Co-infections with Plasmodium knowlesi and other malaria parasites, Myanmar.

To determine the frequency of co-infections with Plasmodium species in southern Myanmar, we investigated the prevalence of P. knowlesi. More than 20% of patients with malaria had P. knowlesi infection, which occurred predominantly as a co-infection with either P. falciparum or P. vivax.

Plasmodium species are co-endemic to regions of Southeast Asia (,). This finding is believed to be underestimated because of insufficient sensitivity of microscopic detection of parasites. The prevalence of mixed infections with malaria parasites in the border regions between Thailand and Myanmar was recently found to be <24% (). Identification of P. knowlesi as the fifth human malaria pathogen, which is prevalent in countries in Southeast Asia, has complicated this situation. P. knowlesi is a parasite that infects mainly long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (M. nemestrina) in Southeast Asia (). The parasite has developed the capacity to naturally infect humans, and infections in some persons have been life-threatening (,). Furthermore, infections with P. knowlesi in travelers to this region have been increasing (,).

P. knowlesi isolates obtained from humans have been frequently misidentified as P. falciparum or P. malariae because of the morphologic similarities of these parasites (). Use of PCRs specific for 18S small subunit (SSU) rRNA genes of malaria parasites has identified suspected cases (,). P. knowlesi infection in humans in the border area between the People’s Republic of China and Myanmar has been reported (), but the prevalence is unknown. We investigated the frequency of co-infections with P. knowlesi and other Plasmodium spp. in this region.

The Study

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 (). 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 (,), 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.

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 (). All sequences clustered with reference sequences of P. falciparum, P. vivax, or P. knowlesi, which suggested that all sequences were species specific (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 with published homologous sequences of P. falciparum, P. vivax, and P. knowlesi. A phylogenetic tree was constructed on the basis of similarities by using MEGA version 4.1 (). Novel sequences identified in this study are indicated in boldface. Scale bar indicates nucleotide substitutions per site.

Conclusions

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 (). 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.

Table

Plasmodium species identified in 146 persons, Myanmar, 2008

Parasite	No. (%) persons
Plasmodium knowlesi	4 (2.7)
P. knowlesi/P. falciparum	13 (8.9)
P. knowlesi/P. vivax	13(8.9)
P. knowlesi/P. falciparum/P. vivax	2(1.4)
P. falciparum	51 (34.9)
P. vivax	53 (36.3)
P. falciparum/P. vivax	10 (6.9)

Our study also emphasizes the need for improvement of current methods for detecting P. knowlesi infection (,). 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 (). 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 ().