Mosquito vectors (Diptera: Culicidae) are responsible for transmission of serious diseases worldwide. Mosquito control is being enhanced in many areas, but there are significant challenges, including increasing resistance to insecticides and lack of alternative, cost-effective, and eco-friendly products. To deal with these crucial issues, recent emphasis has been placed on plant materials with mosquitocidal properties. Furthermore, cancers figure among the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths in 2012. It is expected that annual cancer cases will rise from 14 million in 2012 to 22 million within the next two decades. Nanotechnology is a promising field of research and is expected to give major innovation impulses in a variety of industrial sectors. In this study, we synthesized titanium dioxide (TiO2) nanoparticles using the hydrothermal method. Nanoparticles were subjected to different analysis including UV-Vis spectrophotometry, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), zeta potential, and energy-dispersive spectrometric (EDX). The synthesized TiO2 nanoparticles exhibited dose-dependent cytotoxicity against human breast cancer cells (MCF-7) and normal breast epithelial cells (HBL-100). After 24-h incubation, the inhibitory concentrations (IC50) were found to be 60 and 80 μg/mL on MCF-7 and normal HBL-100 cells, respectively. Induction of apoptosis was evidenced by Acridine Orange (AO)/ethidium bromide (EtBr) and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining. In larvicidal and pupicidal experiments conducted against the primary dengue mosquito Aedes aegypti, LC50 values of nanoparticles were 4.02 ppm (larva I), 4.962 ppm (larva II), 5.671 ppm (larva III), 6.485 ppm (larva IV), and 7.527 ppm (pupa). Overall, our results suggested that TiO2 nanoparticles may be considered as a safe tool to build newer and safer mosquitocides and chemotherapeutic agents with little systemic toxicity.
Malaria remains a major public health problem due to the emergence and spread of Plasmodium falciparum strains resistant to chloroquine. There is an urgent need to investigate new and effective sources of antimalarial drugs. This research proposed a novel method of fern-mediated synthesis of silver nanoparticles (AgNP) using a cheap plant extract of Pteridium aquilinum, acting as a reducing and capping agent. AgNP were characterized by UV-vis spectrophotometry, Fourier transform infrared (FTIR) spectroscopy, energy-dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD). Phytochemical analysis of P. aquilinum leaf extract revealed the presence of phenols, alkaloids, tannins, flavonoids, proteins, carbohydrates, saponins, glycosides, steroids, and triterpenoids. LC/MS analysis identified at least 19 compounds, namely pterosin, hydroquinone, hydroxy-acetophenone, hydroxy-cinnamic acid, 5, 7-dihydroxy-4-methyl coumarin, trans-cinnamic acid, apiole, quercetin 3-glucoside, hydroxy-L-proline, hypaphorine, khellol glucoside, umbelliferose, violaxanthin, ergotamine tartrate, palmatine chloride, deacylgymnemic acid, methyl laurate, and palmitoyl acetate. In DPPH scavenging assays, the IC50 value of the P. aquilinum leaf extract was 10.04 μg/ml, while IC50 of BHT and rutin were 7.93 and 6.35 μg/ml. In mosquitocidal assays, LC50 of P. aquilinum leaf extract against Anopheles stephensi larvae and pupae were 220.44 ppm (larva I), 254.12 ppm (II), 302.32 ppm (III), 395.12 ppm (IV), and 502.20 ppm (pupa). LC50 of P. aquilinum-synthesized AgNP were 7.48 ppm (I), 10.68 ppm (II), 13.77 ppm (III), 18.45 ppm (IV), and 31.51 ppm (pupa). In the field, the application of P. aquilinum extract and AgNP (10 × LC50) led to 100 % larval reduction after 72 h. Both the P. aquilinum extract and AgNP reduced longevity and fecundity of An. stephensi adults. Smoke toxicity experiments conducted against An. stephensi adults showed that P. aquilinum leaf-, stem-, and root-based coils evoked mortality rates comparable to the permethrin-based positive control (57, 50, 41, and 49 %, respectively). Furthermore, the antiplasmodial activity of P. aquilinum leaf extract and green-synthesized AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of P. falciparum. IC50 of P. aquilinum were 62.04 μg/ml (CQ-s) and 71.16 μg/ml (CQ-r); P. aquilinum-synthesized AgNP achieved IC50 of 78.12 μg/ml (CQ-s) and 88.34 μg/ml (CQ-r). Overall, our results highlighted that fern-synthesized AgNP could be candidated as a new tool against chloroquine-resistant P. falciparum and different developmental instars of its primary vector An. stephensi. Further research on nanosynthesis routed by the LC/MS-identified constituents is ongoing.
Blastocystis sp., an intestinal organism is known to cause diarrhea with metronidazole regarded as the first line of treatment despite reports of its resistance. The conflicting reports of variation in drug treatment have been ascribed to subtype differences. The present study evaluated in vitro responses due to metronidazole on ST3 isolated from three symptomatic and asymptomatic patients, respectively. Symptomatic isolates were obtained from clinical patients who showed symptoms such as diarrhea and abdominal bloating. Asymptomatic isolates from a stool survey carried out in a rural area. These patients had no other pathogens other than Blastocystis. Ultrastructural studies using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed drug-treated ST3 from symptomatic patients were irregular and amoebic with surface showing high-convoluted folding when treated with metronidazole. These organisms had higher number of mitochondrion-like organelle (MLO) with prominent cristae. However, the drug-treated ST3 from asymptomatic persons remained spherical in shape. Asymptomatic ST3 showed increase in the size of its central body with the MLO located at the periphery.
Malaria, the most widespread mosquito-borne disease, affects 350-500 million people each year. Eco-friendly control tools against malaria vectors are urgently needed. This research proposed a novel method of plant-mediated synthesis of silver nanoparticles (AgNP) using a cheap seaweed extract of Ulva lactuca, acting as a reducing and capping agent. AgNP were characterized by UV-vis spectrophotometry, Fourier transform infrared (FTIR) spectroscopy, energy-dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), and X-ray diffraction (XRD). The U. lactuca extract and the green-synthesized AgNP were tested against larvae and pupae of the malaria vector Anopheles stephensi. In mosquitocidal assays, LC50 values of U. lactuca extract against A. stephensi larvae and pupae were 18.365 ppm (I instar), 23.948 ppm (II), 29.701 ppm (III), 37.517 ppm (IV), and 43.012 ppm (pupae). LC50 values of AgNP against A. stephensi were 2.111 ppm (I), 3.090 ppm (II), 4.629 ppm (III), 5.261 ppm (IV), and 6.860 ppm (pupae). Smoke toxicity experiments conducted against mosquito adults showed that U. lactuca coils evoked mortality rates comparable to the permethrin-based positive control (66, 51, and 41%, respectively). Furthermore, the antiplasmodial activity of U. lactuca extract and U. lactuca-synthesized AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. Fifty percent inhibitory concentration (IC50) values of U. lactuca were 57.26 μg/ml (CQ-s) and 66.36 μg/ml (CQ-r); U. lactuca-synthesized AgNP IC50 values were 76.33 μg/ml (CQ-s) and 79.13 μg/ml (CQ-r). Overall, our results highlighted out that U. lactuca-synthesized AgNP may be employed to develop newer and safer agents for malaria control.
Sarcocystis nesbitti, using snakes as the definitive host, is a causative agent of acute human muscular sarcocystosis in Malaysia. Therefore, it is important to explore the distribution and prevalence of S. nesbitti in snakes. Nevertheless, epizootiological information of S. nesbitti in snakes remains insufficient because few surveys have assessed Sarcocystis infection in snakes in endemic countries. In Japan, snakes are popular exotic pet animals that are imported from overseas, but the degree of Sarcocystis infection in them remains unclear. The possibility exists that muscular sarcocystosis by S. nesbitti occurs in contact with captive snakes in non-endemic countries. For a total of 125 snake faecal samples from 67 snake species collected at animal hospitals, pet shops and a zoo, this study investigated the presence of Sarcocystis using polymerase chain reaction (PCR) for the 18S ribosomal RNA gene (18S rDNA). Four (3.2%) faecal samples were positive by PCR. Phylogenetic analysis of the 18S rDNA sequences obtained from four amplification products revealed one isolate from a beauty snake (Elaphe taeniura), Sarcocystis zuoi, which uses rat snakes as the definitive host. The isolate from a Macklot's python (Liasis mackloti) was closely related with unidentified Sarcocystis sp. from reticulated pythons in Malaysia. The remaining two isolates from tree boas (Corallus spp.) were closely related with Sarcocystis lacertae, Sarcocystis gallotiae and unidentified Sarcocystis sp. from smooth snakes, Tenerife lizards and European shrews, respectively. This report is the first of a study examining the distribution of Sarcocystis species in captive snakes in Japan.
Blastocystis is one of the most common gut parasites found in the intestinal tract of humans and animals. We have previously reported the irregular shedding of Blastocystis cysts in stools from infected patients. In the present study, we assess the factors influencing shedding patterns from a Blastocystis ST3-infected IBS patient. The stools samples were voluntarily submitted for examination for a period of 30 days from Blastocystis ST3-infected IBS patient. A questionnaire on the factors that could influence the shedding pattern of the cysts was designed to assess the following information: (a) the frequency of frequenting the toilet in a day, (b) the timing of frequenting the toilet, (c) the stool forms, (d) the type of mood the patient was in when frequenting the toilet and (e) food intake. A total of 79 stool samples were collected for 30 days. The highest number of cysts recorded when the patient visited the toilet three times a day was 22.2 × 10(6) cysts/g. Frequenting the toilet between 6 a.m. to 11.59 a.m. showed the highest number of cysts, i.e. 21.7 × 10(6) cysts/g. Semi-solid forms showed the highest cyst count, i.e. 2.00 × 10(6) cysts/g. Irregular shedding of cysts was seen in 10 out of 30 days where the widest range recorded on day 17 was between 0 to 1.2 × 10(6) cysts/g. The average daily cyst count on days of emotional fluctuations was from 0 to 5.13 × 10(6) cysts/g. In conclusion, the study confirms that there are factors influencing shedding patterns of Blastocystis, and these have important implications when it comes to diagnosis and transmission of the parasite.
Dientamoeba fragilis, a trichomonad parasite is usually found in the gastrointestinal tract of human, and it is known to be the cause for gastrointestinal disease. The parasite is globally distributed and mostly found in rural and urban areas. The parasite is found in humans and nonhuman primates such as the macaques, baboons, and gorillas. Often, the parasite is confused with another largely found organism in stools called Blastocystis sp. especially when seen directly under light microscopy on culture samples containing both parasites. Both sometimes are seen with two nuclei with sizes tending to be similar which complicates identification. Stools were collected fresh from nine previously diagnosed persons infected with D. fragilis who also were found to be positive for Blastocystis sp. Samples were then cultured in Loeffler's medium and were stained with Giemsa, iron hematoxylin, and modified Fields' (MF) stain, respectively. D. fragilis was differentiated from Blastocystis sp. when stained with MF stain by the presence of a thinner outer membrane with clearly demarcated nuclei in the center of the cell whilst Blastocystis sp. had a darker and thicker stained outer membrane with the presence of two nuclei. The staining contrast was more evident with modified Fields' stain when compared with the other two. The simplicity in preparing the stain as well as the speed of the staining procedure make MF stain an ideal alternate. The modified Fields' stain is faster and easier to prepare when compared to the other two stains. MF stain provides a better contrast differentiating the two organisms and therefore provides a more reliable diagnostic method to precisely identify one from the other especially when cultures show mixed infections.
Caballeria liewi Lim, 1995, uses adhesive secretions from the head organs and posterior secretory systems to assist in locomotion and attachment. Ultrastructural investigations show that the head organs of C. liewi consist of three pairs of antero-lateral pit-like openings bearing microvilli and ducts leading from two types of uninucleated gland cells (located lateral to the pharynx), one type producing rod-like (S1) bodies with an electron-dense matrix containing less electron-dense vesicles and the second type producing oval (S2) bodies with a homogeneous electron-dense matrix. Interlinking band-like structures are observed between S1 bodies and between S2 bodies. S1 body is synthesised in the granular endoplasmic reticulum, transported to a Golgi complex to be packaged into vesicles and routed into ducts for exudation. The synthesis of the S2 body is unresolved. Haptoral secretions manifested externally as net-like structures are derived from dual electron-dense (DED) secretory body produced in the peduncular gland cells. The DED body consists of a less electron-dense oval core in a homogeneous electron-dense matrix. On exocytosis into the pyriform haptoral reservoir, DED bodies are transformed into a secretion with two types of inclusions (less electron-dense oval and electron-dense spherical inclusions) in an electron-dense matrix. The secretions are further transformed (as small, oval, electron-dense bodies) when transported to the superficial anchor grooves, and on exudation into the gill tissues, the secretions become an electron-dense matrix. Secretory bodies associated with uniciliated structures, anchor sleeves and marginal hooks are also observed.
The WHO adult susceptibility test is in use for insecticide resistance monitoring. Presently, materials are being imported from the Universiti Sains Malaysia, Malaysia and sometimes it is cost prohibitive. As an alternative, we present here a method of bottle bioassay using indigenous material. Different aspects related to the assay were studied and validated in the field. Bottle assay was standardized in the laboratory by using locally sourced material and laboratory-maintained insecticide-susceptible Anopheles stephensi and Aedes aegypti strains against technical grade deltamethrin and cyfluthrin insecticides dissolved in ethanol in a range of different concentrations. The frequency of use of the deltamethrin-coated bottles and shelf-life were determined. Discriminating dose for deltamethrin and cyfluthrin was 10 μg against An. stephensi and 2 μg against Ae. aegypti females. Insecticide-coated bottles stored at 25 to 35 °C can be used for three exposures within 7 days of coating. The study carried out in the laboratory was validated on wild caught An. culicifacies in the states of Odisha and Chhattisgarh against deltamethrin-coated bottles in comparison to WHO adult susceptibility test. Results of the study indicated that deltamethrin-coated bottles were effective up to three exposures within 7 days of coating for field population and 100% mortality was recorded within 35 min as observed in laboratory studies for field collected susceptible population. Also in the WHO adult susceptibility test, 100% knock-down within 35 min and 100% mortality after 24 h holding period were observed in susceptible population, while in it was 50% knock-down in 1 h and 64% mortality after 24 h holding period for resistant population (50% mortality in bottle assay in 60 min). The bottle assay can be used as an alternative to the WHO adult susceptibility test both in the laboratory and field for monitoring insecticide resistance in mosquito vectors using locally sourced material.
In February 2013, forty-seven Notched threadfin bream, the Nemipterus peronii, were sampled from the eastern coastal waters of the South China Sea. The concentration of various elements, namely cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), strontium (Sr), manganese (Mn), selenium (Se), Lead (Pb), nickel (Ni), aluminum (Al), arsenic (As), iron (Fe), and Zinc (Zn) were analyzed in the liver, muscle, and kidney organs of the host, as well as in their parasites Hysterothalycium reliquens (nematode) and the Paraphilometroides nemipteri (nematode), using inductively coupled plasma mass spectrometry (ICP-MS). The former group of parasites showed highest accumulation capacity for Cr, Cu, Fe, Mn, Se, Ni, and Zn while the latter group had high accumulation potential of As, Hg, Cd, Al, Pb, and Sr. The divergence in heavy-metal accumulation profiles of both nematodes is linked with the specificity of microhabitats, cuticle morphology, and interspecific competition. The outcome of this study indicates that both parasite models can be used for biomonitoring of metal pollution in marine ecosystems.
Seaweeds are one of the most widely studied natural resources for their biological activities. Novel seaweed compounds with unique chemical structures have been reported for their pharmacological properties. The urge to search for novel insecticidal compound with a new mode of action for development of botanical insecticides supports the relevant scientific research on discovering the bioactive compounds in seaweeds. The mosquitocidal potential of seaweed extracts and their isolated compounds are documented in this review paper, along with the discussion on bioactivities of the major components of seaweeds such as polysaccharides, phenolics, proteins, terpenes, lipids, and halogenated compounds. The effects of seaweed extracts and compounds toward different life stages of mosquito (egg, larva, pupa, and adult), its growth, development, and reproduction are elaborated. The structure-activity relationships of mosquitocidal compounds are discussed to extrapolate the possible chemical characteristics of seaweed compounds responsible for insecticidal properties. Furthermore, the possible target sites and mode of actions of the mosquitocidal seaweed compounds are included in this paper. The potential synergistic effects between seaweeds and commercial insecticides as well as the toxic effects of seaweed extracts and compounds toward other insects and non-target organisms in the same habitat are also described. On top of that, various factors that influence the mosquitocidal potential of seaweeds, such as abiotic and biotic variables, sample preparation, test procedures, and considerations for a precise experimental design are discussed. The potential of active seaweed extracts and compounds in the development of effective bioinsecticide are also discussed.
Cosmopolitan scuttle fly, Megaselia scalaris (Loew) (Diptera: Phoridae) is one of the commonest forensic species recorded colonizing human corpse indoors and in concealed environment. The occurrence of this species in such environments provides a higher evidential value to assist estimation of postmortem interval (PMI) compared to other forensically important dipterans. However, developmental and size data of M. scalaris are still lacking and they are derived from a limited range of thermal values. The objective of this study is to develop the growth model of M. scalaris by emphasizing the size range of larvae and puparia at different constant temperatures. This species was reared in six replicates at eight varying constant temperatures ranging from 23 to 36 °C and cow's liver was provided as food source. Larvae and puparia were sampled at set time intervals and measured by their length and weight. Because interpretation of forensic entomological evidence is subject to application of different techniques, development of M. scalaris is expressed herein by using developmental table, length/morphological stage diagrams and linear/nonlinear estimation methods. From the findings, it is very important to highlight that sexual dimorphism of M. scalaris during post feeding larva and pupa stage could be observed based on size and developmental periods. Mean length and weight ratios of male to female puparia are approximately 0.8 and 0.3-0.5, respectively, indicating sexual dimorphism of this species. Developmental period in female are 4.0-11.4 h (post feeding larval stage), 3.7-24.0 h (pupal stage), and 3.0-20.1 h (total developmental period) longer in male. Due to this dimorphism, PMI estimation using M. scalaris post feeding larva or puparium specimens must be carried out carefully by to avoid inaccuracy and misinterpretation.
The authors studied the myxosporean infection of wild gobiid fishes (Perciformes: Gobioidei) in the Merang Estuary of Terengganu, Malaysia, and described Myxobolus ophiocarae sp. n. in Ophiocara porocephala. Several myxosporean plasmodia were found intralamellarly within the gill filaments. The spores differed from those of other Myxobolus species previously recorded on gobiid fishes. They were round in valvular view and lens-shaped in sutural view, and had two equal-sized, pyriform polar capsules with polar filaments having six to seven turns. The spores measured 10.34 × 8.79 × 4.53 μm. The 18S rDNA sequence of M. ophiocarae sp. n., based on a contiguous sequence of 1,789 base pairs, differed from any other Myxobolus spp. in GenBank. Phylogenetic analysis of the 18S rDNA gene revealed that this species showed the closest similarity to Myxobolus nagaraensis, Myxobolus lentisuturalis, and Myxobolus cultus.
Blastocystis sp. is a commonly found intestinal microorganism and was reported to cause many nonspecific gastrointestinal symptoms. Various subtypes have been previously reported, and the pathogenicity of different subtypes of Blastocystis is unclear and remains as a controversial issue. A recent study has shown that the Blastocystis antigen isolated from an unknown subtype could facilitate the proliferation of colon cancer cells. Current study was conducted to compare the effect of solubilized antigen isolated from five different subtypes of Blastocystis on colon cancer cells, HCT116. A statistically significant proliferation of these cells was observed when exposed to 1.0 μg/ml solubilized antigen isolated from subtype 3 Blastocystis (37.22%, p < 0.05). Real-time polymerase chain reaction demonstrated the upregulation of Th2 cytokines especially transforming growth factor beta in subtype 3-treated cancer cells (p < 0.01, 3.71-fold difference). Of interest, subtype 3 Blastocystis antigen also caused a significantly higher upregulation of cathepsin B (subtypes 1 and 2, p < 0.01; subtypes 4 and 5, p < 0.001; 6.71-fold difference) which lead to the postulation that it may enhance the exacerbation of existing colon cancer cells by weakening the cellular immune response. The dysregulation of IFN-γ and p53 expression also suggest Blastocystis as a proponent of carcinogenesis. Therefore, it is very likely for subtype 3 Blastocystis to have higher pathogenic potential as it caused an increased propagation of cancer cells and substantial amount of inflammatory reaction compared to other subtypes.
The objective of this study was to assess the physico-chemical parameters and waterborne parasites in selected recreational lakes from Malaysia. Samples were collected from seven stations of Recreational Lake A (RL-A) and six stations of Recreational Lake B (RL-B). The samples were processed to detect the presence of Giardia spp. and Cryptosporidium spp. using immunomagnetic separation kit, helminth eggs or ova by bright field microscopy and Acanthamoeba spp. by cultivation in non-nutrient agar. Chemical parameters such as ammonia, chlorine, fluoride, nitrate and nitrite and physical parameters such as dissolved oxygen, electrical conductivity, pH, salinity, temperature and total dissolved solid were also measured. Both lakes were freshwater with salinity ranging from 0.05 to 0.09 ppt. Most stations of these lakes were contaminated with Cryptosporidium spp., Giardia spp., Ascaris spp. and hookworm. Schistosoma spp. was found in RL-B only, while Acanthamoeba spp. was found in all stations. Of all sampling sites, station 5 of RL-B is the most contaminated. Linear regression and correlation analysis revealed that Giardia spp. and Schistosoma spp. showed a significant negative correlation with turbidity (p
The present study investigated whether people working closely with animals were at higher risk of getting infected with Blastocystis hominis. The prevalence of the parasite was determined in two population groups, i.e., animal handlers and normal healthy individuals who did not work with animals. In all, 105 stool samples were collected from animal handlers from 2 local research institutions, a local zoo, and a local abattoir and 163 stool samples were collected from normal healthy individuals residing in high-rise flats in the city. The in vitro culture method used in the study detected that 41% of 105 animal handlers and 17% of 163 flat-dwellers in the city were positive for Blastocystis. This statistically significant finding (P = 0.0000313) shows that people who work closely with animals do stand at risk of acquiring Blastocystis infection.
Acanthamoeba sp. is a free-living amoeba known to cause chronic central nervous system infection or eye infection in humans. Many cases remain undetected for want of a good detection system. We report for the first time a rapid staining method to facilitate the identification of Acanthamoeba sp. using the modified Field's staining technique. A. castellanii, which was used in the present experiment, is maintained in our laboratory in mycological peptone medium (Gibco). The cultures were pooled together and smears were made on glass slides for staining purposes. Different types of stains such as Field's stain, modified Field's stain, Wright's stain, Giemsa stain, Ziehl-Neelsen stain, and trichrome stain were used to determine the best stain for the identification of this amoeba. The concentration of various stains and the duration of staining were varied to provide the best color and contrast for each stain. Acanthamoeba was also obtained from the brain of experimentally infected mice and was stained with various stains as mentioned above to determine the best stain for use in identifying the presence of this parasite in experimentally infected animals. The modified Field's stain gives a very good color contrast as compared with other stains. Furthermore, it takes only 20 s to be carried out using the least number of reagents, making it suitable for both laboratory and field use.
The shedding pattern of the protozoan parasite, Blastocystis hominis, is investigated in man and in experimental animal infections. The shedding pattern of the vacuolar and cystic forms of Blastocystis hominis in infected individuals have been shown in the present study to be irregular. The study shows that there is marked fluctuation in the shedding of the parasite from day to day, varying from as high as 17 to 0 per x40 microscopic field. The cystic stages when estimated in 8 Blastocystis-infected individuals ranged from as high as 7.4x10(5) cysts per gram of stool to 0. The shedding of cystic and vacuolar forms observed over a period of 20 days in experimentally-infected Wistar rats were not only shown to be irregular but the amount varied from host to host. The study has important diagnostic implications in that the stool samples must be collected more than once from patients showing clinical signs and symptoms to eliminate the cause of it to Blastocystis. The study also shows that there are asymptomatic individuals who pass a large amount of cysts as such individuals should be treated to prevent transmission to others.
A total of 20 isolates of Blastocystis were characterized using a single set of polymerase chain reaction (PCR) primers. The amplification product revealed five types of pattern. All four isolates from Singapore yielded PCR products quite different from those of the local isolates. However, most of the local isolates showed a major product at either 280 or 500 bp, or both. We also suspected that the amplification product detected at 280 bp might be an indicator of the pathogenicity of this parasite. One isolate (M12) obtained from a monkey showed patterns similar to those of human isolates (10203 and KP1) and probably belongs to the same strain. The results indicate that the intraspecific or interstrain variations in these 20 Blastocystis isolates belong to 5 different patterns. The differences among isolates of the same strain revealed by the presence or absence of certain amplification products showed further intrastrain variations in this parasite.
In this study, the genome of the Plasmodium falciparum Gombak A strain was examined for the presence of a gene encoding falcipain-2, a cysteine protease, using homology-based polymerase chain reaction cloning. The nucleotide sequence obtained from the gene cloned (designated pFG1) is approximately 99% homologous to other falcipain-2 genes from different strains. Comparatively, it is 69% homologous to falcipain-3 genes. Direct cloning of the falcipain-2 gene and its resemblance to the reported corresponding mRNA transcript suggests the absence of introns in this gene. Sequence alignment and comparison revealed four amino acid differences at positions 15, 51, 59 and 414 in the falcipain-2 from P. falciparum Gombak A as compared to other falcipain-2 proteins from different strains.