The sulphadoxine/pyrimethamine (SDX/PYR) combination had been chosen to treat uncomplicated falciparum malaria in Malaysia for more than 30 years. Non-silent mutations in dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes are responsible for the resistance to pyrimethamine and sulphadoxine, respectively. This study reports the mutational analysis of pfdhfr and pfdhps in single Plasmodium falciparum infection isolates from the interior division of Sabah, Malaysian Borneo.
Genomic DNA of Blastocystis isolates released into 0.1% Triton X-100 was suitable for amplification and yielded similar results as the genomic DNA extracted with standard kit. The specific B. hominis primers (BH1: GCT TAT CTG GTT GAT CCT GCC AGT and BH2: TGA TCC TTC CGC AGG TTC ACC TAC A) successfully produced the PCR product of about 1,770 bp with all the 7 Blastocystis isolates tested. The restriction fragment length polymorphism (RFLP) patterns yielded by 13 out of 25 restriction endonucleases showed that the 7 isolates could be grouped into 4 subgroups: subgroup-1 consisted of isolate C; subgroup-2 of isolates H4 and H7; subgroup-3 of isolates KP1, Y51 and M12; and subgroup-4 of isolate 27805. The differences between subgroups manifested as clear-cut RFLP patterns. A common band of 230 bp was revealed by Eco R1 in all the Blastocystis isolates tested. The band of about 180 bp was revealed by Alu I, differentiated symptomatic from asymptomatic isolates of this parasite, and might indicate the pathogenicity of this parasite.
Giardia duodenalis causes diarrhoea and malabsorption. The objectives of the study were to detect local isolates of G. doudenalis by polymerase chain reaction (PCR) and to determine their restriction fragment length polymorphisms (RFLP). G. doudenalis isolated from stools of patients from Hospital Orang Asli Gombak were cultured axenically using TYI-S-33 medium with 10% foetal calf serum. The commercially designed primer-pair 432/433 was used to amplify a 0.52 kb segment known to encode the homologous cysteine-rich trophozoite surface antigen (tsp11 and tsa417). Results showed that the primer-pair 432/433 could amplify the target region of the local isolates. RFLP study on the identical isolates showed that all the restriction enzymes tested ( HindIII, ClaI, PstI and Kpn) gave a banding pattern similar to that of the WB strain a reference pathogenic strain from human. The reference pathogenic strain were commercially obtained from the American Type Culture Collection (ATCC).
A nested polymerase chain reaction (PCR) assay that uses Plasmodium genus-specific primers for the initial PCR (nest 1) amplification and either genus- or species-specific primers for the nest 2 amplifications was tested on laboratory and field samples. With in vitro cultured Plasmodium falciparum-infected blood samples, it was capable of detecting six parasites/microl of blood using DNA prepared from 25-microl blood spots on filter paper. The assay was evaluated on fingerprick blood samples collected on filter paper from 129 individuals living in a malaria-endemic area in Malaysia. Malaria prevalence by genus-specific nested PCR was 35.6% (46 of 129) compared with 28.7% (37 of 129) by microscopy. The nested PCR detected seven more malaria samples than microscopy in the first round of microscopic examination, malaria in three microscopically negative samples, six double infections identified as single infections by microscopy and one triple infection identified as a double infection by microscopy. The nested PCR assay described is a sensitive technique for collecting accurate malaria epidemiologic data. When coupled with simple blood spot sampling, it is particularly useful for screening communities in remote regions of the world.
A modified nested polymerase chain reaction (PCR) method for detection of Plasmodium falciparum, P. vivax and P. malariae was combined with a simple blood collection and deoxyribonucleic acid (DNA) extraction method and evaluated in Malaysia. Finger-prick blood samples from 46 hospital patients and 120 individuals living in malaria endemic areas were spotted on filter papers and dried. The simple Chelex method was used to prepare DNA templates for the nested PCR assay. Higher malaria prevalence rates for both clinical (78.2%) and field samples (30.8%) were obtained with the nested PCR method than by microscopy (76.1% and 27.5%, respectively). Nested PCR was more sensitive than microscopy in detecting mixed P. falciparum and P. vivax infections, detected 5 more malaria samples than microscopy on the first round of microscopical examination, and detected malaria in 3 microscopically negative samples. Nested PCR failed to detect parasite DNA in 2 microscopically positive samples, an overall sensitivity of 97.4% compared to microscopy. The nested PCR method, when coupled with simple dried blood spot sampling, is a useful tool for collecting accurate malaria epidemiological data, particularly in remote regions of the world.
This study highlighted the development of a four target nitrocellulose-based nucleic acid lateral flow immunoassay biosensor in a dry-reagent strip format for interpretation of double-labelled double-stranded amplicons from thermostabilised triplex loop-mediated isothermal amplification assay. The DNA biosensor contained two test lines which captured biotin and texas red labelled amplicons; a LAMP internal amplification control line that captured digoxigenin labelled amplicon; and a chromatography control line that validated the functionality of the conjugated gold nanoparticles and membrane. The red lines on detection pad were generated when the gold nanoparticles conjugated antibody bound to the fluorescein labelled amplicons, and the capture agents bound to their specific hapten on the other 5' end of the double-stranded amplicon. The applicability of this DNA biosensor was demonstrated using amoebiasis-causing Entamoeba histolytica simultaneously with the non-pathogenic but morphologically identical Entamoeba dispar and Entamoeba moshkovskii. The biosensor detection limit was 10 E. histolytica trophozoites, and revealed 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. Heat stability test showed that the biosensor was stable for at least 181 days at ambient temperature. This ready-to-use and cold-chain-free biosensor facilitated the post-LAMP analysis based on visualisation of lines on strip instead of observation of amplicon patterns in agarose gel.
Strains of a dinoflagellate from the Salton Sea, previously identified as Protoceratium reticulatum and yessotoxin producing, have been reexamined morphologically and genetically and Pentaplacodinium saltonense n. gen. et sp. is erected to accommodate this species. Pentaplacodinium saltonense differs from Protoceratium reticulatum (Claparède et Lachmann 1859) Bütschli 1885 in the number of precingular plates (five vs. six), cingular displacement (two widths vs. one), and distinct cyst morphology. Incubation experiments (excystment and encystment) show that the resting cyst of Pentaplacodinium saltonense is morphologically most similar to the cyst-defined species Operculodinium israelianum (Rossignol, 1962) Wall (1967) and O. psilatum Wall (1967). Collections of comparative material from around the globe (including Protoceratium reticulatum and the genus Ceratocorys) and single cell PCR were used to clarify molecular phylogenies. Variable regions in the LSU (three new sequences), SSU (12 new sequences) and intergenic ITS 1-2 (14 new sequences) were obtained. These show that Pentaplacodinium saltonense and Protoceratium reticulatum form two distinct clades. Pentaplacodinium saltonense forms a monophyletic clade with several unidentified strains from Malaysia. LSU and SSU rDNA sequences of three species of Ceratocorys (C. armata, C. gourreti, C. horrida) from the Mediterranean and several other unidentified strains from Malaysia form a well-supported sister clade. The unique phylogenetic position of an unidentified strain from Hawaii is also documented and requires further examination. In addition, based on the V9 SSU topology (bootstrap values >80%), specimens from Elands Bay (South Africa), originally described as Gonyaulax grindleyi by Reinecke (1967), cluster with Protoceratium reticulatum. The known range of Pentaplacodinium saltonense is tropical to subtropical, and its cyst is recorded as a fossil in upper Cenozoic sediments. Protoceratium reticulatum and Pentaplacodinium saltonense seem to inhabit different niches: motile stages of these dinoflagellates have not been found in the same plankton sample.
Hepatozoon canis has been widely reported in dogs. Its prevalence in ticks, however, has not been well-established. Here we determine the occurrence of Hepatozoon DNA in the brown dog tick Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) sensu lato (s.l.) and domestic dogs from Peninsular Malaysia using a polymerase chain reaction (PCR) assay based on amplification of the 18S ribosomal RNA coding sequence. Our results revealed a relatively low prevalence of H. canis DNA in both R. sanguineus s.l. (0.7%) and dogs (3.33%). This study represents the first report of H. canis DNA in R. sanguineus s.l. in Malaysia, highlighting the risk of this infection in dogs.
The genus Gymnodinium includes many morphologically similar species, but molecular phylogenies show that it is polyphyletic. Eight strains of Gymnodinium impudicum, Gymnodinium dorsalisulcum and a novel Gymnodinium-like species from Chinese and Malaysian waters and the Mediterranean Sea were established. All of these strains were examined with light microscopy, scanning electron microscopy and transmission electron microscopy. SSU, LSU and internal transcribed spacers rDNA sequences were obtained. A new genus, Wangodinium, was erected to incorporate strains with a loop-shaped apical structure complex (ASC) comprising two rows of amphiesmal vesicles, here referred to as a new type of ASC. The chloroplasts of Wangodinium sinense are enveloped by two membranes. Pigment analysis shows that peridinin is the main accessory pigment in W. sinense. Wangodinium differs from other genera mainly in its unique ASC, and additionally differs from Gymnodinium in the absence of nuclear chambers, and from Lepidodinium in the absence of Chl b and nuclear chambers. New morphological information was provided for G. dorsalisulcum and G. impudicum, e.g., a short sulcal intrusion in G. dorsalisulcum; nuclear chambers in G. impudicum and G. dorsalisulcum; and a chloroplast enveloped by two membranes in G. impudicum. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference with independent SSU and LSU rDNA sequences. Our results support the classification of Wangodinium within the Gymnodiniales sensu stricto clade and it is close to Lepidodinium. Our results also support the close relationship among G. dorsalisulcum, G. impudicum, and Barrufeta. Further research is needed to assign these Gymnodinium species to Barrufeta or to erect new genera.
Entamoeba histolytica is the only Entamoeba species that causes amoebiasis in humans. Approximately 50 million people are infected, with 100, 000 deaths annually in endemic countries. Molecular diagnosis of Entamoeba histolytica is important to differentiate it from the morphologically identical Entamoeba dispar to avoid unnecessary medication. Conventional molecular diagnostic tests require trained personnel, cold-chain transportation and/or are storage-dependent, which make them user-unfriendly. The aim of this study was to develop a thermostabilized, one-step, nested, tetraplex PCR assay for the detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba species in cold-chain-free and ready-to-use form. The PCR test was designed based on the Entamoeba small subunit rRNA (SSU-rRNA) gene, which detects the presence of any Entamoeba species, and simultaneously can be used to differentiate Entamoeba histolytica from Entamoeba dispar. In addition, a pair of primers was designed to serve as an internal amplification control to help identify inhibitors in the samples. All PCR reagents together with the designed primers were thermostabilized by lyophilization and were stable at 24 °C for at least 6 months. The limit of detection of the tetraplex PCR was found to be 39 pg DNA or 1000 cells for Entamoeba histolytica and 78 pg DNA or 1000 cells for Entamoeba dispar, and the specificity was 100 %. In conclusion, this cold-chain-free, thermostabilized, one-step, nested, multiplex PCR assay was found to be efficacious in differentiating Entamoeba histolytica from other non-pathogenic Entamoeba species.
Plasmodium knowlesi is a newly described zoonosis that causes malaria in the human population that can be severe and fatal. The study of P. knowlesi parasites from human clinical isolates is relatively new and, in order to obtain maximum information from patient sample collections, we explored the possibility of generating P. knowlesi genome sequences from archived clinical isolates. Our patient sample collection consisted of frozen whole blood samples that contained excessive human DNA contamination and, in that form, were not suitable for parasite genome sequencing. We developed a method to reduce the amount of human DNA in the thawed blood samples in preparation for high throughput parasite genome sequencing using Illumina HiSeq and MiSeq sequencing platforms. Seven of fifteen samples processed had sufficiently pure P. knowlesi DNA for whole genome sequencing. The reads were mapped to the P. knowlesi H strain reference genome and an average mapping of 90% was obtained. Genes with low coverage were removed leaving 4623 genes for subsequent analyses. Previously we identified a DNA sequence dimorphism on a small fragment of the P. knowlesi normocyte binding protein xa gene on chromosome 14. We used the genome data to assemble full-length Pknbpxa sequences and discovered that the dimorphism extended along the gene. An in-house algorithm was developed to detect SNP sites co-associating with the dimorphism. More than half of the P. knowlesi genome was dimorphic, involving genes on all chromosomes and suggesting that two distinct types of P. knowlesi infect the human population in Sarawak, Malaysian Borneo. We use P. knowlesi clinical samples to demonstrate that Plasmodium DNA from archived patient samples can produce high quality genome data. We show that analyses, of even small numbers of difficult clinical malaria isolates, can generate comprehensive genomic information that will improve our understanding of malaria parasite diversity and pathobiology.
Despite frequent reports on the presence of Blastocystis hominis in human intestinal tract, its pathogenicity remains a matter of intense debate. These discrepancies may be due to the varying pathogenic potential or virulence of the isolates studied. The present study represents the first to investigate both phenotypic and genotypic characteristics of B. hominis obtained from symptomatic and asymptomatic individuals. Symptomatic isolates had a significantly greater size range and lower growth rate in Jones' medium than asymptomatic isolates. The parasite cells of symptomatic isolates exhibited rougher surface topography and greater binding affinity to Canavalia ensiformis (ConA) and Helix pomatia (HPA). The present study also identifies further phenotypic characteristics, which aided in differentiating the pathogenic forms from the non-pathogenic forms of B. hominis. Blastocystis subtype 3 was found to be correlated well with the disease.
Giardia duodenalis is an intestinal parasite that causes diarrhoea and malabsorption in children. The parasite also infects AIDS patients with a weak immune system. A study was carried out on six local isolates of Giardia duodenalis (110, 7304, 6304, M007, 2002 and 6307) from faeces of Orang Asli patients admitted to the Gombak Hospital. WB, a reference pathogenic strain from human and G. muris from a wild mouse, were commercially obtained from the American Type Culture Collection (ATCC). All the isolates were cultured axenically in TYI-S-33 medium. Two sets of primers were used for the techniques: primers LP1 and RP1 and primers LP2 and RP2. The sets of primers amplified giardine gene of 171 bp and 218 bp in sizes respectively. The study showed that the two sets of primers could detect G. duodenalis to the genus and species level specifically.
Dried Anopheles farauti mosquitos caught in Solomon Islands in 1990 were examined for malaria sporozoites by ELISA and nested polymerase chain reaction (PCR). Only heads and thoraces were used. Plasmodium genus-specific nested PCR amplifications were carried out on all samples. Of the 402 pools of mosquitos that were processed, 30 were positive for malaria. Nest 1 products of positive samples were subjected to further PCR amplifications with species-specific primers for P. falciparum and P. vivax. Twenty pools were positive for P. vivax by PCR while only 7 were positive by ELISA. For P. falciparum 2 pools were positive by both ELISA and PCR, and one of these was a pool which was positive for P. vivax by PCR and ELISA. Thus the sensitivity of PCR for P. vivax was 100% while the specificity was 96.7%. For P. falciparum the sensitivity and specificity were 100%. The PCR technique is highly sensitive and can be used on dried mosquitos which makes it a valuable tool for determining sporozoite rates of mosquitos, even in remote areas.
One of the most important routes of transmission for Toxoplasma gondii infection is the ingestion of foods contaminated with cat feces containing sporulated oocysts. The diagnosis of T. gondii infection by fecal microscopy is complicated, as other similar coccidian oocysts are often present in the same fecal specimen. This study aimed to identify T. gondii oocysts in cat feces using a novel PCR technique. Feline fecal specimens (n = 254) were screened for coccidian oocysts by light microscopy using the Sheather's flotation method. PCR analysis performed on the same specimens targeted a 529 bp repeat element and internal transcribed spacer-1 (ITS-1) regions were used to confirm the presence of Toxoplasma oocysts. By light microscopy, 49/254 (19.3%) of specimens contained coccidian oocysts. PCR analysis demonstrated 2/254 (0.8%) and 17/254 (6.7%) positive results using Tox and ITS-1 primers, respectively. However, coccidian oocysts were not identified on microscopic examination of specimens that were PCR-positive by Tox primers. Coccidian oocysts were identified on microscopic examination of 6/17 (35.3%) of the PCR-positive fecal specimens using ITS-1 primers. The BLAST results of 16 ITS-1 sequences were identified as T. gondii (n = 12; 4.7%) and Hammondia hammondi (n = 4; 1.6%). There was slight agreement between the 529 bp and ITS-1 PCR results (κ = 0.148). This is the first report of the detection of Toxoplasma oocysts using PCR analysis on feline fecal specimens from Southern Thailand. The ITS-1 region has potential as an alternative marker to identify T. gondii oocysts in feline fecal specimens.
A cross-sectional study was carried out to identify species and determine the prevalence of Cryptosporidium sp. shedding in pre-weaned and post-weaned dairy calves and to identify management factors that may be contributing to disease. A total of 240 calf faecal samples were collected from 16 farms in two districts in Johor, Malaysia, and screened by PCR. The overall Cryptosporidium prevalence was 27.1%. The prevalence of Cryptosporidium species in pre-weaned calves was 32.4% for C. parvum, 26.5% for C. bovis, followed by C. andersoni (20.6%), C. ryanae (11.8%) and mixed sp. (8.8%). The prevalence of Cryptosporidium species in post-weaned calves was 35% for C. bovis followed by C. andersoni and C. ryanae (30% each) and mixed sp. (5%). Subtyping analysis of 8 of the 11 C. parvum isolates at the gp60 locus identified five isolates as IIdA15G1, one as IIa18A3R1 and two isolates as IIa17G2R1. Management factors that increased the risk of Cryptosporidium infection included having other cattle farms close by, feeding calves with saleable milk, keeping pre-weaned calves in pens with slatted floors and keeping post-weaned calves in pens with a sand floor.
Four human deaths caused by Plasmodium knowlesi, a simian malaria species, are stimulating a surge of public health interest and clinical vigilance in vulnerable areas of Southeast Asia. We, and other colleagues, emphasize that these cases, identified in Malaysia, are a clear warning that health facilities and clinicians must rethink the diagnosis and treatment of malaria cases presumed to be caused by a less virulent human malaria species, Plasmodium malariae.
The intraspecific variation of four laboratory-reared isolates of Taenia taeniaformis the SRN and KRN isolates from Norway rats, Rattus norvegicus, captured in Japan and Malaysia, respectively; the BMM isolated from a house mouse, Mus musculus, captured in Belgium; and the ACR isolate from a gray red-backed vole, Clethrionomys rufocanus bedfordiae, captured in Japan was examined by various criteria. Eggs of each of the four isolates were orally inoculated into several species of intermediate host. They were most infective to the rodent species from which the original metacestode of each isolate had been isolated in the field, and only the ACR isolate was infective to the gray red-backed vole. Although little difference was found between the SRN, KRN, and BMM isolates by the other criteria, including the morphology of rostellar hooks, the protein composition of the metacestode, and restriction endonuclease analysis of DNA, the ACR isolate was clearly different from the others. It was considered that the ACR isolate was independent as a strain distinct from the other three isolates.
Entamoeba histolytica is a causative agent of amoebic liver abscess (ALA) and is endemic in many underdeveloped countries. We investigated antigenic E. histolytica proteins in liver abscess aspirates using proteomics approach. Pus samples were first tested by real-time PCR to confirm the presence of E. histolytica DNA and the corresponding serum samples tested for E. histolytica-specific IgG by a commercial ELISA. Proteins were extracted from three and one pool(s) of pus samples from ALA and PLA (pyogenic liver abscess) patients respectively, followed by analysis using isoelectric focussing, SDS-PAGE and Western blot. Unpurified pooled serum samples from infected hamsters and pooled human amoebic-specific IgG were used as primary antibodies. The antigenic protein band was excised from the gel, digested and analysed by MALDI-TOF/TOF and LC-MS/MS. The results using both primary antibodies showed an antigenic protein band of ∼14kDa. Based on the mass spectrum analysis, putative tyrosine kinase is the most probable identification of the antigenic band.
During 2009 to 2010, a total of 408 blood samples collected from malaria patients in Ranong (149) and Yala (259) Provinces, Thailand were investigated for Plasmodium spp using microscopic examination. There are no statistical differences in the prevalence of P. falciparum and P. vivax in samples collected from Ranong and Yala (46% vs 52%, and 54% vs 45%, respectively). Single nucleotide polymorphism of codon 86 in pfmdr1 (encoding P. falciparum multidrug resistance protein 1) was investigated among 75 samples of P. falciparum and 2 samples of P. knowlesi. A pfmdr1 N86Y mutation was detected in 1 out of 29 samples and 45 out of 46 samples obtained from Ranong and Yala Provinces, respectively. It is interesting that pfmdr1 was detected in two P. knowlesi DNA samples obtained previously from Ranong Province which was 99% homologous to pfmdr1 obtained from falciparum parasites in the same area but the mutation was not observed. The difference in multidrug resistance protein in Plasmodium obtained from those two border areas of Thailand will be of use in monitoring drug resistance in these border regions of the country.