Veeranoot Nissapatorn

Since more than a decade, DNA barcoding has been widely used to examine biological samples and differentiate species, as well as employed in ecological and conservational studies. There is a growing interest of DNA barcoding, particularly in medical parasitology, but its potential utility in soil-transmitted helminths (STHs) remains unclear. Therefore, in this article, we review the studies using DNA barcoding and its applications in medical parasitology with special focus on STHs such as Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, and Trichuris trichiura. DNA barcoding is reliable for identifying STH species as well as its cryptic species. In addition, epidemiological data and the impacts of STH infections on children are discussed. This article further discusses the paucity of STH DNA barcodes (partial cytochrome oxidase subunit 1 [COI] mitochondrial DNA [mtDNA] sequences) in two gene banks; National Center for Biotechnology Information (NCBI) and Barcode of Life Data (BOLD) Systems. It also highlights the future prospects of DNA barcoding including primer designing and reference library on STHs.

Data on the distribution of free-living amoebae is still lacking especially in Southeast Asian region. The aquatic environment revealed a high occurrence of free-living amoebae (FLA) due to its suitable condition and availability of food source, which subsequently causes infection to humans. A total of 94 water samples consisted of both treated and untreated from Laos (31), Myanmar (42), and Singapore (21) were investigated for the presence of pathogenic FLA. Each water sample was filtered and cultured onto non-nutrient agar seeded with live suspension of Escherichia coli and incubated at room temperature. Morphological identification was conducted for both trophozoites and cysts via microscopic stains (Giemsa and immunofluorescence). The presence of Naegleria-like structures was the most frequently encountered in both treated and untreated water samples, followed by Acanthamoeba-like and Vermamoeba-like features. To identify the pathogenic isolates, species-specific primer sets were applied for molecular identification of Acanthamoeba, Naegleria, and Vermamoeba. The pathogenic species of Acanthamoeba lenticulata and A. triangularis were detected from untreated water samples, while Vermamoeba vermiformis was found in both treated and untreated water samples. Our results suggested that poor water quality as well as inadequate maintenance and treatment might be the cause of this alarming problem since chlorine disinfection is ineffective in eradicating these amoebas in treated water samples. Regular monitoring and examination of water qualities are necessary in order to control the growth, hence, further preventing the widespread of FLA infections among the public.

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. 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. 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. 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.

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.

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. 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. 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). 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.

Plasmodium is a blood protozoan parasite that is responsible for malaria. To date, Plasmodium falciparum has shown multi-drug resistance, particularly in Thailand, Myanmar and Malaysia. The aim of the study is to screen the plant extracts that can effectively inhibit P. falciparum 3D7, a common lab strain malaria parasite. Nine plants were collected and processed through maceration using hexane, chloroform and ethanol, resulting in 24 crude plant extracts. Of these, extracts from Artabotrys crassifolius, Pericampylus glacus and Leuconotis eugeniifolia showed promising antiplasmodial activities at IC50 of 15.32 to 39.75 mu g/mL in a modified schizont maturation assay. Further studies are warranted to explore its efficacies and lead compounds of these three plant extracts for the development of antiplasmodial drugs.

Toxoplasmosis is one of the most common opportunistic parasitic diseases in patients living with HIV/AIDS. This study aimed to determine the seroprevalence of Toxoplasma infection in HIV-infected patients and to identify associated risk factors in Toxoplasma seropositive patients. This study was conducted at a regional public hospital in Hat Yai, southern Thailand during October 2009 to June 2010. Blood samples were collected from 300 HIV-infected patients. Each subject also answered a socio-demographic and risk factors associated with Toxoplasma infection. The prevalence of anti-Toxoplasma IgG antibodies in HIV-infected patients was 109 (36.3%), of which 83 (76.2%) had past infection and 26 (23.9%) had recently acquired Toxoplasma infection as indicated by their IgG avidity. Multivariate analysis using logistic regression showed that gender difference (adjusted OR=1.69, 95% CI=1.05-2.72) was the only factor associated with Toxoplasma infection. From the results obtained, these HIV-infected patients could be at high risk of developing clinical evidence of severe toxoplasmosis. Therefore, it is necessary to introduce primary behavioral practices to prevent Toxoplasma infection among HIV-infected patients.

A cross-sectional study was conducted to determine the seroepidemiology of Toxoplasma gondii infection among individuals who have close contact with animals. The association between plausible risk factors and the seropositivity for Toxoplasma was examined. A total of 312 blood samples were collected form veterinarians(38), veterinary technicians(45), veterinary students(194) and pet owners(35) from several veterinary hospitals and clinics in the area of Klang Valley, Malaysia. About 4cc 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.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 where 7(18.4%) veterinarians, 15(33.3%)veterinary technicians, 29(14.9%)veterinary students and 11(31.4%) pet owners were infected. Out of 19.9% positive samples for toxoplasmosis, 18.3% of these samples were positive for IgG antibody, 1.0% was positive for IgM antibody and 0.6% were positive for both IgG and IgM antibodies.Positive samples for IgM antibody and for both IgG and IgM antibodies were further analysed by IgG avidity test. Out of 5 samples tested for avidity test, 4 samples showed a high avidity results which means infection occurred in the past whereas only 1 sample showed a recently acquired infection. Univariate analysis showed that age group, gender,study population, gardening, task performance and working duration were significantly associated with seropositivity of toxoplasmosis. Further analysis by multivariate analysis using logistic regression showed that age group more than or equal to 30 years old (OR=0.34, 95% CI=0.18-0.63, p=0.001)and working or study duration more than 10 years in handling with animals(OR=5.07, 95% CI=1.80-14.24, p=0.002)were identified as significant

This study investigated the distribution of parasites as main contaminants in water environments of peninsular Malaysia (October 2011–December 2011) and the southeastern coast of Thailand (June 2012). Sixty-four water samples, 33 from Malaysia and 31 from Thailand, of various water types were examined according to U.S. Environmental Protection Agency guidelines. Drinking or household water types from both countries were free from parasitic contamination. The recreational/environmental (except a swimming pool in Malaysia) and effluent water types from these two countries were contaminated with waterborne parasites: Giardia (0.04–4 cysts/L), Cryptosporidium (0.06–2.33 oocysts/L), hookworm (6.67–350 ova/L), Ascaris (0.33–33.33 ova/L), and Schistosoma (9.25–13.33 ova/L). The most contaminated sites were recreational lake garden 3 in Malaysia and river 2 in Thailand. Higher concentrations of Giardia, Cryptosporidium, and hookworm were found in samples from Malaysia than in samples from Thailand. The presence of Giardia cysts showed a significant association with the presence of Cryptosporidium oocysts (P < 0.005).

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 < 0.01). Based on the preliminary data obtained, it is clearly shown that there is a necessity to implement the detection of waterborne parasites and physico-chemical analysis in Malaysia. Future work on heavy metals (chromium, copper, mercury and zinc) is recommended to enhance the overall water quality monitoring and to take appropriate safety measures to ensure maintenance of good water standards.