METHODOLOGY/PRINCIPAL FINDINGS: An established ultra-sensitive Plasmodium genus quantitative-PCR (qPCR) assay targeting the 18S rRNA gene underwent LOD evaluation with and without reverse transcription (RT) for P. knowlesi, P. cynomolgi and P. vivax using total nucleic acid preserved (DNA/RNA Shield) isolates and archived dried blood spots (DBS). LODs for selected P. knowlesi-specific assays, and reference P. vivax- and P. cynomolgi-specific assays were determined with reverse transcription (RT). Assay specificities were assessed using clinical malaria samples and malaria-negative controls. The use of reverse transcription improved Plasmodium species detection by up to 10,000-fold (Plasmodium genus), 2759-fold (P. knowlesi) and 1000-fold (P. vivax and P. cynomolgi). The Kamau et al. Plasmodium genus RT-qPCR assay was highly sensitive for P. knowlesi detection with a median LOD of ≤0.0002 parasites/μL compared to 0.002 parasites/μL for P. cynomolgi and P. vivax. The LODs with RT for P. knowlesi-specific PCRs were enhanced for the Imwong et al. 18S rRNA (0.0007 parasites/μL) and Divis et al. real-time 18S rRNA (0.0002 parasites/μL) assays, but not for the Lubis et al. hemi-nested SICAvar (1.1 parasites/μL) and Lee et al. nested 18S rRNA (11 parasites/μL). The LOD for P. vivax- and P. cynomolgi-specific assays with RT were moderately improved at 0.02 and 0.002 parasites/μL, respectively (1000-fold change). For DBS P. knowlesi samples the use of RT also markedly improved the Plasmodium genus qPCR LOD from 19.89 to 0.08 parasites/μL (249-fold change); no LOD improvement was demonstrated in DBS archived beyond 6 years. The Plasmodium genus and P. knowlesi-assays were 100% specific for Plasmodium species and P. knowlesi detection, respectively, from 190 clinical infections and 48 healthy controls. Reference P. vivax-specific primers demonstrated known cross-reactivity with P. cynomolgi.
CONCLUSIONS/SIGNIFICANCE: Our findings support the use of an 18S rRNA Plasmodium genus qPCR and species-specific nested PCR protocol with RT for highly-sensitive surveillance of zoonotic and human Plasmodium species infections.
METHODS: A total of 473 faecal samples were collected: 256 (54.1%) and 217 (45.9%) samples were obtained during the wet (October-November 2014) and the dry season (June 2015), respectively. All fresh faecal samples were subjected to molecular analysis for subtype and allele identification.
RESULTS: Of the 473 samples, 42.6% and 37.8% were positive for Blastocystis ST1, ST2, ST3 and ST4 during wet and dry seasons, respectively. Prevalence of Blastocystis ST1 was significantly higher during the wet season compared to the dry season (Z = 2.146, P
METHODS: In the present study, we included a larger sample size of P. knowlesi (83 samples) covering eight states of Malaysia to determine the genetic polymorphism, natural selection and haplotype groups of the gene fragment coding PkMSP-142. The region flanking PkMSP-142 was amplified by PCR and directly sequenced. Genetic diversity, haplotype diversity, population genetic differentiation and natural selection were determined in order to study the polymorphic characteristic of PkMSP-142.
RESULTS: A high level of genetic diversity (Hd = 0.970 ± 0.007; л = 0.01079 ± 0.00033) was observed among the 83 P. knowlesi samples, confirming the extensive genetic polymorphism exhibited among the P. knowlesi population found in Malaysia. A total of 18 distinct haplotypes with 17 amino acid changes were identified, whereby 15 were new haplotypes. High population differentiation values were observed within samples from Peninsular Malaysia and Malaysian Borneo. The 42 kDa fragments of P. knowlesi from Malaysian Borneo were found to be acting on balancing selection whilst purifying selection was suggested to act on isolates from Peninsular Malaysia. The separation of PkMSP-142 haplotypes into two main groups based on geographical separation has further supported the existence of two distinct P. knowlesi lineages.
CONCLUSIONS: A high level of genetic diversity was observed among PkMSP-142 in Malaysia, whereby most of the polymorphisms were found within the 33 kDa region. Taken together, these data will be useful in order to understand the nature of P. knowlesi population in Malaysia as well as the design and development of a MSP-142 based knowlesi malaria vaccine.