as follows: initial denaturation at 98°C for 30 sec followed by 40 cycles of amplification at 94°C for 7 sec, 51°C for 20 sec, 72°C for 20 sec followed by a final extension step of 10 min. The expected size of the PCR products is approximately 1.2 kb and amplicons from each isolate were excised from the gel and purified using Perfectprep gel cleanup kit (Eppendorf, Germany), following the manufacturer's recommendation. The purified products were cloned as previously described . At least 20 of transformants from each PCR were screened using the csp primers mentioned above. Amplification was done in a 20 μl reaction mixtures containing 1× reaction buffer (5× Green Go Taq Flexi Buffer, Promega Madison USA) 2 mM MgCl2(Promega), 200 mM of each deoxynucleoside triphosphate (Promega), 300 nM of each primers and 0.5 U Go Taq DNA polymerase (Promega,). PCR conditions were as follows: initial denaturation of 94°C for 10 min followed by 30 cycles of amplification at 94°C at 1 min, annealing at 53°C at 1 min, extension at 72°C for 1 min 20 sec, followed by a final extension step at 72°C at 5 min. Ten microliters of the amplicons were digested with Eco R1 (Promega) and analyzed by gel electrophoresis. Plasmids from clones having the correct inserts were extracted using S.N.A.P. plasmid extraction kit (Invitrogen, USA) following the manufacturer's protocol. Purified plasmids was sent to Solgent Company Limited (Daejeon, South Korea) for sequencing to obtain the entire csp gene sequence of P. knowlesi
Analysis of sequence data
Analysis of the csp genes was performed as previously described . Sequences of the 456 nucleotide that encodes non – repeat N- terminal (first 195 nucleotides of coding sequence) and C- terminal (the last 261 nucleotides of the csp gene coding sequence) region of the protein were aligned by CLUSTAL W using Megalign (Lasergene, DNASTAR, USA). Regions of the protein were aligned using Megalign software (Lasergene). The csp gene sequences from patients, mosquitoes and monkey samples were compared with those obtained from the GenBank data base. Phylogenetic trees were performed by the neighbour-joining (NJ)  and Bayesian methods . The NJ method was analyzed using the Kimura-2 parameter with 1000 bootstrap replicates and was carried out using the MEGA version 4.0 software . On the other hand, the Bayesian method was analysed using the Hasegawa-Kishino-Yano (HKY) model with the following parameters: the search was performed at 1,000,000 generations, sampling every 100 generations and the first 2000 trees were discarded in the burn-ins. The analysis was carried using the Mr. Bayes 3.1 software .
Nucleotide sequences reported in this study have been deposited in Gen Bank under accession numbers: EU687467–EU687470, EU708437 (human samples), EU821335 (mosquito sample), EU821336 (monkey sample). The other malaria csp gene sequences used were obtained from GenBank: P. knowlesi (M11031), P. knowlesi (K0082), KH35 (AH013332), KH43 (AH013333), KH50 (AH013334), KH107 (AH013336), KH115 (AH013337), P. coatneyi (AY135360), P. cynomolgi (M15104), P. simiovale (U09765), P. simium (L05068), P. inui (FJ009512) P. vivax (M34697), P. malariae (U09766), P. malariae (J03992), P. falciparum (K02194) and P. vinckei lentum (AF162331).
Trapping of monkeys
Monkeys were trapped in the vicinity of Kuala Lumpur, Selangor State and Kuala Lipis in Pahang State with help from the wild-life department. The captured monkeys were anesthetized by intramuscular injection with ketamine hydrochloride. Ten ml blood was collected, after which the monkeys were tagged with an electronic identification system (Trovan Ltd London). After they recovered they were released into the deep forest.
Preparation of blood films
Both thick and thin blood films were prepared followed by staining with Giemsa. Microscopic examinations were carried out using compound microscope under oil immersion under 100× magnification.
DNA extraction from whole blood, PCR and sequencing
DNA was extracted from the whole blood using the Dneasy Tissue Extraction Kit (Qiagen, Germany) following the manufacturer's recommendation. All monkeys were screened for malaria parasites using primers rPLU3 and rPLU4 , and for P. knowlesi using the primers Pmk8 and Pmk9 . Nested PCR, cloning and sequencing of all samples positive for P. knowlesi were performed as described above.
Study Sites for mosquito collection
The study site for mosquito collection was in Kuala Lipis district in the State of Pahang. Pre-surveys for mosquito collections were carried out to determine suitable sites for long term study based on presence of monkeys and occurrence of cases. Based, on pre-surveys two sites were selected for the study. One is Serunai Mela village [4° 7.0'N, 102° 11.9'E ] and the other is a fruit farm in Sungai Ular [4° 15.7'N, 102°4.8'E ]. In Serunai Mela, a case of P. knowlesi had occurred and the case house is situated at the forest fringe. Sungai Ular was selected as a patient had reported that he visited the farm before falling ill and it was also frequented by monkeys
All night mosquito collections using bare-leg catch method  were performed from July 2007 to November 2007. In each area 4 nights of collection were carried out every month by three men working outdoors from 18.00 to 06.00 hours.
In order to compare the number of mosquitoes attracted to humans and monkeys, a monkey- baited – trap was constructed in Serunai Mela Village as previously described [26, 27].
Mosquito identification and dissection
All mosquitoes were identified morphologically in the field laboratory. The keys of Reid  were used for the identification of Anopheles mosquitoes and keys of Sallum  were used for leucosphyrus group in particular. Anopheles mosquitoes were dissected to extract ovaries to determine parity and the midguts and salivary glands were examined for oocysts and sporozoites, respectively. All the positive salivary glands were placed in 1.5 microcentrifuge tubes (Axygen, USA) containing absolute alcohol and were labeled accordingly.
DNA extraction, PCR and sequencing
Ethanol used in the preservation of the salivary glands were allowed to evaporate completely by placing the tubes in a Thermomixer (Eppendorf, Germany) set at 70°C. DNA was then extracted using the Qiagen D Neasy Blood Tissue Kit (Hilden, Germany) as described above. PCR and sequencing were also carried out as mentioned above.
This project was approved by the Institute for Medical Research & Ethical Committee Ministry of Health Malaysia and the Animal Use Committee of the Institute.