High Prevalence of Haplorchiasis in Nan and Lampang Provinces, Thailand, Proven by Adult Worm Recovery from Suspected Opisthorchiasis Cases

Opisthorchiasis, a risk factor for cholangiocarcinoma in humans, is of public health importance in Thailand. The Annual Surveillance Reports from Nan and Lampang Provinces, Thailand, for the year 2011 showed an opisthorchiasis prevalence of over 70% by recovery of eggs in the feces. This study investigated whether most cases are actually due to minute intestinal flukes (MIF) rather than Opisthorchis viverrini, as the eggs of both can hardly be differentiated by morphology. Fifty and 100 cases from residents in Nan and Lampang, respectively, had stools positive for eggs initially assumed to be those of O. viverrini. Each patient was given praziquantel at 40 mg/kg in a single dose. After 2 hr, 30-45 ml of the purgative magnesium sulfate was given, and stools were collected up to 4 times sequentially. The stools were examined for adult worms by simple sedimentation. It was found that 39 of 50 cases (78.0%) from Nan Province had Haplorchis taichui, with intensities ranging from 5 to 1,250 with an average of 62 worms/case. Taenia saginata (7 cases) and Enterobius vermicularis (1 case) were other helminths recovered as the co-infectants. In Lampang Province, H. taichui was recovered from 69 cases (69.0%). The number of flukes recovered ranged from 1 to 4,277, with an average of 326 worms/case. Four cases had Phaneropsolus bonnei, and 10 T. saginata as the co-infectants. Adult specimens of O. viverrini were not recovered from any stool. Clearly, MIF infection, especially haplorchiasis, is more common in northern Thailand. These findings should encourage the Public Health Office to employ more specific tools than Kato's method for surveillance of opisthorchiasis in Thailand.

Prevalence of Metagonimus Metacercariae in Sweetfish, Plecoglossus altivelis, from Eastern and Southern Coastal Areas in Korea

The present study was performed to determine the current infecion status of Metagonimus yokogawai metacercariae in sweetfish, Plecoglossus altivelis, collected from several streams in eastern and southern coastal areas of Korea. The sweetfish collected were artificially digested with pepsin-HCl solution and examined under a stereomicroscope in August and September, 2007. Out of 145 sweetfish collected from 10 streams in eastern coasts (Gangwon-do and Gye-ongsangbuk-do), 88 (60.7%) were infected with Metagonimus metacercariae. The average metacercarial density was 61 per infected fish. Among 141 sweetfish collected from 10 streams in southern coasts (Gyeongsangnam-do, Ulsan Metropolitan city, and Jeollanam-do), 140 (99.3%) were infected with Metagonimus metacercariae, and their average density was 949 per infected fish. The present study confirmed that M. yokogawai metacercariae are still prevalent in the sweetfish from several eastern and southern coastal localities. The prevalence and metacercarial density were much higher in the sweetfish from streams of southern coasts than in those of eastern coasts. Therefore, attention should be paid to this small fluke infection, and consumption of raw sweetfish naturally produced in these areas should be prohibited.

Haplorchis taichui as a Possible Etiologic Agent of Irritable Bowel Syndrome-Like Symptoms

The aim of this study is to clarify the clinical features of Haplorchis taichui infection in humans in Nan Province, Thailand, and to correlate the clinical features with irritable bowel syndrome (IBS)-like symptoms. In this study area, only H. taichui, but neither other minute intestinal flukes nor small liver flukes were endemic. The degree of infection was determined by fecal egg counts and also by collecting adult worms after deworming. The signs and symptoms of individual patients together with their hematological and biochemical laboratory data were gathered to evaluate the relationship between the clinical features and the severity of infection. Special emphasis was made to elucidate the possible similarities of the clinical features of H. taichui infection and IBS-like symptoms. The results showed useful clinical information and the significant (> 50%) proportion of haplorchiasis patients complained of abdominal pain, lassitude, and flatulence, which were the important diagnostic symptoms of IBS. This study has reported a possible link between H. taichui and IBS, and H. taichui might probably play a role in the etiology of these IBS-like symptoms.

Prevalence of Haplorchis taichui in Field-Collected Snails: A Molecular Approach

The prevalence of the cercarial stage of an intestinal trematode, Haplorchis taichui, in thiarid snails (Gastropoda: Thiaridae) was investigated using light microscope and species-specific PCR procedures. A total of 988 snails were collected from Mae Taeng district, Chiang Mai province, northern Thailand, which comprised of 3 species; Melanoides tuberculata, Tarebia granifera, and Thiara scabra. The overall prevalence of pleurolophocercous cercariae was 21.7% as determined by the morphology. For genetic detection of H. taichui infection in snails, 2 primers Hapt_F (5'-GGCCAACGCAATCGTCATCC-3') and Hapt_R (5'-GCGTCGGGTTTCAGACATGG-3'), were used. The genomic DNA of H. taichui, which was used as a positive control, gave an amplification of the 256 bp fragment. The overall prevalence of H. taichui from specific PCR was 9.7%. The proportion of H. taichui among the pleurolophocercous cercariae in this study was 44.9%.

Egg Laying Capacity of Haplorchis taichui (Digenea: Heterophyidae) in Humans

Quantitative fecal egg counts represented as the number of eggs per gram of feces (EPG) are generally a reliable parameter to estimate the worm burden of intestinal and hepatic parasitoses. Although Haplorchis taichui (Digenea: Heterophyidae) is one of the most common minute human intestinal flukes, little is known about the relationship between EPG and the actual worm burden in patients or the severity of the disease. In the present study, fecal samples were collected from 25 villagers in northern Thailand before and after praziquantel treatment. The EPG values of each participant were determined by the modified cellophane thick smear method, and adult worms were collected from the whole stool after the treatment. Eggs per day per worm (EPDPW) of H. taichui were estimated 82 from egg counts and expelled worms. The EPG was not well correlated with the worm burden, and a reverse correlation was observed between the EPDPW and the worm burden.

Prevalence of Haplorchis taichui and Haplorchoides sp. Metacercariae in Freshwater Fish from Water Reservoirs, Chiang Mai, Thailand

A parasitological investigation on trematode metacercariae was made on 62 freshwater fishes of 13 species in northern Thailand; Cyclocheilichthys apogon, Puntioplites proctozysron, Labiobarbus siamensis, Barbodes gonionotus, Barbodes altus, Henicorhynchus siamensis, Osteochilus hasselti, Notopterus notopterus, Mystacoleucus marginatus, Anabas testudineus, Systomus orphoides, Morulius chrysophykadian, and Hampala macrolepidota. The fish were caught over the summer period (February-May 2007) from 2 Chiang Mai water reservoirs, i.e., the Mae Ngad (UTM 47Q E 503200, 47Q N 2119300) and the Mae Kuang Udomtara (UTM 47Q E 513000, 47Q N 2092600) Reservoirs in Chiang Mai province, Thailand. The prevalence of heterophyid (Haplorchis taichui and Haplorchoides sp.) metacercariae in these fish was 83.9% and 74.2% in the Mae Ngad and Mae Kuang Udomtara Reservoirs, respectively. The highest intensity of heterophyid metacercariae in H. siamensis in the Mae Ngad was 120.4 and that in P. proctozysron in the Mae Kuang Udomtara was 180.0. The fish, A. testudineus, C. apogon, and M. chrysophykadian, were not found to be infected with H. taichui metacercariae. The results show that the freshwater fish in Chiang Mai water reservoirs are heavily infected with H. taichui and Haplorchoides sp. metacercariae.

Natural infection of fresh and brackish water fish with heterophyid encysted metacercariae in Dakahlia Governorate Egypt

The encysted metacercariae [EMC] of heterophyids in the fish species; Mugil caphalus, M. capito, M. auratus, Oreochromis, n.niloticus [=Tilapia nilotica], and Tilapia zillii trapped brackish and fresh water fishes were isolated by the artificial digestion method and identified. The EMC by morphometric analysis proved to be seven phenotypical distinct types. Cross-matched seven groups of clean laboratory bred puppies each was experimentally infected with a distinct EMC type. The recovered adult flukes were compatible to seven species of six genera of family Heterophyidae. These were Heterophyes heterophyes, H. aequalis, Pygidiopsis genata, Haplorchis yokogawai, Prohemostomum vivax, Phagicola sp. and Stictodora sp. The EMC of the seven heterophyids were recovered from all the brackish water fishes, M. caphalus, M. capito, M. auratus, O. niloticus and T. zillii. On the other hand, the EMC of P. genata, Haplorchis yokogawai and Phagicola sp. were recovered only from the fresh water fish, O. niloticus and T. zillii. All the EMC was re-described and photographed

A new endemic focus of Heterophyes nocens and other heterophyid infections in a coastal area of Gangjin-gun, Jeollanam-do

To know the prevalence of heterophyid trematodes among inhabitants of a southern coastal village, i.e., Sacho-ri, Gangjin-gun, Jeollanam-do (Province), 82 stool samples were examined on helminth eggs and protozoan cysts using Kato-Katz and formalin-ether sedimentation techniques. Total 33 people (40.2%) were positive for trematodes (Heterophyes nocens; 15 people, Pygidiopsis summa; 3, Metagonimus sp.; 4, Clonorchis sinensis; 7, Gymnophalloides seoi; 6) and/or protozoa (Entamoeba coli; 3). Among intestinal trematode egg positive cases, 17 were treated with praziquantel and their whole diarrheic stools were collected after purgation. Adult flukes of H. nocens (number of specimens=1,294), P. summa (386), Stellantchasmus falcatus (5), Stictodora lari (4), and Heterophyopsis continua (1) were collected using a stereomicroscope. To know the source of human H. nocens infections in this village, metacercarial infections in mullets (10) were examined and most H. nocens metacercariae (101/105, 96.2%) were found in the trunk portion. From above results, the surveyed coastal village has been newly known as an endemic focus of human H. nocens infection and consuming raw mullets was the presumable source of human heterophyid infections.

A survey of Heterophyes nocens and Pygidiopsis summa metacercariae in mullets and gobies along the coastal areas of the Republic of Korea

The infection status of mullets Mugil cephalus (n = 139) and gobies (n = 35) Acanthogobius flavimanus with metacercariae of Heterophyes nocens and Pygidiopsis summa was examined in 11 western, southern, and eastern coastal areas of the Republic of Korea, using a digestion technique. Heterophyid metacercariae were highly prevalent in mullets from western and southern coastal areas; Shinan-gun (100% for H. nocens and 100% for P. summa), Muan-gun (93% and 100%), Buan-gun (42% and 75%), Seocheon-gun (73% and 53%), Ganghwa-gun (47% and 100%), Sacheon-shi (47% and 77%), and Gangjin-gun (50% and 70%, respectively). Only 1 (10%) of 10 mullets from an eastern coastal area, i.e., Donghae-shi, was positive for P. summa metacercariae. Metacercarial densities were the highest in the trunk of mullets for H. nocens and the gill for P. summa. Gobies from Muan-gun were positive for H. nocens (40%) and P. summa metacercariae (40%), and gobies from Seocheon-gun revealed H. nocens metacercariae (20%). The metacercarial density was remarkably higher in mullets than in gobies. The results revealed that H. nocens and P. summa metacercariae are prevalent in mullets and gobies from coastal areas of the Republic of Korea, and the prevalence and intensity of infection vary according to geographical locality.

Prevalence of Gymnophalloides seoi infection in coastal villages of Haenam-gun and Yeongam-gun, Republic of Korea

One coastal village in Haenam-gun and two in Yeongam-gun, Jeollanam-do were surveyed for intestinal parasite infections by fecal examination. The egg positive rates of Gymnophalloides seoi were high, 24.1% (14/58) in Haenam-gun and 9.3% (11/118) in Yeongam-gun. The egg positive rates of heterophyids, including Heterophyes nocens, and of Clonorchis sinensis were 10.3% and 6.9% in Haenam-gun, and 14.4% and 8.5% in Yeongam-gun, respectively. After praziquantel treatment and purgation, a total of 37,761 fluke specimens were recovered from 17 patients; 11 in Haenam-gun and 6 in Yeongam-gun. Gymnophalloides seoi was the most commonly recovered species, with 37,489 specimens in total (2,205 per person). Other recovered flukes included Heterophyes nocens, Stictodora fuscata, Heterophyopsis continua, Pygidiopsis summa, and undetermined species. These results indicate that the areas surveyed are new endemic foci of G. seoi.

Survival of Haplorchis taichui metacercariae in Lab-Pla, Thai traditional food preparation.

Lab-Pla is a famous kind of Thai traditional food in northern and northeasthern Thailand. Consumption of uncooked Lab-Pla can lead to trematode infection because cyprinoid fish serve as the 2nd intermediate hosts of trematodes. The preparation of Lab-Pla can be made in different ways, depending on seasonings used in different places. The effect of the seasonings used in Lab-Pla on the survival of Haplorchis taichui metacercariae were, therefore, determined. The methodology was done by the following: 100 g of chopped Siamese mud carp fish (Henicorhynchus siamensis) were used for each experiment of seasonings used for completely mixed Lab-Pla. Mixed materials were blended and digested by 1% pepsin solution to obtain metacercariae and then checked for the movement under a stereomicroscope. The results showed that metacercariae remained active in 5 experimental groups: fermented fish treated, fish sauce treated, chili powder treated, ground roasted rice treated, and complete mixed treated indicated by average as follows: 27, 38.7, 33.3, 42.7 and 21%, respectively. In conclusion, fermented fish and completely mixed methods have more effectiveness in killing metacercariae in Lab-Pla preparation than other seasonings (p < 0.05).

Melanoides tuberculatus (Gastropoda: Thiaridae) as intermediate host of Heterophyidae (Trematoda: Digenea) in Rio de Janeiro metropolitan area, Brazil

No final da década de 60, caramujos da espécie Melanoides tuberculatus, originários do nordeste africano e sudeste asiático, foram introduzidos no Brasil. Os primeiros registros de espécimes infectados com cercárias foram feitos no Estado do Rio de Janeiro em 2001. O presente estudo relata a ocorrência de M. tuberculatus infectados com larvas de trematódeos na cidade do Rio de Janeiro. O sedimento dos criadouros foi coletado e peneirado através de malhas de 0,25 polegadas. Os caramujos foram transportados em recipientes com água dos criadouros, sendo medidos e posteriormente individualizados. Eles foram expostos à luz e temperatura artificiais para induzir a emergência cercariana. As cercárias mais ativas foram processadas pelos métodos de coloração diferencial e de impregnação por nitrato de prata. Os caramujos negativos foram subsequentemente dissecados. Aproximadamente 700 caramujos foram coletados. Seu comprimento variou de 1,2 a 3,3 cm. A taxa de prevalência foi de 15,76% apesar de 53,76% dos caramujos estarem infectados em uma das áreas. Os caramujos estavam infectados com rédias e cercárias do tipo pleurolofocerca. A morfologia e a quetotaxia cercarianas foram compatíveis com as da família Heterophyidae principalmente devido à presença de nadadeiras medianas dorsais e ventrais e à ausência de receptores sensoriais CI dorsais.

High prevalence of Haplorchis taichui metacercariae in cyprinoid fish from Chiang Mai Province, Thailand.

This study aimed to investigate Haplorchis taichui metacercarial infection in fish collected from the Chom Thong and Mae Taeng districts, Chiang Mai Province during November 2001 to October 2002. A total 617 cyprinoid fish of 15 species were randomly collected and examined for H. taichui metacercariae. All the species of fish were found to be infected with H. taichui. The infection rates were 91.4% (266/290) and 83.8% (274/327), with mean intensities of 242.9 and 107.4 in the Chom Thong and Mae Taeng districts, respectively. The portion of the fish body with the highest metacercarial density was the muscles, and second, the head, in both districts. In addition, the fish had mixed-infection with other species of trematodes, namely: Centrocestus caninus, Haplorchoides sp, and Haplorchis pumilio.

Rare case of Metagonimus yokogawai.

Intestinal Heterophyid infections are almost unknown in India with only one documented case report. We report a case of six year old child with diarrhoea without any other associated gastrointestinal symptoms. Examination of the faeces revealed eggs of Metagonimus yokogawai. However, the patient left against medical advice and was lost to follow up. The case is being reported because of its rarity.

Epidemiological study of clonorchiasis and metagonimiasis along the Geum-gang (River) in Okcheon-gun (County), Korea

The endemic status of clonorchiasis and metagonimiasis along the Geum-gang (River) in Okcheon-gun (County) in Korea was examined. From February to December 2000, stools of total 1,081 inhabitants living in 5 villages were examined. Each stool specimen was examined by both the cellophane thick smear method and the formalin-ether sedimentation technique. Egg-positive cases were further analyzed by Stoll's egg-counting technique, and praziquantel was administered to positive cases. The egg-positive rates for Clonorchis sinensis and Metagonimus species were 9.3% and 5.5%, respectively, and the double infection rate was 3.5%. The numbers of eggs per gram (EPG) of feces of C. sinensis and Metagonimus sp. were 918 +/-1.463 and 711+/-947, respectively. The egg-positive rates for C. sinensis and Metagonimus sp. in the riverside area were 14.2% and 8.4%, respectively, which were significantly higher than those of the inland area (3.2% and 1.7%, respectively). The egg-positive rates of C. sinensis and Metagonimus sp. in males (16.7% and 10.0%) were significantly higher than those of females (3.5% and 1.8%). However, there were no significant differences of EPG values between localities and sexes. The prevalence of clonorchiasis and metagonimiasis in this survey was significantly lower than that in the previous reports. However, there is still a high prevalence of infection with C. sinensis and Metagonimus sp. in this region, especially in the riverside area.

Occurrence of two heterophyid metacercariae Haplorchis and Haplorchoides in cyprinoid fish of some districts in Chiang Mai and Lumphun Province.

According to currently available information, the metacercaria of one heterophyid trematode, Haplorchis, is commonly found in freshwater fishes, especially the cyprinoid group. Thus, in an investigation into the level of Haplorchis metacercaria infection in cyprinoid fishes, comprising ten species, 811 cyprinoid fishes were captured from canals, reservoirs and rivers in six districts of Chiang Mai Province and 4 districts of Lumphun Province, Thailand, 43.03% of the fishes comprising seven species viz Puntius leiacanthus, P. gonionotus, P. orphoides, P. stolickkae, Cirrhina spp, Rasbora spp and R. lateristriata, were found infected, with the prevalence of infection at 77.29, 70.31, 56.52, 47.14, 18.48, 7.94 and 5.18% respectively. Infected fishes were collected from Mae Rim and Sansai districts of Chiang Mai, and Pasang, Ban Hong and Li districts of Lumphun. It should be noted that the Haplorchis metacercariae are commonly found along with Haplorchoides metacercariae. Both minute heterophyids metacercariae were mainly localized in scales, a few were found in fins and gills of fishes.

Metacercariae isolated from cyprinoid fishes in Khon Kaen District by digestion technic.

Several kinds of fresh-water fishes collected from natural water resources in Khon Kaen District were digested with 0.25% pepsin A to obtain the infective stage or metacercariae of fish-borne trematodes. Most of them harbored at least 3 species of trematodes. Two families of medical trematodes, i.e., Family heterophyidae and Family Opisthorchiidae, were isolated. The heterophyid flukes consisted of Haplorchis taichui, H. pumilio and Centrocestus formosanus. Opisthorchis viverrini was the only parasite of the Family Opisthorchiidae found in these fish. Among these four species, H. taichui was predominant and found in all kinds of fish examined, especially Hampala dispar.

Metacercariae in fishes of Sun Moon lake which is an endemic area for Clonorchis sinensis in Taiwan.

The Sun Moon lake in Central Taiwan is a known endemic area for clonorchiasis. Of the 45 fresh water fish, Hemiculter leucisculus, from the lake that were examined by artificial gastric juice digestion in October 1995, all were found to harbor metacercariae in their muscle. The number of metacercariae isolated from each fish ranged from 2 to 2,185, with an average of 254. A total of 11,443 metacercariae was collected from the 45 fish. Of the 4,223 metacercaria that were examined under light microscope, 4,064 (96.23%) were found to belong to Haplorchis taichui, 90(2.13%) to H. pumilio, 2(0.05%) to C. sinensis and 67 (1.59%) to unknown species due to the metacercariae being not yet developed or immature. The 2 C. sinensis metacercariae were obtained from 2 out of 45 fish examined. Our results contrast with reports of a decade ago which stated that all the fish of the Sun Moon lake examined were positive for C. sinensis. Possible reasons for the decrease of C. sinensis metacercariae are the disappearance of pig farms around the lake, increased awareness of the trematode by the lakeside inhabitants and probably the exclusive use of mammals as its definitive host by C. sinensis. In contrast, besides mammals, Haplorchis spp also use birds as their definitive hosts.

Application of hazard analysis critical control point (HACCP) as a possible control measure for Opisthorchis viverrini infection in cultured carp (Puntius gonionotus).

Opisthorchiasis due to Opisthorchis viverrini and transmitted through infected freshwater cyprinoid fish (carps) affects more than 8 million people in Thailand, People's Democratic Republic of Lao, and Vietnam. The Hazard Analysis Critical Control Point (HACCP)-concept has been recommended by FAO and WHO to be included in programs to control foodborne trematode infections (FBT). HACCP is a multifactorial approach to control food hazards through surveillance of diseases, foods, and operations and education. This study describes the first attempt to apply HACCP to the prevention and control of Opisthorchis viverrini in pond culture carp (Puntius gonionotus). The experiment was designed and carried out by a multidisciplinary "HACCP team" including experts in the field of public health, parasitology, epidemiology, aquaculture, fisheries extension and fish inspection. The investigation was performed in two fish ponds in the District of Sun Pa Tong, Chiang Mai, Thailand. In the experimental pond, fish was cultured according to HACCP principles and compared with the control pond, which followed conventional aquaculture practices. Water supply to the pond, fish fry, fish feed and pond conditions during the growing period were identified as critical control points (CCPs). Hazards were identified and analyzed, as well as control measures, critical limits, monitoring procedures, corrective actions, and record keeping developed for each one of the above CCPs. Complete pond preparation, particularly aiming to eliminate contamination of pond water with O. viverrini eggs, fish infected with parasite meacercariae and the first intermediate host (Bithynia spp), was conducted. After the pond was filled with water, O. viverrini metacercaria-free fry were released into the pond. The preliminary results obtained indicate that HACCP-based principles applied to carp pond culture could be used as a strategy to prevent and control O. viverrini. Further studies should be undertaken aiming to confirm these preliminary results.