The study was reviewed and approved by the Ethic Committee of the Institute for Parasitic Disease Control of Yunnan Province and local administration authority in Myanmar. One hundred forty-six blood samples were obtained in 2008 from randomly selected patients with uncomplicated malaria in southern Myanmar near Yunnan Province of China, where pig-tailed macaques are also present. Written consent was obtained from each person before blood samples were obtained. A drop (20 µL–50 µL) of fingerprick blood was placed directly on premarked filter paper. Malaria infection was identified by microscopic analysis of Giemsa-stained blood films made from blood spotted on the paper.

DNA templates for a nested PCR were prepared from whole blood spots on filter paper according to a previously reported method (11). Genomic DNA of P. falciparum was obtained from in vitro–proliferated 3D7 clone. Genomic DNA of P. vivax was obtained from a patient from the study region previously identified by using PCR and the primer sets used in this study. As reported (2,12), DNA (3 μL/sample and 1 ng of each positive control DNA) from each sample was amplified with the Plasmodium genus–specific primer pair rPLU1 and rPLU5.

Two microliters of PCR product from each amplification was subjected to a second PCR amplification with species-specific primer pairs rFAL1 and rFAL2 for P. falciparum, rMAL1 and rMAL2 for P. malariae, rVIV1 and rVIV2 for P. vivax, rOVA1 and rOVA2 for P. ovale, and Pmk8 and Pmkr9 for P. knowlesi. PCR products amplified with nested primers were analyzed by agarose gel electrophoresis. DNA bands were removed from the gel, purified by using the QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA, USA) and ligated to T-cloning vector (Invitrogen, Carlsbad, CA, USA) according to protocols provided by the manufacturers. Plasmid inserts were then sequenced.

Figure

Figure. Phylogenetic analysis of A-type and S-type 18S small subunit (SSU) rRNA gene sequences of Plasmodium spp., Myanmar, 2008. Fragments of 18S SSU rRNA gene sequences of samples were analyzed by aligning...

Sequence identity was confirmed by random basic local alignment search tool analysis of sequences in GenBank (http://blast.ncbi.nlm.nih.gov/). Novel sequences were deposited in GenBank with accession nos. GU816242–GU816250. Phylogenetic relationships of unique sequences amplified by using nested primers with corresponding reference sequences were constructed by using the neighbor-joining method in MEGA version 4.0 (13). All sequences clustered with reference sequences of P. falciparum, P. vivax, or P. knowlesi, which suggested that all sequences were species specific (Figure).

Three parasite species (P. falciparum, P. vivax, and P. knowlesi) were identified in 146 infected persons. Phylogenetic analysis showed that amplified products were species specific (Figure). Monoinfection with P. falciparum, P. vivax, and P. knowlesi accounted for 34.9% (51/146), 36.3% (53/146), and 2.7% (4/146), respectively, of the infections. Mixed infections of P. knowlesi with P. falciparum or P. vivax accounted for 6.9% (10/146) of the infections, and mixed infections with P. knowlesi and either P. falciparum or P. vivax accounted for 8.9% (n = 13 in both groups) of the infections. Only 2 samples (1.4%) had mixed infections with P. falciparum, P. vivax, and P. knowlesi (Table). Thus, the prevalence of mixed infections in southern Myanmar was lower than that in northern Myanmar near the border with Thailand (3). The prevalence of P. knowlesi was 21.9%. In most cases, this parasite showed co-infection with either P. falciparum or P. vivax, which indicated that P. knowlesi may have not fully adapted to the human host or that humans who were infected with other malaria parasites may be more vulnerable to P. knowlesi infection.

Our study also emphasizes the need for improvement of current methods for detecting P. knowlesi infection (14,15). A recent report found that the primer pair Pmk8 and Pmkr9, which is specific for the 18S SSU rRNA gene of P. knowlesi, can cross-hybridize with the corresponding sequence of P. vivax (14). We observed weak amplifications in 16 samples (11%); all were from P. vivax–infected blood. All amplicons made by using Pmk8 and Pmkr9 primers were sequenced and compared with the homologous sequences. Because of the high similarity of 18S SSU rRNA gene sequences among these parasites, more specific sequences are needed for establishing a reliable PCR-based method for routine diagnosis of P. knowlesi infection (14).