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.
Blastocystis sp. is a gastrointestinal (GI) protozoan parasite reported to cause non-specific GI symptoms including diarrhea, flatulence, abdominal pain, and nausea. Complete eradication of Blastocystis sp. is rather challenging even with the drug of choice, i.e., metronidazole. Here, we report on two Blastocystis sp.-infected individuals, who presented increased parasite load and exacerbated symptoms upon treatment with the usual recommended dosage and regime of metronidazole. The two studies uniquely demonstrate for the first time a cyst count as high as fivefold more than the original cyst count before treatment and show an exacerbation of GI symptoms despite treatment. The study provides additional support in recognizing metronidazole resistance in Blastocystis sp. and its consequences towards the pathogenicity of the parasite.
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.
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.
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.
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.
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
Vaccine development against the blood-stage malaria parasite is aimed at reducing the pathology of the disease. We constructed a recombinant Mycobacterium bovis bacille Calmette Guerin (rBCG) expressing the 19 kDa C-terminus of Plasmodium falciparum merozoite surface protein-1 (MSP-1(19)) to evaluate its protective ability against merozoite invasion of red blood cells in vitro. A mutated version of MSP-1(19), previously shown to induce the production of inhibitory but not blocking antibodies, was cloned into a suitable shuttle plasmid and transformed into BCG Japan (designated rBCG016). A native version of the molecule was also cloned into BCG (rBCG026). Recombinant BCG expressing the mutated version of MSP-1(19) (rBCG016) elicited enhanced specific immune response against the epitope in BALB/c mice as compared to rBCG expressing the native version of the epitope (rBCG026). Sera from rBCG016-immunized mice contained significant levels of specific IgG, especially of the IgG2a subclass, against MSP-1(19) as determined by enzyme-linked immunosorbent assay. The sera was reactive with fixed P. falciparum merozoites as demonstrated by indirect immunofluorescence assay (IFA) and inhibited merozoite invasion of erythrocytes in vitro. Furthermore, lymphocytes from rBCG016-immunized mice demonstrated higher proliferative response against the MSP-1(19) antigen as compared to those of rBCG026- and BCG-immunized animals. rBCG expressing the mutated version of MSP-1(19) of P. falciparum induced enhanced humoral and cellular responses against the parasites paving the way for the rational use of rBCG as a blood-stage malaria vaccine candidate.
Plasmodium knowlesi is a simian malaria parasite that causes significant zoonotic infections in Southeast Asia, particularly in Malaysia. The Plasmodium thrombospondin-related apical merozoite protein (TRAMP) plays an essential role in the invasion of the parasite into its host erythrocyte. The present study investigated the genetic polymorphism and natural selection of the full length PkTRAMP from P. knowlesi clinical isolates from Malaysia. Blood samples (n = 40) were collected from P. knowlesi malaria patients from Peninsular Malaysia and Malaysian Borneo. The PkTRAMP gene was amplified using PCR, followed by cloning into a plasmid vector and sequenced. Results showed that the nucleotide diversity of PkTRAMP was low (π: 0.009). Z-test results indicated negative (purifying) selection of PkTRAMP. The alignment of the deduced amino acid sequences of PkTRAMP of Peninsular Malaysia and Malaysian Borneo revealed 38 dimorphic sites. A total of 27 haplotypes were identified from the amino acid sequence alignment. Haplotype analysis revealed that there was no clustering of PkTRAMP from Peninsular Malaysia and Malaysian Borneo.
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, 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.
A main challenge in parasitology is the development of reliable tools to prevent or treat mosquito-borne diseases. We investigated the toxicity of magnetic nanoparticles (MNP) produced by Magnetospirillum gryphiswaldense (strain MSR-1) on chloroquine-resistant (CQ-r) and sensitive (CQ-s) Plasmodium falciparum, dengue virus (DEN-2), and two of their main vectors, Anopheles stephensi and Aedes aegypti, respectively. MNP were studied by Fourier-transform infrared spectroscopy and transmission electron microscopy. They were toxic to larvae and pupae of An. stephensi, LC50 ranged from 2.563 ppm (1st instar larva) to 6.430 ppm (pupa), and Ae. aegypti, LC50 ranged from 3.231 ppm (1st instar larva) to 7.545 ppm (pupa). MNP IC50 on P. falciparum were 83.32 μg ml(-1) (CQ-s) and 87.47 μg ml(-1) (CQ-r). However, the in vivo efficacy of MNP on Plasmodium berghei was low if compared to CQ-based treatments. Moderate cytotoxicity was detected on Vero cells post-treatment with MNP doses lower than 4 μg ml(-1). MNP evaluated at 2-8 μg ml(-1) inhibited DEN-2 replication inhibiting the expression of the envelope (E) protein. In conclusion, our findings represent the first report about the use of MNP in medical and veterinary entomology, proposing them as suitable materials to develop reliable tools to combat mosquito-borne diseases.
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.
Thus far, Entamoeba species have been classified based on morphology such as the number of nuclei in mature cysts and their hosts. Using recently developed molecular tools, ruminant Entamoeba spp. are currently classified into four species/genotypes: E. bovis and Entamoeba ribosomal lineages (RL) 1, 2, and 4. However, the distribution or pathogenicity of ruminant Entamoeba has not been well documented. In the present study, we examined a total of 25 fecal and seven environmental samples collected from six farms in Japan from 2016 to 2017 by the floatation method and PCR and sequencing analyses. Consequently, we detected Entamoeba cysts in 18 of 25 cattle samples and four of the seven environmental samples, including soil and drinking water, by microscopic examinations. In sequential examinations, Entamoeba-positive cattle were found to shed cysts without any clinical symptoms for more than 8 months. By PCR for molecular identification, isolates in ten cattle and one soil sample were successfully sequenced and formed a cluster of E. bovis, which was separated from those of other Entamoeba species/genotypes such as RL1-4 in phylogenetic analysis. To our knowledge, this is the first report about E. bovis in Japan, and our results may implicate that E. bovis is not pathogenic.
Ivermectin at single doses of 0.2-1.0 mg/kg body weight reduced the microfilarial counts of subperiodic Brugia malayi in Presbytis cristata by 59.9%-89.6% of initial counts, 4 weeks after treatment. Adult filaricidal activity was poor, live adult worms being recovered from all animals at autopsy. There was no serious side effect at these doses.
Presbytis cristata monkeys infected through the inoculation of between 200 and 400 subperiodic Brugia malayi infective larvae (L3) in the right thigh, in both thighs or in the dorsum of the right foot were followed up for varying periods of up to about 8 months after infection. All 148 inoculated animals became patent, with mean prepatent periods being between 66 and 76 days. In animals injected in the thigh, the patterns of microfilaraemia were similar, there being a rapid rise in the geometric mean counts (GMCs) of microfilariae during the first 10-12 weeks of patency, which then plateaued at levels of greater than 1000/ml. Adult worm recovery, expressed as the percentage of the infective dose, was significantly higher in animals injected with 100 L3 in each thigh, being 9.4% as compared with 2.8%-4.8% in other groups. It is therefore recommended that animals should be injected with 100 L3 in each thigh and that the testing of potential filaricides in this model be carried out during the phase of rapid increase in microfilaraemia to ensure that any microfilaricidal effect can easily be detected.
This study investigated the polymorphism in the P. falciparum chloroquine resistance transporter (pfcrt) gene 11 years after chloroquine (CQ) cessation in Jazan region, southwestern Saudi Arabia. Two hundred and thirty-five P. falciparum isolates were amplified to detect mutations in the pfcrt gene. The pfcrt 76 T molecular marker for CQ resistance was detected in 66.4% (156/235) of the isolates, while the K76 CQ-sensitive wild type was detected in 33.6%. The pfcrt 74I and pfcrt 75E point mutations were each found to be present in 56.2% of isolates, while only four isolates (1.7%) were found to carry the pfcrt 72S mutation. Moreover, four pfcrt haplotypes were identified as follows: the CVIET triple-allele (56.2%), SVMET double-allele (1.7%) and CVMNT single-allele (8.5%) mutant haplotypes and the CVMNK wild haplotype (33.6%). The analysis also revealed significant associations between the prevalence of mutant pfcrt alleles and haplotypes and the age group, governorate and nationality of the patients as well as the parasitaemia level (p
The Simulium rufibasis subgroup is one of three subgroups of the Simulium (Simulium) tuberosum species-group; it is characterized by a pair of clustered stout hairs on the ventral surface of female abdominal segment 7. A member of the S. rufibasis subgroup in Taiwan was investigated morphologically and genetically using the universal cytochrome c oxidase subunit I (COI) barcoding gene and polytene chromosomal banding pattern. The Taiwanese material is morphologically similar to S. rosliramlii Takaoka & Chen from Vietnam and represents the second species of the S. rufibasis subgroup known from Taiwan. It also represents a novel molecular lineage that is distinct from three other primary lineages identified as S. doipuiense, S. doipuiense/S. rufibasis, and S. weji previously reported from Thailand. The mitochondrial evidence for a distinct lineage in Taiwan is supported by chromosomal analysis, which revealed unique sex chromosomes. For nomenclatural stability, we associate the name S. arisanum Shiraki with the Taiwanese entity. Originally described from females from Taiwan, S. arisanum until now has remained an enigmatic species.
Nine 50-l surface water samples from a Malaysian recreational lake were examined microscopically using an immunomagnetisable separation-immunofluorescent method. No Cryptosporidium oocysts were detected, but 77.8% of samples contained low numbers of Giardia cysts (range, 0.17-1.1 cysts/l), which were genetically characterised by SSU rRNA gene sequencing. Genotype analyses indicated the presence of Giardia duodenalis assemblage A suggesting potential risk to public health. The present study represents the first contribution to our knowledge of G. duodenalis assemblages in Malaysian recreational water.