Molecular Differentiation of Opisthorchis viverrini and Clonorchis sinensis Eggs by Multiplex Real-Time PCR with High Resolution Melting Analysis

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4+/-0.09degrees C and 85.9+/-0.08degrees C for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.

Field investigation of Bithynia funiculata, intermediate host of Opisthorchis viverrini in northern Thailand.

A survey of freshwater snail, Bithynia funiculata, was conducted in four locations, Doi Saket, Mueang, Saraphi and Mae Rim Districts of Chiang Mai Province, northern Thailand, between June and October, during the rainy season of 2004. A total of 2,240 snails was collected and classified into 7 families and 15 genera; of which 352 B. funiculata were obtained. B. funiculata was found most abundant in July and September. The infection rate of trematode cercariae in B. funiculata was 9.6% (19/352), while that of pleurolophocercous was 0.3% (1/352). Virgulate cercaria was the most common type, followed by lophocercous, monostome and pleurolophocercous. B. funiculata prefers habitats with clear water, which was less than 30 cm depth, temperatures between 24.48 and 31.78 degrees C, dissolved oxygen 2.03-7.66 mg/l, saturated dissolved oxygen 26.70-95.00%, conductivity 0.000-0.2642 mS/cm, turbidity 16.00-288.00 NTU and pH 6.58-7.56. Geographic Information System (GIS) analysis using loose soil mixed with clay revealed that the breeding grounds of this snail species was in the paddy fields and village environs of the Ping, Kuang and Fang river basins.

Methylene blue staining method for identification of Opisthorchis viverrini egg.

Methylene blue staining method was used to distinguish O. viverrini eggs from Haplorchis taichui and Prosthodendrium molenkampi eggs. All eggs were obtained from dissected adult worms, fixed in 10% formalin, and stained with methylene blue prior to light microscopy observation. The distinct musk-melon-like texture of the O. viverini eggshell surface and the thread-like texture of H. taichui eggshell surface were recognized, while P. molenkampi eggs showed a smooth eggshell. We also evaluated the sensitivity and specificity of the method by training investigators to differentiate surface textures. After training, the investigators were randomly tested with 10 slides containing fluke eggs. The sensitivity and specificity were 95% and 95%, respectively.

Opisthorchis viverrini: the effects of colchicine and cytochalasin B on the adult tegument.

The roles of the tegumental cytoskeleton were tested by treating adult flukes with colchicine and cytochalasin B. Following a short incubation period (10-20 minutes), colchicine disrupted microtubules in the tegumental cells' processes which, in turn, affected the transport of dense granules from the cells' soma to the tegument; as a result some of these granules were fused together to form membrane-bound vacuoles. In addition, at many spots microtrabeculae were also depolymerized, which resulted in the formation of non-membrane-bound vacuoles and the distension of microvilli to form blebs, some of which were disrupted. After prolonged incubation (120 minutes), general breakdown of the tegumental cytoskeleton occurred, and parts of it were sloughed off. In cytochalasin B treatment, the responses were similar to those of colchicine but with less severity. After a short incubation period (10-20 minutes), the microtrabeculae were depolymerized which led to the formation of non-membrane-bound vacuoles in the apical and middle zones of the tegument. Later, the tegumental microvilli were distended to form blebs but no evidence of tegumental sloughing occurred even in prolonged incubation. From these observations, it was concluded that microtubules played a role in the translocation of granules from the tegumental cells to the tegument which modulated the synthesis of membrane and glycocalyx, while microtrabeculae were involved in the maintenance of the structure and integrity of the tegument.

Opisthorchis viverrini: effect of praziquantel on the adult tegument.

Ultrastructural changes of the tegument of adult liver flukes, Opisthorchis viverrini, after in vitro incubation in Minimal Essential Medium containing 0, 0.1, 1.0 and 10.0 micrograms/ml of anthelminthic praziquantel for 5, 15, 30, 45 and 60 minutes were investigated by scanning (SEM) and transmission (TEM) electron microscopy. SEM observations showed that the surface damage was composed of blebbing due to the swelling of microvilli, followed later by the disruption of these structures to form lesions that caused the erosion and desquamation of the surface. Sensory papillae, by contrast, appeared relatively unaffected. The surface changes could be observed at all doses but the extent of damage increased with increasing duration of incubation and concentration of the drug. The ventral as well as the dorsal surfaces exhibited similar change, whereas the anterior part tended to be damaged less than the posterior part. Under TEM observations, the earliest sign of changes was the depolymerization of the microtrabecular network in scattered foci, which resulted in the formation of non-membrane-bound vacuoles under microvilli. The basal infoldings also became dilated, and some turned into membrane-bound vacuoles in the basal zone. Subsequently, microvilli became enlarged, and eventually formed blebs that later rupture to form lesion spots as observed in the SEM. Finally, the microtrabecular network in all regions broke down, creating vacuoles of various sizes throughout the tegument, leading to its total disintegration and detachment. The sequence of morphological changes was generally similar at all doses; however, the changes occurred faster at the higher doses and the longer incubation times. In addition, at the longer durations myofilaments in most muscle cells also became depolymerized, while microtubules were unchanged by the drug. Therefore, it is possible that praziquantel, through its induction of Ca2+ influx, causes depolymerization of the microtrabecular network that leads to the vacuolization, swelling, blebbing, and eventually the disruption and detachment of the tegument, and the breakdown of myofilaments in the muscle cells.