Echinostoma mekongi n. sp. (Digenea: Echinostomatidae) from Riparian People along the Mekong River in Cambodia.

Echinostoma mekongi n. sp. (Digenea: Echinostomatidae) is described based on adult flukes collected from humans residing along the Mekong River in Cambodia. Total 256 flukes were collected from the diarrheic stool of 6 echinostome egg positive villagers in Kratie and Takeo Province after praziquantel treatment and purging. Adults of the new species were 9.0-13.1 (av. 11.3) mm in length and 1.3-2.5 (1.9) mm in maximum width and characterized by having a head collar armed with 37 collar spines (dorsal spines arranged in 2 alternative rows), including 5 end group spines. The eggs in feces and worm uterus were 98-132 (117) µm long and 62-90 (75) µm wide. These morphological features closely resembled those of Echinostoma revolutum, E. miyagawai, and several other 37-collar-spined Echinostoma species. However, sequencing of the nuclear ITS (ITS1-5.8S rRNA-ITS2) and 2 mitochondrial genes, cox1 and </>nad1, revealed unique features distinct from E. revolutum and also from other 37-collar-spined Echinostoma group available in GenBank (E. bolschewense, E. caproni, E. cinetorchis, E. deserticum, E. miyagawai, E. nasincovae, E. novaezealandense, E. paraensei, E. paraulum, E. robustum, E. trivolvis, and Echinostoma sp. IG). Thus, we assigned our flukes as a new species, E. mekongi. The new species revealed marked variation in the morphology of testes (globular or lobulated), and smaller head collar, collar spines, oral and ventral suckers, and cirrus sac compared to E. revolutum and E. miyagawai. Epidemiological studies regarding the geographical distribution and its life history, including the source of human infections, remain to be performed.

Artyfechinostomum malayanum: Metacercariae Encysted in Pila sp. Snails Purchased from Phnom Penh, Cambodia.

The metacercariae of Artyfechinostomum malayanum (Leiper, 1911) Mendheim, 1943 were discovered in Pila sp. snails purchased from a market in Phnom Penh, Cambodia. They were isolated from the snails using the artificial digestion technique and were orally fed to 2 hamsters, 1 rat, and 2 mice to obtain the adult flukes. The metacercariae were round, 145-165 µm in diameter, having a cyst wall of 6-10 µm in thickness, a head collar and collar spines, and characteristic features of excretory granules. Adult flukes were recovered in the small intestines of the animals at days 14 and 32 post infection and were morphologically observed using a light microscope and a scanning electron microscope. They were plump or elongated, ventrally curved, 6.0-8.1×1.6-2.0 mm in size, and characterized by the head collar bearing 43 collar spines, including 5 end group ones on each side, a long cirrus sac extending beyond the posterior margin of the ventral sucker, a submedian ovary, and 2 deeply lobed testes. Eggs in uteri were operculate, ovoid to ellipsoid, and 120-135×68-75 µm in size. In scanning electron microscopy, the head collar was prominent with collar spines looking like horns. Scale-like tegumental spines were densely distributed on the ventral surface between the head collar and ventral sucker. Sensory papillae were distributed mainly on the tegument around suckers. By this study, it has been first confirmed that the life cycle of A. malayanum exists in Cambodia.

Stellantchasmus falcatus (Digenea: Heterophyidae) in Cambodia: Discovery of Metacercariae in Mullets and Recovery of Adult Flukes in an Experimental Hamster.

Stellantchasmus falcatus (Digenea: Heterophyidae) is first reported from Cambodia through recovery of the metacercariae from mullet fish and adult flukes from an experimentally infected hamster. We purchased 7 mullets, Chelon macrolepis, in a local market of Phnom Penh, Cambodia, and each of them was examined by the artificial digestion method on May 2010. The metacercariae of S. falcatus were detected in all mullets (100%) examined, and their average density was 177 per fish. They were elliptical, 220×168 µm in average size. They were orally infected to an hamster to obtain adult flukes. Adults recovered at day 10 post infection were observed with a light microscope and a scanning electron microscope (SEM). They were small, 450×237 µm in average size, had a small oral sucker (41×50 µm), subglobular pharynx (29×21 µm), slender esophagus (57 µm), long and thick-walled expulsor (119×32 µm), spherical ovary (58×69 µm), and 2 ovoid testes (right: 117×74 µm; left: 114×63 µm). Eggs were small, yellow, and 23×12 µm in average size. In SEM observations, tegumental spines were densely distributed on the whole tegument, and single small type I sensory papillae were distributed around the lip of oral sucker. The small ventral sucker was dextrally located and had 8 type I sensory papillae on the left margin. It has been first confirmed in the present study that the mullet, C. macrolepis, is playing the role of a second intermediate host of S. falcatus in Cambodia.

Prevalence of intestinal helminths among inhabitants of Cambodia (2006-2011).

In order to investigate the status of intestinal helminthic infections in Cambodia, epidemiological surveys were carried out on a national scale, including 19 provinces. A total of 32,201 fecal samples were collected from schoolchildren and adults between 2006 and 2011 and examined once by the Kato-Katz thick smear technique. The overall egg positive rate of intestinal helminths was 26.2%. The prevalence of hookworms was the highest (9.6%), followed by that of Opisthorchis viverrini/minute intestinal flukes (Ov/MIF) (5.7%), Ascaris lumbricoides (4.6%), and Trichuris trichiura (4.1%). Other types of parasites detected were Enterobius vermicularis (1.1%), Taenia spp. (0.4%), and Hymenolepis spp. (0.2%). The northwestern regions such as the Siem Reap, Oddar Meanchey, and Banteay Meanchey Provinces showed higher prevalences (17.4-22.3%) of hookworms than the other localities. The southwestern areas, including Koh Kong and Preah Sihanouk Provinces showed higher prevalences of A. lumbricoides (17.5-19.2%) and T. trichiura (6.1-21.0%). Meanwhile, the central and southern areas, in particular, Takeo and Kampong Cham Provinces, showed high prevalences of Ov/MIF (23.8-24.0%). The results indicate that a considerably high prevalence of intestinal helminths has been revealed in Cambodia, and thus sustained national parasite control projects are necessary to reduce morbidity due to parasitic infections in Cambodia.

High prevalence of Opisthorchis viverrini infection in a riparian population in Takeo Province, Cambodia.

Opisthorchis viverrini infection was found to be highly prevalent in 3 riverside villages (Ang Svay Chek A, B, and C) of the Prey Kabas District, Takeo Province. This area is located in the southern part of Cambodia, where the recovery of adult O. viverrini worms was recently reported. From May 2006 until May 2010, fecal examinations were performed on a total of 1,799 villagers using the Kato-Katz thick smear technique. In the 3 villages, the overall positive rate for helminth eggs ranged from 51.7 to 59.0% (av. 57.4%), and the percentage positive for O. viverrini was 46.4-50.6% (47.5%). Other helminths detected included hookworms (13.2%), echinostomes (2.9%), Trichuris trichiura (1.3%), Ascaris lumbricoides (0.6%), and Taenia spp. (0.06%). The prevalence of O. viverrini eggs appeared to reflect a lower infection in younger individuals (<20 years) than in the adult population (>20 years). Men (50.4%) revealed a significantly higher (P=0.02) prevalence than women (44.3%). The Ang Svay Chek villages of the Prey Kabas District, Takeo Province, Cambodia have been confirmed to be a highly endemic area for human O. viverrini infection.

Echinostoma revolutum infection in children, Pursat Province, Cambodia.

To determine the prevalence of helminthic infections in Pursat Province, Cambodia, we tested fecal specimens from 471 children, 10-14 years of age, in June 2007. The prevalence of infection with echinostome flukes ranged from 7.5% to 22.4% in 4 schools surveyed. Adult worms were identified as Echinostoma revolutum.

Efficacy of sodium metaperiodate (SMP)-ELISA for the serodiagnosis of schistosomiasis mekongi.

Schistosomiasis mekongi is an important public health issue in endemic countries. In this study, we evaluated an indirect immunodiagnostic ELISA method using Schistosoma mekongi soluble egg antigen. Sodium metaperiodate (SMP)-ELISA was utilized in order to remove the glycosylated epitopes responsible for false positive reactions and the results using this method were compared with those using conventional ELISA (conv-ELISA). Forty-two serum samples from schistosomiasis mekongi egg-positive patients and 100 serum samples from schistosomiasis-negative Cambodian subjects were tested using both ELISA methods. The ranges of ELISA values for positive and negative sera were distinct on SMP-ELISA, but the ranges of the two groups of sera overlapped on conv-ELISA. Therefore, diagnostic criteria may be established based on the highest ELISA value on negative sera and the lowest ELISA value on positive sera. In the present study, both the sensitivity and specificity of SMP-ELISA reached 100% using the criteria in which an ELISA value > or = 0.2 was positive.

Molecular identification of Taenia tapeworms by Cox1 gene in Koh Kong, Cambodia.

We collected fecal samples from 21 individuals infected with Taenia tapeworms in Koh Kong Province, Cambodia, and performed nucleotide sequencing of the cox1 gene and multiplex PCR on the eggs for DNA differential diagnosis of human Taenia tapeworms. Genomic DNA was extracted from the eggs of a minimum number of 10 isolated from fecal samples. Using oligonucleotide primers Ta7126F, Ts7313F, Tso7466F, and Rev7915, the multiplex PCR assay proved useful for differentially diagnosing Taenia solium, Taenia saginata, and Taenia asiatica based on 706, 629, and 474 bp bands, respectively. All of the Taenia specimens from Kho Kong, Cambodia, were identified as either T. saginata (n=19) or T. solium (n=2) by cox1 sequencing and multiplex PCR.

Echinostoma ilocanum infection in Oddar Meanchey Province, Cambodia.

Fecal examinations using the Kato Katz technique were performed on a total of 1,287 villagers (945 students and 342 general inhabitants) of Oddar Meanchey Province, Cambodia in May 2007 and November 2009. The overall intestinal helminth egg positive rate was 23.9%, and the most prevalent helminth species was hookworms (21.6%). Other helminth eggs detected included echinostomes (1.0%), Enterobius vermicularis (0.8%), small trematode eggs (0.7%), which may include Opisthorchis viverrini and Haplorchis spp., and Hymenolepis nana (0.4%). In order to recover adult echinostomes, we treated 2 patients with 10-15 mg/kg praziquantel and purged. Total 14 adult echinostomes, 1 and 13 worms from each patient, were collected. The echinostomes characteristically had 49-51 collar spines and 2 round or slightly lobated testes. They were identified as Echinostoma ilocanum (Garrison, 1908) Odhner, 1911. So far as literature are concerned, this is the first record on the discovery of human E. ilocanum infection in Cambodia.

The evaluation of control measures against Schistosoma mekongi in Cambodia by a mathematical model.

We constructed a mathematical model for the transmission of Schistosoma mekongi in Cambodia. The simulation of the model will be instrumental in planning schistosomiasis control measures. The model includes two definitive hosts, humans and dogs, as animal reservoirs. Dogs are recognized to play an important role in schistosomiasis transmission in Cambodia. For the purpose of dealing with age-specific prevalence and intensity of infection, the human population was classified into eight age categories in the model. To describe the seasonal fluctuation of the intermediate host population of S. mekongi, the "Post-Spate Survival" hypothesis was adopted for the population dynamics of Neotricula aperta present in the Mekong River. We carried out simulations to evaluate the effect of universal treatment (UT) and targeted mass treatment (TT) with praziquantel on the reduction in prevalence of S. mekongi. The simulations indicated that biyearly UT for 8 years or yearly TT for 5 years after three courses of yearly UT could reduce the prevalence to below 5% when a UT or TT coverage of 85% of inhabitants was achieved. The simulation suggested that the suppression of S. mekongi in Cambodia would be possible by UT or TT with a high coverage rate.

Invasive Pomacea snails as important intermediate hosts of Angiostrongylus cantonensis in Laos, Cambodia and Vietnam: Implications for outbreaks of eosinophilic meningitis.

The rat lungworm Angiostrongylus cantonensis causes human eosinophilic meningitis and it is endemic in Southeast Asia, but little is known about its distribution in Laos, Cambodia and Vietnam. We conducted a multi-country survey for A. cantonensis in these countries to estimate its prevalence in snails along the Mekong River and the east coast of Vietnam. We identified Angiostrongylus species by morphological and molecular analysis. We found A. cantonensis in the invasive snail, Pomacea spp. The wide accessibility of Pomacea snails, along with their infection by A. cantonensis, indicates that this snail species could be used in surveillance for preventing outbreaks of eosinophilic meningitis.

Pfcrt genotypes and related microsatellite DNA polymorphisms on Plasmodium falciparum differed among populations in the Greater Mekong Subregion.

Malaria morbidity and mortality have decreased gradually in the Greater Mekong Subregion (GMS). Presently, WHO sets a goal to eliminate malaria by 2030 in the GMS. However, drug-resistant malaria has been reported from several endemic areas. To achieve the goal of elimination, the status of the emergence and spread of drug resistance should be monitored. In this study, the genotype of the Plasmodium falciparum chloroquine (CQ) resistance transporter gene (pfcrt) and 6 microsatellite DNA loci flanking the gene were examined. P. falciparum isolates (n = 136) was collected from malaria patients in Thailand (n = 50, 2002-2005), Vietnam (n = 39, 2004), Laos (n = 15, 2007) and Cambodia (n = 32, 2009). Amino acid sequences at codons 72-76 on the gene were determined. All of the isolates from Thailand were CQ-resistant (CVIET), as were all of the isolates from Cambodia (CVIET, CVIDT). Thirteen of the 15 isolates (87%) from Laos were CQ-resistant (CVIET, CVIDT), whereas the other 2 (13%) were CQ-susceptible (CVMNK). In contrast, 27 of the 39 isolates (69%) from Vietnam were CQ-susceptible (CVMNK), whereas the other 12 (31%) were CQ-resistant (CVIET, CVIDT, CVMDT) or mixed (CVMNK/CVIDT). The mean of expected heterozygosity of the microsatellite loci was 0.444 in the Thai population, 0.482 in the Cambodian population, and 0.734 in the Vietnamese population. Genetic diversity in the Thai population was significantly lower than that in the Vietnamese population. These results suggested that chloroquine selective pressure on P. falciparum populations is heterogeneous in the GMS. Therefore, further examination to understand the mechanisms behind the emergence and spread of drug-resistant malaria are needed.

Schistosoma mekongi and Schistosoma japonicum: Differences in the distribution of eggs in the viscera of mice.

The difference in the distribution of Schistosoma eggs in the viscera has not been clearly elucidated in the two closely related species Schistosoma japonicum and Schistosoma mekongi. In this study, we quantitatively compared the distribution of eggs in mice infected with the two species. In S. mekongi-infected mice, 56.6% to 69.4% of total eggs were found in the distal small intestine 9 to 15 weeks after infection, while in S. japonicum-infected mice, 48.8% to 71.8% of eggs were found in the proximal small intestine during the same period. There were significantly more eggs in the liver in mice infected with S. japonicum than in those infected with S. mekongi. The number of adult worms recovered did not differ between the two species during the study period. The total number of eggs laid in the tissues also did not differ between the two species at 12 to 15 weeks postinfection, but in the earlier period the total number of eggs was significantly fewer in S. mekongi-infected than in S. japonicum-infected mice, suggesting the delayed maturation of the former compared with the latter. These results clearly show that S. japonicum and S. mekongi exhibit different oviposition behavior in their hosts.

High susceptibility of Neotricula aperta gamma-strain from Krakor and Sdau in Cambodia to Schistosoma mekongi from Khong Island in Laos.

Neotricula aperta gamma-strain snails collected from Krakor and Sdau in Cambodia were found to have the same or higher susceptibility to Schistosoma mekongi as N. aperta originally isolated from Khong in Laos. Infection rates of N. aperta gamma-strain snails exposed to 3 miracidia at week 8 were: Khong gamma-strain, 22.6%; Krakor gamma-strain, 33.3%; and Sdau gamma-strain, 67.4%. At week 10, the Sdau gamma-strain showed the highest infection rate of 83.3%. We thus found significantly high susceptibility of the Sdau gamma-strain to S. mekongi originally isolated from Khong. However, in another experiment, susceptibility of the Sdau gamma-strain was rather comparable to that of Khong and Krakor gamma-strain. We also found no significant differences in infection and survival rates between the Khong and Krakor gamma-strain when the snails were exposed to 3 or 6 miracidia. This is the first report to confirm the high susceptibility in the laboratory of N. aperta gamma-strain snails from endemic areas in Cambodia to S. mekongi originally isolated from Laos. The high susceptibility of N. aperta gamma-strain snails to S. mekongi in distant areas may be an important factor in the endemic transmission of human schistosomiasis.

Effects of repeated praziquantel treatment on schistosomiasis mekongi morbidity as detected by ultrasonography.

Schistosomiasis mekongi is endemic in the Mekong River basin; about 80,000 people are at risk of infection in Cambodia. We conducted ultrasonographic studies of patients with schistosomiasis mekongi in Kratie province, Cambodia, focusing especially on the relationship between the frequency of praziquantel treatment and findings of ultrasonographic imaging. The frequency of praziquantel treatment in the period from 1995 to 2002 was classified into four groups: 1-2, 3-4, 5-6, and 7-8 times. Ultrasonographic images were examined to determine the presence of thickening of the portal vein wall and formation of meandering collateral circulation of the splenic vein. We selected these parameters because they are unaffected by interobserver bias. The results showed that thickening of the portal vein wall may have potential to improve with frequent praziquantel treatment. On the other hand, established hard splenomegaly and meandering collateral circulation of the splenic vein, improved very little with praziquantel treatment.

The High Prevalence of Anemia in Cambodian Children and Women Cannot Be Satisfactorily Explained by Nutritional Deficiencies or Hemoglobin Disorders.

BACKGROUND: Anemia is highly prevalent in Cambodian women and children, but data on causes of anemia are scarce. We performed a national micronutrient survey in children and women that was linked to the Cambodian Demographic Health Survey 2014 (CDHS-2014) to assess the prevalence of micronutrient deficiency, hemoglobin disorders and intestinal parasite infection. METHODS: One-sixth of households from the CDHS-2014 were selected for a follow-up visit for the micronutrient survey. Households were visited from two weeks to two months after the CDHS-2014 visit. Data on micronutrient status were available for 1512 subjects (792 children and 720 women). RESULTS: Anemia was found in 43% of the women and 53% of the children. Hemoglobin disorders affected >50% of the population, with Hemoglobin-E the most prevalent disorder. Deficiencies of iron (ferritin < 15 g/L), vitamin A (retinol-binding-protein (RBP) < 0.70 mol/L) or vitamin B12 (<150 pmol/L) were not prevalent in the women (<5% for all), whereas 17.8% of the women had low concentrations of folic acid (<10 nmol/L). In the children, the prevalence of iron, vitamin A, vitamin B12 or folic acid deficiency was <10%. Zinc deficiency, hookworm infection and hemoglobinopathy were significantly associated with anemia in children, whereas in the women none of the factors was significantly associated with anemia. Iron deficiency anemia (IDA) was more prevalent in children <2 years, but in older children and women, the prevalence of IDA was <5%. The most prevalent, preventable causes of anemia were hookworm infection and zinc and folic acid deficiency. Over 40% of the anemia was not caused by nutritional factors. CONCLUSION: The very high prevalence of anemia in Cambodian women and children cannot be explained solely by micronutrient deficiencies and hemoglobin disorders. Micronutrient interventions to improve anemia prevalence are likely to have limited impact in the Cambodian setting. The focus of current interventions to reduce the high prevalence of anemia in children and women should be broadened to include zinc and folic acid as well as effective anti-hookworm measures.

Cognitive Performance and Iron Status are Negatively Associated with Hookworm Infection in Cambodian Schoolchildren.

Soil-transmitted helminth (STH) infection has been associated with lower cognitive performance of schoolchildren. To identify pathways through which STH infection might affect school performance, baseline data from a large rice-fortification trial in Cambodian schoolchildren were used to investigate associations between STH infection, micronutrient status, anemia, and cognitive performance. Complete data on anthropometry, cognitive performance, and micronutrient status were available for 1,760 schoolchildren, 6-16 years of age. STH infection was identified using Kato-Katz, whereas cognitive performance was assessed using Raven's Colored Progressive Matrices (RCPM), block design, and picture completion. STH infection was found in 18% of the children; almost exclusively hookwork infection. After adjusting for age and gender, raw cognitive test scores were significantly lower in hookworm-infected children (-0.65; -0.78; -2.03 points for picture completion, RCPM, and block design, respectively; P < 0.05 for all). Hookworm infection was associated with iron status (total body iron), but not with vitamin A and zinc status, nor with inflammation or anthropometry. Body iron was negatively associated with increased intensity of hookworm infection (R = 0.22, P < 0.001). Hookworm infection in Cambodian schoolchildren was associated with lower cognitive performance, an effect most likely mediated through lower body iron. Interventions that are more effective against hookworm infection are needed to contribute to better health and improvement of cognitive performance.

Weekly iron and folic acid supplementation as a tool to reduce anemia among primary school children in Cambodia.

The prevalence of anemia decreased from 62% to 12% and from 57% to 26% in children 5 to 11 years of age in two rural primary schools in Kampot Province, Cambodia, after oral weekly supplementation with iron-folic acid tablets for 20 weeks and with vitamin A and mebendazole twice per year. In 12- to 15-year-old children, success was less marked. The prevalence of hookworm infestation did not change, but the number of eggs in the stool decreased drastically. The intervention had no significant influence on stunting and wasting. An integrated community approach including mass deworming, health education, and multi-micronutrient supplementation was very effective in reducing anemia in Cambodian schoolchildren and should be adopted on a larger scale.

Financial costs of deworming children in all primary schools in Cambodia.

In 2002, Cambodia's Ministry of Health launched a deworming programme to deliver an anthelmintic drug (mebendazole 500 mg) and health education to 75% of its school children twice a year. Cambodia's school population is approximately 2.8 million. The deworming programme was organized into two phases: the first phase (December 2002-March 2003) targeted more than one million school children from 11 provinces; and the second phase (July 2003-January 2004) targeted the entire school population. The cost to treat each child was 12 cents (0.11 USD) during the first phase, 6 cents during the second phase, and 3 cents for re-treatment in areas where the campaign was conducted for the second time. The Cambodian experience demonstrates that, with political commitment, high coverage for deworming is achievable even in a country with minimal resources. Cambodia's deworming programme represents a successful model for other developing countries.