Detection of the Plasmodium falciparum Kelch-13 gene P553L mutation in sporozoites isolated from mosquito salivary glands in South-Central Vietnam

Plasmodium falciparum has developed resistance against artemisinin in Southeast Asia. Mutations in the P. falciparum Kelch-13 (Pfk13) gene are associated with artemisinin resistance in vitro and in vivo. We investigated the prevalence of mutations in PfK13 from sporozoite-stage parasites isolated from the salivary glands of Anopheles dirus mosquitoes. Mosquitoes were caught by human-landing catches at two locations within the Khanh Phu commune, South-Central Vietnam. Identification of Anopheles species was performed based on morphological features and nucleotide sequence analysis. Sporozoite-infected salivary glands were stored on filter paper and at 4–6 °C. A nested-PCR targeting the small subunit ribosomal RNA gene was used for Plasmodium species identification. Pfk13 was amplified by nested PCR, and subjected to nucleotide sequencing. Five of 33 P. falciparum sporozoite samples carried the P553L mutation at the PfK13 locus. This mutation has been recorded previously in Vietnam, but not in Khanh Hoa province, were surveys of K13 polymorphism have not previously been carried out. These results demonstrate the utility of mosquito-stage malaria parasite samples for studies on the molecular epidemiology of drug resistance.


Background
Artemisinin-based combination therapy (ACT) against Plasmodium falciparum is currently the most common and effective first-line therapy in most malaria-endemic countries. Recently, however, clinical cases of artemisinin resistance have been reported from various countries in Southeast Asia [1][2][3][4][5][6] and Africa [7].
Genome-wide analysis of artemisinin resistance in P. falciparum has demonstrated that mutations in the propeller domain of the gene encoding the Kelch 13 (K13) protein (Pfk13) are associated with delayed parasite clearance in vitro and in vivo [8,9]. In Southeast Asia, a number of mutations in the Pfk13 gene linked to artemisinin resistance have been identified, including C580Y, Y493H, R539T, I543T and others. In Vietnam, the C580Y, P574L, V568G, P553L, I543T and Y493H mutations have been recorded, all from the Southern part of the country on the border with Cambodia [10].
In this report, we characterized the polymorphism at the Pfk13 locus using parasite DNA extracted from sporozoites isolated from the salivary glands of humanbiting Anopheles dirus mosquitoes in South-central Vietnam.

Methods
Collection of mosquitoes was carried out through human-baited landing catches in and around the forest near Nga Hai village in the south of Khanh Phu commune, Khanh Vinh district, Khanh Hoa province, Vietnam. Collection of mosquitoes was carried out as previously described [11,12] from January 2008 to October 2012. Mosquito collectors provide informed consent and were regularly screened for malaria and treated with ACT if a malaria infection was detected. Identification of Anopheles species was carried out based on morphological characteristics [13] and through analysis of nucleotide sequences [12]. Female mosquitoes were dissected and examined by microscopy for sporozoites. Sporozoite-infected salivary glands were stored on filter paper kept in closed vials at 4-6°C until analysis [13].
Genomic DNA (gDNA) was extracted from preserved filter paper with sporozoite-positive salivary glands [11,14]. The 18S rRNA gene-based nested PCR was used for the detection of P. falciparum and other Plasmodium species [12,15]. Amplification of the Pfk13 gene was carried out by nested PCR as previously described [8], and the products sequenced with BigDye Terminator v3.1 Cycle Sequencing Premix Kit (ABI, Foster city, CA, USA). Sequencing products were run on an ABI/Hitachi 3130x1 Genetic Analyzer (ABI) and nucleotide sequences were analysed using Genetyx (Genetyx Corporation, Tokyo, Japan).

Results and discussion
A total of 11,464 female An. dirus ( Figure 1) were captured; of these, 11,437 mosquitoes were dissected and the presence of sporozoites in salivary glands determined by microscopic examination. One hundred and sixtyeight (1.47%) showed Plasmodium sporozoites infection ( Table 1). Of the sporozoites infected mosquitoes, 152 out of 168 (90.5%) were used for this study. By nested PCR analysis, 42 out of 152 stored sporozoite-positive mosquitoes (27.6%) were identified as harbouring P. falciparum (Table 2).
Nucleotide sequencing was performed on the nested PCR amplified portion of Pfk13 (nucleotide positions 1279-2127) for 34 out of the 42 P. falciparum sporozoite-positive mosquitoes, of which 33 were successfully assayed ( Table 2). Single nucleotide polymorphisms (SNPs) with respect to the Pfk13 sequence of the 3D7 clone (PF3D7_1343700) were detected at low frequencies (Table 3). Synonymous mutations were not observed, and only a single non-synonymous mutation, P553L, was recorded. This mutation was present in 5 of the 33 P. falciparum sporozoite-  (Table 3). Samples containing mutant alleles were captured both in the forest and at the forest fringe. Three mutant samples were observed in P. falciparum single species infections, and two were found in P. falciparum infections co-infecting mosquito salivary glands with other malaria parasite species (Table 3).
The prevalence of malaria in the study area has declined sharply since 1998 and was considered to be 'residual forest malaria' during the study period, while the proportion of P. falciparum among infections has remained substantial; densities of An. dirus typically peak in March and April [11]. The sample sizes of P. falciparum infections analyzed for K13 from 2008 to 2012 ( Table 2) preclude analysis of prevalence trends through time.
There have been no previous reports of ARTresistance associated mutations in the Pfk13 gene from this region of Vietnam. We observed only an azygous non-synonymous mutation in Pfk13, P553L, and this was present at low frequencies. This mutant allele has been identified previously, in samples collected from the Vietnam-Cambodia border, Southern China and Kenya [10,[16][17][18][19]. It is not currently known whether it is linked resistance to ART [8].
The Vietnamese national malaria treatment guidelines proscribe that patients are treated with 2.4 mg/ kg of body weight dihydroartemisinin and 18 mg/kg piperaquine once a day for 3 days, and these guidelines were in effect in Khanh Hoa province during this study. The first record of the protracted parasite clearance after treatment with artesunate monotherapy or dihydroartemisinin-piperaruine was from the Bu Dang district of Binh Phuoc province in 2009 [20]. In that area, five types of mutant allele, including P553L, have been found Pfk13 [10]. Our study demonstrates the presence of the Pfk13 P553L SNP in 2009 and 2010 in Khanh Hoa province, some 200 km distant from Binh Phuoc. This result could suggest that the parasite populations of Southcentral Vietnam are contiguous over large distances, and that mutant allele gene flow is possible between relatively distant regions, although more evidence is required to support this hypothesis.

Conclusions
In conclusion, this study demonstrates the presence of mono non-synonymous nucleotide mutations in the Pfk13 gene in P. falciparum sporozoites isolated from the salivary glands of from An. dirus. The