First case of human infection with Plasmodium knowlesi in Laos

1 Department of Tropical Medicine and Malaria, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan, 2 SATREPS project (JICA/AMED) for Parasitic Diseases, Vientiane, Lao PDR, 3 Parasitology Laboratory, Institut Pasteur du Lao PDR, Ministry of Health, Vientiane, Lao PDR, 4 National Institute of Public Health, Ministry of Health, Vientiane, Lao PDR, 5 Department of Tropical Medicine and Parasitology, Dokkyo Medical University, Tochigi, Japan, 6 Center of Malariology, Parasitology and Entomology, Ministry of Health, Vientiane, Lao PDR, 7 Institut Pasteur du Lao PDR, Ministry of Health, Vientiane, Lao PDR


DNA analysis
DNA was extracted from the dried blood spot on the filter paper with a Maxwell RSC Instrument (Promega, United States of America) in accordance with the manufacturer's instructions with minor modification (S1 Table).
The Plasmodium species present in the patient's blood were identified by real-time PCR using species-specific primers for 5 different Plasmodium species (S1 Table). Based on the realtime PCR, it was suspected that the DNA sample contained P. knowlesi. To confirm the P. knowlesi infection, DNA sequencing was conducted. First, 2 gene regions (partial cytochrome b gene [cytb] of the mitochondrial genome and merozoite surface protein-1 gene [msp1] of the nuclear genome) of P. knowlesi were amplified by nested PCR [1] (S1 Table). Second, the PCR amplicons were purified with Performa DTR Gel Filtration Cartridges (Edge Bio, United States of America) and sequenced by an ABI Genetic Analyzer model 3130XL (Life Technologies, Japan). Molecular phylogenetic trees were constructed based on polymorphic sites in the partial sequences of both gene regions using the neighbor-joining method in molecular evolutionary genetics analysis (MEGA) software version 7.0.21 [2]. In addition to sequences from the patient, those from P. knowlesi isolates from other countries and some other Plasmodium species that appear in GenBank were included in the tree.
In the phylogenetic analysis, the partial DNA sequences of the genes from the patient isolate clustered with the sequences of P. knowlesi isolates from Thailand or Malaysia (Fig 1). The cytb sequence of the Lao isolate (DNA Data Bank of Japan [DDBJ] accession no. LC327233) was 99% (221/223 bp) identical to that of a Malaysian isolate (GenBank accession no. EU880463), whereas the msp1 sequence of the Lao isolate (DDBJ accession no. LC327234) was 99% (473/479 bp) identical to that of a Thai isolate (GenBank accession no. JF837343) (Fig 1).

Case discussion
In this study, we identified the first documented case of a human infection with P. knowlesi in Laos, although it was a very rare case (1/2,698 cases). In this study, the predominant species were P. falciparum (42%), P. vivax (39%), and mixed infection with P. falciparum and P. vivax (4%).
The simian malaria parasite P. knowlesi is prevalent in macaque monkeys in Southeast Asian countries. Human infections with this parasite were thought to be extremely rare until multiple human infections were reported in 2004 from Malaysian Borneo [3]. Thereafter, several cases of human infections, including fatal cases, were reported in several Southeast Asian countries other than Laos [3][4][5][6][7]. Recently, however, a malaria study of long-tailed macaques in Southeast Asian countries demonstrated that 1 of 44 (2.3%) macaques originating from Laos was infected with P. knowlesi, but the exact geographic location where the infected macaque was captured in Laos was not given [8].
Presently, in Laos, the majority of malaria patients are adult males [9]. Higher numbers of malaria cases in men are usually associated with occupational activities, such as logging, mining, and dam or road construction in and through the forest [9]. In the present case, we speculate that the 12-year-old boy contracted P. knowlesi while going to the forest or somewhere near his village, which is surrounded by the forest and where wild monkeys had been observed previously (personal communication, father of the patient to the author). This case suggests that P. knowlesi is being transmitted in the forest near the village, but some malaria patients in the area may be contracting it without their knowledge, because no PCR analysis is available at the local health center. In addition, nowadays, ecotourism activities such as elephant riding, kayaking, trekking, caving, observing wild animals, and staying overnight in treehouses in the forest have become very popular among foreign travelers in Laos. These trends increase the number of foreign travelers who are in close contact with the forest and its fauna, including wild monkeys. Furthermore, a recent study reported that 63% (28/44) of long-tailed macaques in Laos were infected with P. cynomolgi, which can also be infectious to humans [8,10]. Therefore, an investigation of human infection with P. cynomolgi is also needed in Laos.
In this study, the patient was first diagnosed with P. vivax infection by the RDT kit. The positive P. vivax RDT result could have originated from a cross-reaction. In fact, previous studies demonstrated that false-positive results for P. vivax and P. falciparum have been observed for P. knowlesi mono-infection with certain RDTs [11,12]. The RDT sensitivity to P. knowlesi should be investigated further.
To better understand the epidemiology of simian malaria in Laos, further investigations, such as mass blood surveys among the villagers, entomological surveys to identify the species of Anopheles, and wild monkey surveys (using blood, urine, or feces), are needed. Supporting information S1 Table. PCR primers, master mix, and condition of assay. Table footnotes: DNA extraction: DNA was extracted from the dried blood spots on the filter papers with a Maxwell RSC Instrument (Promega, United States of America) in accordance with the manufacturer's instructions with minor modification. Three punched-out circles of 3.175-mm (1/8-inch) diameter from the dried blood spot on the filter paper were used for DNA extraction, which was equivalent to 15-20 μL of whole blood. The punched-out filter paper circles were incubated in 30 μL of proteinase-K and 180 μL of incubation buffer from the kit at 70˚C for 90 minutes and then followed with the extraction instructions. The extracted DNA was eluted with 50 μL of elution buffer and preserved until use at −30˚C. PCR: For identification of P. knowlesi, partial cytb and partial msp1 of P. knowlesi were amplified by nested PCR. For primary PCR of the cytb, real-time PCR was performed using a primer set of PCBF and PCBR. For secondary PCR of the cytb, conventional PCR was performed using a primer set of PKCBF and PKCBR. For primary PCR of the msp1, real-time PCR was performed using a primer set of Pk_MSP1_F3 and Pk_MSP1_R2. For secondary PCR of the msp1, conventional PCR was performed using a primer set of Pk_MSP1_F2 and Pk_MSP1_R2. For identification of P. falciparum, real-time PCR was performed using a primer set of Pf F1 and Pf R1. For identification of P. vivax, real-time PCR was performed using a primer set of PvF11 and PvR7. For identification of P. ovale, real-time PCR was performed using a primer set of POCBF and POCB_R1. For identification of P. malariae, real-time PCR was performed using a primer set of PMCBF and PMCBR. Abbreviations: cytb, cytochrome b gene; msp1, merozoite surface protein-1 gene. (XLSX)