Establishing a protocol for water sample processing for the detection of Blastocystis sp.

This study was aimed at establishing a protocol for water sample processing for the detection of Blastocystis sp. using distilled water spiked with Blastocystis sp. cysts. The study established a protocol involving eight technical aspects, namely, storage temperature, storage duration, minimum water sample volume, optimum relative centrifugal force, centrifugation duration, minimum number of cyst for inoculation in Jones' medium and turn-around-time for the detection of vacuolar forms of Blastocystis sp. Results showed a minimum of 1.0 L water sample should be collected and processed on the same day. Otherwise, it should be stored at 4 °C and processed within 3 days. Water sample should be centrifuged at 1400×g for 10 min. For the isolation of Blastocystis sp. cysts, parasite pellet could be layered on top of Ficoll-Paque™ PLUS, centrifuged at 1400×g for 20 min and washed twice using 0.9% saline with centrifugation at 1400×g for 10 min. A minimum of 1 × 105 cysts could then be inoculated in Jones' medium supplement with 10% horse serum, incubated at 37 °C and examined for any presence of vacuolar forms of Blastocystis sp. after 3 days of inoculation. A protocol for water sample processing for the detection of Blastocystis sp. has successfully been established. The protocol was validated using 106 various water samples. This protocol will be very useful in determining the extent of Blastocystis sp. contamination in water sources in order to identify the seriousness of contamination.

Acanthamoebicidal activity of periglaucine A and betulinic acid from Pericampylus glaucus (Lam.) Merr. in vitro.

Acanthamoeba species are pathogenic protozoa which account for amoebic keratitis, conjunctivitis and granulomatous amoebic encephalitis. These amoebae form cysts which resist drugs and more effective acanthamoebicidal agents are needed. Medicinal plants could be useful in improving the current treatment strategies for Acanthamoeba infections. In the present study, we examined the amoebicidal effects of Pericampylus glaucus (Lam.) Merr., a medicinal plant used for the treatment of conjunctivitis in Malaysia. Pathogenic Acanthamoeba triangularis were isolated from environmental water samples and treated with different concentrations of fractions obtained from Pericampylus glaucus (Lam.) Merr. as well as main constituents for 24-72 h. Chlorhexidine was used as a reference drug. Ethanol fraction of stem showed significant (p < 0.05) inhibition of trophozoites survival. Betulinic acid and periglaucine A from this plant at 100 µg/mL inhibited more than 70% survival of both cysts and trophozoites. The calculated therapeutic index for betulinic acid and periglaucine A was 170 and 1.5 for trophozoites stage and 3.75 and 8.5 for cysts stage. The observed amoebicidal efficacies indicate the beneficial aspects of this plant in the treatment of Acanthamoeba infection. Periglaucine A could also be of value for the treatment of Acanthamoeba infection.

Anti-encystment and amoebicidal activity of Lonicera japonica Thunb. and its major constituent chlorogenic acid in vitro.

OBJECTIVE: To examine the acanthamoebicidal effects of ethyl acetate, aqueous and butanol fractions of dried flower buds of Lonicera japonica (L. japonica) Thunb. (Flos Lonicerae) in vitro. METHODS: Acanthamoeba triangularis isolates were obtained from environmental water samples and identified by PCR. They were exposed to ethyl acetate, water and butanol fractions of L. japonica Thunb. at concentrations ranging from 0.5 mg/mL to 1.5 mg/mL. The extracts were evaluated for growth inhibition at 24, 48 and 72 h, respectively. Chlorogenic acid at a concentration of 1 mg/mL was examined for inhibition of encystment. RESULTS: Ethyl acetate fraction at a concentration of 1.5 mg/mL evoked a significant reduction of trophozoite viability by 48.9% after 24 h, 49.2% after 48 h and 33.7% after 72 h chlorogenic acid, the major active constituent of L. japonica Thunb. at the concentration of 1 mg/mL reduced the cysts/trophozoite ratio by 100% after 24 h, 84.0% after 48 h and 72.3% after 72 h. This phenolic compound at concentration of 1 mg/mL concurrent with 0.6% hydrogen peroxide inhibited hydrogen peroxide-induced encystment by 92.8% at 72 h. CONCLUSIONS: Results obtained from this study show that ethyl acetate fraction at 1.5 mg/mL is the most potent fraction of L. japonica Thunb. and its major constituent chlorogenic acid showed the remarkable inhibition of encystment at a concentration of 1 mg/mL.

Monitoring of Waterborne Parasites in Two Drinking Water Treatment Plants: A Study in Sarawak, Malaysia.

The occurrence of waterborne parasites coupled with water parameters at various processing sites of two drinking water treatment plants (A and B) and seven distribution system (DS) sites in Sarawak, Malaysia were studied. Ten liters of water underwent immunomagnetic separation (IMS) technique to detect the presence of Giardia and Cryptosporidium (oo)cysts. The remaining supernatant was used to detect other parasites whilst 50 mL of water sample was each used in the detection of free-living amoebae and fecal coliforms. Sampled water was positive for Giardia (32.9%; 28/85), Cryptosporidium (18.8%; 16/85) followed by Spirometra ova-like (25.9%; 22/85), Blastocystis-like (25.9%; 22/85), nematode larvae-like (8.2%; 7/85) and Taenia ova-like (1.2%; 1/85). Meanwhile, 90.2% (55/61) samples were positive for Acanthamoeba and Naegleria via cultivation and of these, 11 isolates were confirmed as Acanthamoeba genotype T3 (5/7) and T4 (2/7) followed by Naegleria sp. (4/11), Naegleria italica (2/11), Naegleria australiensis (1/11), Naegleria angularis (1/11) and Vahlkampfia sp. (3/11). Cryptosporidium, Acanthamoeba and Naegleria were also detected in one of the seven tested DS sites. Only Giardia and Cryptosporidium showed significant correlations with fluoride and fecal coliforms. These results describe the occurrence of waterborne parasites that will assist key stakeholders in mitigating contamination at the specific sites.

Granular Formation during Apoptosis in Blastocystis sp. Exposed to Metronidazole (MTZ).

The role and function of the granular life cycle stage in Blastocystis sp, remains uncertain despite suggestions being made that the granules are metabolic, reproductive and lipid in nature. This present study aims to understand granular formation by triggering apoptosis in Blastocystis sp. by treating them with metronidazole (MTZ). Blastocystis sp.cultures of 4 sub-types namely 1, 2, 3 and 5 when treated with 0.01 and 0.0001 mg/ml of metronidazole (MTZ) respectively showed many of the parasites to be both viable and apoptotic (VA). Treated subtype 3 isolates exhibited the highest number of granular forms i.e. 88% (p<0.001) (0.0001 mg/ml) and 69% (p<0.01) (0.01 mg/ml) respectively at the 72 h in in vitro culture compared to other subtypes. These VA forms showed distinct granules using acridine orange (AO) and 4',6-diamino-2-phenylindole (DAPI) staining with a mean per cell ranging from 5 in ST 5 to as high as 16 in ST 3. These forms showed intact mitochondria in both viable apoptotic (VA) and viable non-apoptotic (VNA) cells with a pattern of accumulation of lipid droplets corresponding to viable cells. Granular VA forms looked ultra-structurally different with prominent presence of mitochondria-like organelle (MLO) and a changed mitochondrial trans-membrane potential with thicker membrane and a highly convoluted inner membrane than the less dense non-viable apoptotic (NVA) cells. This suggests that granular formation during apoptosis is a self-regulatory mechanism to produce higher number of viable cells in response to treatment. This study directs the need to search novel chemotherapeutic approaches by incorporating these findings when developing drugs against the emerging Blastocystis sp. infections.

Presence of Cryptosporidium parvum and Giardia lamblia in water samples from Southeast Asia: towards an integrated water detection system.

BACKGROUND: Access to clean and safe drinking water that is free from pathogenic protozoan parasites, especially Cryptosporidium parvum and Giardia lamblia that cause gastrointestinal illness in humans, is still an issue in Southeast Asia (SEA). This study is the first attempt to detect the aforementioned protozoan parasites in water samples from countries in SEA, using real-time polymerase chain reaction (qPCR) assays. METHODS: A total of 221 water samples of 10 l each were collected between April and October 2013 from Malaysia (53), Thailand (120), the Philippines (33), and Vietnam (15). A physicochemical analysis was conducted. The water samples were processed in accordance with the US Environmental Protection Agency's methods 1622/1623.1, microscopically observed and subsequently screened using qPCR assays. RESULTS: Cryptosporidium oocysts were detected in treated water samples from the Philippines (1/10), with a concentration of 0.06 ± 0.19 oocyst/L, and untreated water samples from Thailand (25/93), Malaysia (17/44), and the Philippines (11/23), with concentrations ranging from 0.13 ± 0.18 to 0.57 ± 1.41 oocyst/L. Giardia cysts were found in treated water samples from the Philippines (1/10), with a concentration of 0.02 ± 0.06 cyst/L, and in untreated water samples from Thailand (20/93), Vietnam (5/10), Malaysia (22/44), and the Philippines (16/23), with concentrations ranging from 0.12 ± 0.3 to 8.90 ± 19.65 cyst/L. The pathogens C. parvum and G. lamblia were detected using using qPCR assays by targeting the 138-bp fragment and the small subunit gene, respectively. C. parvum was detected in untreated water samples from the Philippines (1/23) and Malaysia (2/44), whilst, G. lamblia detected was detected in treated water samples from the Philippines (1/10) and in untreated water samples from Thailand (21/93), Malaysia (12/44), and the Philippines (17/23). Nitrate concentration was found to have a high positive correlation with (oo)cyst (0.993). CONCLUSION: The presence of (oo)cysts in the water samples means that there is potential risk for zoonotic disease transmission in the studied countries. Detection using qPCR is feasible for quantifying both pathogenic C. parvum and G. lamblia in large water samples.

Seroepidemiology of Toxoplasmosis among People Having Close Contact with Animals.

A cross-sectional study was conducted to determine the seroepidemiology of Toxoplasma infection and its risk association among people having close contact with animals. A total of 312 blood samples were collected from veterinary personnel (veterinarian, technicians, and students) and pet owners from veterinary clinics and hospitals in the area of Klang Valley, Malaysia. About 4 cc of blood samples drawn from agreed participants were processed for measurement of anti-Toxoplasma IgG and IgM antibodies as well as avidity test of Toxoplasma IgG by ELISA I, II, and III kits. Meanwhile, the demographic profiles and possible risk factors of these participants were also recorded in the standardized data collection sheets. Overall seroprevalence of toxoplasmosis was observed in 62 (19.9%) participants being 7 (18.4%) in veterinarians, 15 (33.3%) in veterinary technicians, 29 (14.9%) in veterinary students, and 11 (31.4%) in pet owners. Of 19.9% Toxoplasma seropositive samples, 18.3% was positive for IgG antibody, 1.0% for IgM antibody, and 0.6% for both IgG and IgM antibodies. Of three different IgG avidity ELISA kits, ELISA III showed high avidity in all five seropositive samples (IgM and IgG/IgM antibodies) indicating chronic Toxoplasma infection which is consistent with no evidence of clinical toxoplasmosis diagnosed during the time of this study. Univariate analysis showed that age group, gender, study population, gardening, task performance, and working duration were significantly associated with Toxoplasma seropositivity. Further analysis by multivariate analysis using logistic regression showed that age group of ≥30 years old (OR = 0.34, 95% CI = 0.18-0.63, p = 0.001) and working or study duration of >10 years having close contact with animals (OR = 5.07, 95% CI = 1.80-14.24, p = 0.002) were identified as significant risks for Toxoplasma infection. Based on the results obtained, a comprehensive Toxoplasma screening and health surveillance program on toxoplasmosis should be implemented among people having close contact with animals in general and confirmed Toxoplasma seronegative individuals in particular to prevent seroconversion.

A reproducible method for extraction of Plasmodium falciparum DNA by microwave irradiation and its potential for rapid molecular diagnosis.

Malaria remains one of the most important communicable diseases. A rapid, simple and accurate method is a crucial part of malaria diagnosis. The aim of this study was to reevaluate the microwave irradiation method to extract DNA from Plasmodium falciparum and compare with six other existing DNA extraction methods such as QIAamp DNA mini kit (Qiagen), FTA elute card, phenol-chloroform, Chelex, Chelex without proteinase-K and Rapid boiling. Two different P. falciparum isolates were used: (i) Laboratory strains with 0.3% parasitemia and (ii) clinical isolate with 0.6% parasitemia. Each DNA extraction method was validated for the presence of P. falciparum by a routine nested and real time PCR. In order to evaluate the sensitivity of the DNA extraction by microwave, double serial dilution of P. falciparum from in vitro culture at parasitemia that ranged from 0.0001 to 0.17% were used to extract the DNA by microwave and the P. falciparum DNA was then detected by nested and real-time PCR. The nested and real-time PCR were able to detect. P. falciparum DNA at the parasitemia level as low as 0.0003% and 0.0001%, respectively. Our results can reproduce the results from earlier studies and reveal microwave as a rapid and simple tool to extract P. falciparum DNA and subsequent molecular diagnosis of malaria.

Comparative study on Toxoplasma infection between Malaysian and Myanmar pregnant women.

BACKGROUND: Toxoplasma gondii, an obligate intracellular protozoan parasite, causes a disease called toxoplasmosis which can sometimes be acquired congenitally by a newborn from an infected mother. This study aimed to determine the seroprevalence of Toxoplasma infection and its associated risks among 219 and 215 pregnant women from Malaysia and Myanmar, respectively. METHODS: Anti-Toxoplasma IgG and IgM antibodies were screened by using standard commercial ELISA kits. The socio-demographic, obstetrics and risk factors associated with Toxoplasma infection data were compared between the two countries. RESULTS: The overall prevalence of Toxoplasma infection in Malaysian pregnant women (42.47%; 95% CI = 36.11-49.09) was significantly higher (p < 0.05) than Myanmar pregnant women (30.70%; 95% CI = 27.92-37.16). By univariate analysis, this study identified that age group, education, parity, awareness on toxoplasmosis and consumption of undercooked meat were significantly associated (p < 0.05) with Toxoplasma seropositive Malaysian pregnant women but none of these factors associated with Toxoplasma seropositive Myanmar pregnant women. In comparison using univariate analysis between the two countries, it was found that Toxoplasma seropositive Malaysian pregnant women was associated with aged 30 years and above, secondary or lower-secondary level of education, the third trimester of pregnancy, having one child or more, lacking awareness of toxoplasmosis, absence of bad obstetrics history, having no history of close contact with cats or soil, living on a farm and also consumption of undercooked meat, unpasterized milk or untreated water. Avidity measurement was used to confirm the stages of Toxoplasma infection in pregnant women who were positive for both IgG and IgM antibodies and found all were infected in the past. CONCLUSION: From our study, Toxoplasma screening and its risk measurement in pregnant women is firmly recommended for monitoring purposes and assisting proper management, including diagnosis and treatment during antenatal period. Also, it is necessary to initiate preventive measures for Toxoplasma infection among reproductive-age women in general and seronegative pregnant women in particular. Avidity measurement should be incorporated in Toxoplasma routine screening, especially with the availability of a single serum sample to assist in the diagnosis.

Waterborne parasites: a current status from the Philippines.

BACKGROUND: Despite the amount of awareness created, waterborne disease still poses threat, especially in developing countries. Due to the scarcity of reported data on waterborne parasites, the consumption of unsafe water prolongs. Thus, the occurrences of waterborne parasites from various samples were investigated from one of the Southeast Asian country, the Philippines. METHODS: A total of thirty three samples, each consisting of twelve liters, were collected and processed to obtain the sediment. Ten liters of sample each was processed to detect Cryptosporidium spp. and Giardia spp. using an immunomagnetic separation method prior to enumeration via fluorescence microscope. Meanwhile, the remaining two liters were cultured to detect Acanthamoeba and Naegleria through microscopy examination and polymerase chain reaction (PCR) analysis. RESULTS: Twelve samples (36.4%) from river (5), swimming pool (1), pond (3), rain tank (1), and natural lake (2) were positive for Cryptosporidium spp., 17 (45.5%) samples from river (9), pond (2), swimming pool (1), rain tank (1), and natural lake (4) were positive for Giardia spp. while, 13 (33.3%) samples from river (3), swimming pool (2), pond (2), dispenser (1), well (1), tap (2) and natural lake (2) were positive for Acanthamoeba spp. and 5 (18.2%) samples from river (1), natural lake (1), tap (1), dispenser (1) and mineral (1) were Naegleria spp. positive. Physical parameters such as temperature, conductivity, total dissolved solid (TDS), salinity, dissolved oxygen (DO), pH, and turbidity and chemical parameters such as ammonia, chlorine, fluoride, nitrate and nitrite were also measured. The highest chemical contamination was observed at pond 2. A good correlation was observed between Giardia and nitrite (r = 0.736, p < 0.01) and Giardia and nitrate (r = 0.502, p < 0.01). CONCLUSION: This study was aimed to create greater awareness of parasitic contamination in the water environment in the Philippines and also to act as a platform of the current scenario for policymakers as water pollution is a key health issue in this region.

Increased detection of Plasmodium knowlesi in Sandakan division, Sabah as revealed by PlasmoNex™.

BACKGROUND: Plasmodium knowlesi is a simian malaria parasite that is widespread in humans in Malaysian Borneo. However, little is known about the incidence and distribution of this parasite in the Sandakan division, Malaysian Borneo. Therefore, the aim of the present epidemiological study was to investigate the incidence and distribution of P. knowlesi as well as other Plasmodium species in this division based on a most recent developed hexaplex PCR system (PlasmoNex™). METHODS: A total of 189 whole blood samples were collected from Telupid Health Clinic, Sabah, Malaysia, from 2008 to 2011. All patients who participated in the study were microscopically malaria positive before recruitment. Complete demographic details and haematological profiles were obtained from 85 patients (13 females and 72 males). Identification of Plasmodium species was conducted using PlasmoNex™ targeting the 18S ssu rRNA gene. RESULTS: A total of 178 samples were positive for Plasmodium species by using PlasmoNex™. Plasmodium falciparum was identified in 68 samples (38.2%) followed by 64 cases (36.0%) of Plasmodium vivax, 42 (23.6%) cases of P. knowlesi, two (1.1%) cases of Plasmodium malariae and two (1.1%) mixed-species infections (i e, P. vivax/P. falciparum). Thirty-five PlasmoNex™ positive P. knowlesi samples were misdiagnosed as P. malariae by microscopy. Plasmodium knowlesi was detected in all four districts of Sandakan division with the highest incidence in the Kinabatangan district. Thrombocytopaenia and anaemia showed to be the most frequent malaria-associated haematological complications in this study. CONCLUSIONS: The discovery of P. knowlesi in Sandakan division showed that prospective studies on the epidemiological risk factors and transmission dynamics of P. knowlesi in these areas are crucial in order to develop strategies for effective malaria control. The availability of advanced diagnostic tool PlasmoNex™ enhanced the accuracy and accelerated the speed in the diagnosis of malaria.

Review of forensically important entomological specimens collected from human cadavers in Malaysia (2005-2010).

Forensic entomological specimens collected from human decedents during crime scene investigations in Malaysia in the past 6 years (2005-2010) are reviewed. A total of 80 cases were recorded and 93 specimens were collected. From these specimens, 10 species of cyclorrphagic flies were identified, consisting of Chrysomya rufifacies (Macquart) -38 specimens (40.86%), Chrysomya megacephala (Fabricius) -36 specimens (38.70%), Chrysomya villeneuvi (Patton) -2 specimens (2.15%), Chrysomya nigripes (Aubertin) -2 specimens (2.15%), Chrysomya pinguis (Walker) -1 specimen (1.08%), Hermetia illucens (Linnaeus) -1 specimen (1.08%), Hemipyrellia liguriens (Wiedemann) -5 specimens (5.37%), Synthesiomyia nudiseta (Wulp) -1 specimen (1.08%), Megaselia scalaris (Loew)-1 specimen (1.08%) and Sarcophaga ruficornis (Fabricius) -4 specimens (4.30%). In two specimens (2.15%), the maggots were not identifiable. Ch. megacephala and Ch. rufifacies were the commonest species found in human decedents from three different ecological habitats. S. nudiseta is an uncommon species found only on human cadavers from indoors. A total of 75 cases (93.75%) had a single fly infestation and 5 cases (6.25%) had double fly infestation. In conclusion, although large numbers of fly species were found on human decedents, the predominant species are still those of Chrysomya.

Genetic diversity of caprine Blastocystis from Peninsular Malaysia.

Blastocystis sp. is a common intestinal parasite found in humans and animals. The possibility of zoonotic transmission to humans from livestock especially goats led us to investigate the genetic diversity of caprine Blastocystis sp. obtained from five different farms in Peninsular Malaysia. Moreover, there is a lack of information on the prevalence as well as genetic diversity of Blastocystis sp. in goat worldwide. Results showed that 73/236 (30.9 %) of the goats were found to be positive for Blastocystis infection. The most predominant Blastocystis sp. subtype was ST1 (60.3 %) followed by ST7 (41.1 %), ST6 (41.1 %), and ST3 (11.0 %) when amplified by PCR using sequenced-tagged site (STS) primers. Four farms had goats infected only with ST1 whereas the fifth showed mixed infections with multiple STs. The proximity of the fifth farm to human dwellings, nearby domesticated animals and grass land as opposed to a sterile captive environment in the first four farms may account for the multiple STs seen in the fifth farm. Since ST1, ST3, ST6 and ST 7 were previously reported in human infection worldwide in particular Malaysia, the potential of the zoonotic transmission of blastocystosis should not be disregarded. The implications of different farm management systems on the distribution of Blastocystis sp. STs are discussed.

Molecular identification of blow flies recovered from human cadavers during crime scene investigations in Malaysia.

Forensic entomology applies knowledge about insects associated with decedent in crime scene investigation. It is possible to calculate a minimum postmortem interval (PMI) by determining the age and species of the oldest blow fly larvae feeding on decedent. This study was conducted in Malaysia to identify maggot specimens collected during crime scene investigations. The usefulness of the molecular and morphological approach in species identifications was evaluated in 10 morphologically identified blow fly larvae sampled from 10 different crime scenes in Malaysia. The molecular identification method involved the sequencing of a total length of 2.2 kilo base pairs encompassing the 'barcode' fragments of the mitochondrial cytochrome oxidase I (COI), cytochrome oxidase II (COII) and t-RNA leucine genes. Phylogenetic analyses confirmed the presence of Chrysomya megacephala, Chrysomya rufifacies and Chrysomya nigripes. In addition, one unidentified blow fly species was found based on phylogenetic tree analysis.

Molecular identification of blow flies recovered from human cadavers during crime scene investigations in Malaysia

Forensic entomology applies knowledge about insects associated with decedent in crime scene investigation. It is possible to calculate a minimum postmortem interval (PMI) by determining the age and species of the oldest blow fly larvae feeding on decedent. This study was conducted in Malaysia to identify maggot specimens collected during crime scene investigations. The usefulness of the molecular and morphological approach in species identifications was evaluated in 10 morphologically identified blow fly larvae sampled from 10 different crime scenes in Malaysia. The molecular identification method involved the sequencing of a total length of 2.2 kilo base pairs encompassing the 'barcode' fragments of the mitochondrial cytochrome oxidase I (COI), cytochrome oxidase II (COII) and t-RNA leucine genes. Phylogenetic analyses confirmed the presence of Chrysomya megacephala, Chrysomya rufifacies and Chrysomya nigripes. In addition, one unidentified blow fly species was found based on phylogenetic tree analysis.