METHODS: The PCR technique was established based on published information and validated using blood sample of laboratory animals of which their whole gene sequences are available in GenBank. PCR was next performed to compile gene sequences of different species of wild rodents. The primers used were complementary to the conserved region of the cytb gene of vertebrate's mtDNA. A total of 100 blood samples, both from laboratory animals and wild rodents were collected and analyzed. The obtained unknown sequences were compared with those in the GenBank database using BLAST program to identify the vertebrate animal species.
RESULTS: Gene sequences of 11 species of wild animals caught in 9 localities of Peninsular Malaysia were compiled using the established PCR. The animals involved were Rattus (rattus) tanezumi, Rattus tiomanicus, Leopoldamys sabanus, Tupaia glis, Tupaia minor, Niviventor cremoriventor, Rhinosciurus laticaudatus, Callosciurus caniseps, Sundamys muelleri, Rattus rajah and Maxomys whiteheadi. The BLAST results confirmed the host with exact or nearly exact matches (>89% identity). Ten new gene sequences have been deposited in GenBank database since September 2010.
CONCLUSIONS: This study indicates that the PCR direct sequencing system using universal primer sets for vertebrate cytb gene is a promising technique for blood meal identification.
METHODS: A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the P. knowlesi small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing P. knowlesi DNA and genomic DNA of P. falciparum, P. knowlesi, P. malariae, P. ovale and P. vivax isolated from clinical samples. DNA samples of the simian malaria parasites Plasmodium cynomolgi and Plasmodium inui that can infect humans under experimental conditions were also examined together with human DNA samples.
RESULTS: Analytical sensitivity of the P. knowlesi-specific assay was 10 copies/μL and quantitation was linear over a range of 10-106 copies. The sensitivity of the assay is equivalent to nested PCR and P. knowlesi DNA was detected from all 40 clinical P. knowlesi specimens, including one from a patient with a parasitaemia of three parasites/μL of blood. No cross-reactivity was observed with 67 Plasmodium DNA samples (31 P. falciparum, 23 P. vivax, six P. ovale, three P. malariae, one P. malariae/P. ovale, one P. falciparum/P. malariae, one P. inui and one P. cynomolgi) and four samples of human DNA.
CONCLUSIONS: This test demonstrated excellent sensitivity and specificity, and adds P. knowlesi to the repertoire of Plasmodium targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA copy number provides a useful advantage over other molecular assays to investigate the correlation between levels of infection and the spectrum of disease.
METHODS: Water samples were collected from rivers, water tanks, wells and seawater on Tioman Island over the course of April to October 2015. Water samples were indirectly screened for Sarcocystis species by obtaining sediment from respective water sources. PCR amplification of the 18S rRNA gene region was conducted to identify positive samples. Microscopy was used in an attempt to reappraise PCR results, but no sporocysts were detected in any of the samples.
RESULTS: A total of 157 water samples were obtained and 19 were positive for various Sarcocystis species. Through BLASTn and phylogenetic analysis, these species were found to be S. singaporensis, S. nesbitti, Sarcocystis sp. YLL-2013 and one unidentified Sarcocystis species.
CONCLUSIONS: This is the first positive finding of S. nesbitti in water samples on Tioman Island, which was found in a water tank and in river water samples. This finding supports the hypothesis that water was a potential medium for the transmission of S. nesbitti during the outbreak. This will potentially identify areas in which preventive measures can be taken to prevent future outbreaks.