A filtration-based rapid test using a partially purified third-stage larval antigen to detect specific antibodies for the diagnosis of gnathostomiasis.

Human gnathostomiasis is an emerging food-borne parasitic disease caused by nematodes of the genus Gnathostoma. Currently, serological tests are commonly applied to support clinical diagnosis. In the present study, a simple and rapid filtration-based test, dot immune-gold filtration assay (DIGFA) was developed using a partially purified antigen of Gnathostoma third-stage larvae (L3). A total of 180 serum samples were tested to evaluate the diagnostic potential of DIGFA for gnathostomiasis. The diagnostic sensitivity and specificity were 96.7% (29/30) and 100% (25/25), respectively. The cross-reactivity with sera from other helminthiasis patients ranged from 0 to 4%, with an average of 1.6% (2/125). DIGFA using a partially purified L3 antigen was not only simple and rapid, but also more accurate than standard assays for the diagnosis of human gnathostomiasis. DIGFA may represent a promising tool for application in laboratories or in the field, without requiring any instrumentation.

Angiostrongylus mackerrasae and A. cantonensis (Nematoda: Metastrongyloidea) belong to same genetic lineage: evidence from mitochondrial protein-coding genes.

Angiostrongylus mackerrasae is a parasitic nematode of rats found in Australia. When first reported, it was referred to as A. cantonensis. Recent molecular studies, including the mitochondrial genome, indicate that it is highly similar to A. cantonensis. These studies did not include A. malaysiensis, another member of the A. cantonensis species complex, for comparison. The present study examined the genetic distance and phylogenetic relationship between the component taxa (A. cantonensis, A. mackerrasae and A. malaysiensis) of the A. cantonensis species complex, based on the 12 protein-coding genes (PCGs) of their mitochondrial genome. Both the nucleotide and amino acid sequences were analysed. Angiostrongylus mackerrasae and A. cantonensis are members of the same genetic lineage and both are genetically distinct from A. malaysiensis. The genetic distance based on concatenated nucleotide sequences of 12 mt-PCGs between A. mackerrasae and A. cantonensis from Thailand is p = 1.73%, while that between the Thai and Chinese taxa of A. cantonensis is p = 3.52%; the genetic distance between A. mackerrasae and A. cantonensis from China is p = 3.70%. The results indicate that A. mackerrasae and A. cantonensis belong to the same genetic lineage, and that A. mackerrasae may be conspecific with A. cantonensis. It remains to be resolved whether A. mackerrasae is conspecific with A. cantonensis or undergoing incipient speciation.

Cytochrome c oxidase subunit I haplotype reveals high genetic diversity of Angiostrongylus malaysiensis (Nematoda: Angiostrongylidae).

The rat lungworm Angiostrongylus malaysiensis is a metastrongyloid nematode parasite. It has been reported in Malaysia, Thailand, Laos, Myanmar, Indonesia and Japan. In this study, A. malaysiensis adult worms recovered from the lungs of wild rats in different geographical regions/provinces in Thailand were used to determine their haplotype by means of the mitochondrial partial cytochrome c oxidase subunit I (COI) gene sequence. The results revealed high COI haplotype diversity of A. malaysiensis from Thailand. The geographical isolates of A. malaysiensis from Thailand and other countries formed a monophyletic clade distinct from the closely related A. cantonensis. In the present study, five new haplotypes were identified in addition to the four haplotypes reported in the literature. Phylogenetic analysis revealed that four of these five new haplotypes - one from Mae Hong Song (northern region), two from Tak (western region) and one from Phang Nga (southern region) - formed a distinct clade with those from Phatthalung (southern region) and Malaysia. The haplotype from Malaysia was identical to that of Phatthalung (haplotype AM1). In general, the COI sequences did not differentiate unambiguously the various geographical isolates of A. malaysiensis. This study has confirmed the presence of high COI genetic diversity in various geographical isolates of A. malaysiensis. The COI gene sequence will be suitable for studying genetic diversity, population structure and phylogeography.

Genetic diversity of infective larvae of Gnathostoma spinigerum (Nematoda: Gnathostomatidae) in freshwater swamp eels from Thailand.

Human gnathostomiasis is a food-borne zoonosis caused by a tissue nematode of the genus Gnathostoma. The disease is highly endemic in Asia, including Thailand. The freshwater swamp eel (Monopterus albus), the second intermediate host of the gnathostome nematode, has an important role in transmitting the infection in Thailand. Surveys on the infective larvae of Gnathostoma spinigerum based on morphological features in freshwater swamp eels have been performed continuously and reported in Thailand. However, there is still limited molecular data on intra-species variations of the parasite. In this study, a total of 19 third-stage larvae of morphologically identified G. spinigerum were collected from 437 liver samples of freshwater swamp eels purchased from a large wholesale market in Bangkok, Thailand. Molecular characterization based on mitochondrial cytochrome c oxidase subunit I (COI) sequences was performed to elucidate their genetic variations and phylogenetic relationship. Among the 19 infective larvae recovered from these eels, 16 were sequenced successfully. Phylogenetic analyses inferred from the partial COI gene showed the presence of three distinct COI haplotypes. Our findings confirm the presence of G. spinigerum as the main species in Thailand.

Phylogenetics and systematics of Angiostrongylus lungworms and related taxa (Nematoda: Metastrongyloidea) inferred from the nuclear small subunit (SSU) ribosomal DNA sequences.

The Angiostrongylus lungworms are of public health and veterinary concern in many countries. At the family level, the Angiostrongylus lungworms have been included in the family Angiostrongylidae or the family Metastrongylidae. The present study was undertaken to determine the usefulness and suitability of the nuclear 18S (small subunit, SSU) rDNA sequences for differentiating various taxa of the genus Angiostrongylus, as well as to determine the systematics and phylogenetic relationship of Angiostrongylus species and other metastrongyloid taxa. This study revealed six 18S (SSU) haplotypes in A. cantonensis, indicating considerable genetic diversity. The uncorrected pairwise 'p' distances among A. cantonensis ranged from 0 to 0.86%. The 18S (SSU) rDNA sequences unequivocally distinguished the five Angiostrongylus species, confirmed the close relationship of A. cantonensis and A. malaysiensis and that of A. costaricensis and A. dujardini, and were consistent with the family status of Angiostrongylidae and Metastrongylidae. In all cases, the congeneric metastrongyloid species clustered together. There was no supporting evidence to include the genus Skrjabingylus as a member of Metastrongylidae. The genera Aelurostrongylus and Didelphostrongylus were not recovered with Angiostrongylus, indicating polyphyly of the Angiostrongylidae. Of the currently recognized families of Metastrongyloidea, only Crenosomatidae appeared to be monophyletic. In view of the unsettled questions regarding the phylogenetic relationships of various taxa of the metastrongyloid worms, further analyses using more markers and more taxa are warranted.

Dot immunogold filtration assay (DIGFA) for the rapid detection of specific antibodies against the rat lungworm Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) using purified 31-kDa antigen.

A rapid dot immunogold filtration assay (DIGFA) was adopted for specific immunodiagnosis of human cerebral angiostrongyliasis, using purified 31-kDa glycoprotein specific to Angiostrongylus cantonensis as diagnostic antigen and protein A colloidal gold conjugate as antigen-antibody detector. A total of 59 serum samples were assayed - 11 samples from clinically diagnosed patients with detectable A. cantonensis-specific antibody in immunoblotting; 23 samples from patients with other related parasitic diseases, i.e. gnathostomiasis (n= 8), cysticercosis (n= 5), toxocariasis (n= 2), filariasis (n= 4), paragonimiasis (n= 2) and malaria (n= 2); and 25 samples from normal healthy subjects. The sensitivity and specificity of DIGFA to detect anti-A. cantonensis specific antibodies in serologically confirmed angiostrongyliasis cases, were both 100%. No positive DIGFA was observed in cases with other parasitic diseases, and the healthy control subjects. The 3-min DIGFA is as sensitive and specific as the 3-h immunoblot test in angiostrongyliasis confirmed cases that revealed a 31-kDa reactive band. The gold-based DIGFA is more rapid and easier to perform than the traditional enzyme-linked immunosorbent assay (ELISA). The test utilizing purified A. cantonensis antigen is reliable and reproducible for specific immunodiagnosis of human infection with A. cantonensis - thus can be applied as an additional routine test for clinical diagnostic support. Large-scale sero-epidemiological studies in endemic communities in north-east Thailand are under way to evaluate its usefulness under field conditions.

Needlescopy-assisted resection of pulmonary nodule after dual localisation.

The aim of this study was to evaluate the feasibility of dual localisation with hookwire and lipiodol before needlescopy-assisted resection for pulmonary nodule. Computed tomography-guided dual marking was performed on 36 pulmonary nodules of 32 patients and needlescopy-assisted resection was performed monitored by C-arm fluoroscopy. The mean age of the patients was 58 ± 12 (range 12-77) yrs. The mean size of the nodules was 7.5 ± 3.7 (3-17) mm. Their mean distance from the pleural surface was 7.3 ± 7.5 (0-35) mm. There were nine pure ground-glass opacity lesions, five semi-solid lesions and 22 solid lesions. The time of the dual localisation procedure was 13.1 ± 4.8 (7-23) min. Complications of the marking were pneumothorax in nine patients, and intrapulmonary bleeding in three. One hookwire dislodged during the operation. All nodules were successfully resected under needlescopy without conversion to a conventional thoracoscopy (5 mm or 10 mm thoracoscopy) or a minithoracotomy. There was no complication related to needlescopy-assisted resection. Dual marking with hookwire and lipiodol is a safe and none time consuming procedure, and needlescopy-assisted lung resection for small nodules is technically feasible and useful for histological diagnosis and treatment.

Genetic variation of NADH dehydrogenase subunit 1 (nad1) mitochondrial gene sequence in adult Necator americanus hookworms recovered from a female patient in Thailand.

Two female and one male adult hookworms were recovered from a female patient in Thailand. Based on gross and microscopic morphology, the three hookworms are members of Necator americanus. Phylogenetic reconstruction based on partial NADH dehydrogenase subunit 1 (nad1) mitochondrial gene sequences shows that these hookworms belong to the same genetic lineage as N. americanus adult worm from Zhejiang, China. The male and female hookworms were genetically distinct, belonging to two different nad1-haplotypes. This is the first report targeting the nad1 gene on the identification and genetic characterization of the human hookworms originated from infected patient. The nad1 gene marker is useful for species and higher taxa differentiation of hookworms.

Genetic differentiation of Anisakis species (Nematoda: Anisakidae) in marine fish Priacanthus tayenus from Gulf of Thailand.

Members of the genus Anisakis are parasitic nematodes of the family Anisakidae. They are potential zoonotic parasites, causing anisakiasis in humans who consume raw or undercooked seafood (fish or squid) infected with the larvae of this nematode. In the present study, anisakid nematodes collected from the marine fish Priacanthus tayenus (Purplespotted big-eye) caught from the Gulf of Thailand were examined morphologically and characterized genetically by DNA sequence analysis. Sequence data from the mitochondrial cytochrome c oxidase subunit II (mtDNA cox2) gene were used to identify these nematodes to species level and to evaluate the phylogenetic relationship among various taxa. All the 15 third-stage larvae of Anisakis nematodes investigated in this study belonged to the same genetic lineage as the A. typica species complex (named here as A. typica sp. T - T for Thailand). Eight mtDNA cox2 haplotypes were revealed in the 15 isolates of this Anisakis from Thailand. The mtDNA cox2 haplotypes of A. typica sp. T from Thailand were genetically distinct from those of the A. typica sensu stricto. Taxonomic description of this A. typica sp. T as a distinct species however awaits the availability of adult specimens.

Restriction enzyme digestion analysis of PCR-amplified DNA of Blastocystis hominis isolates.

Genomic DNA of Blastocystis isolates released into 0.1% Triton X-100 was suitable for amplification and yielded similar results as the genomic DNA extracted with standard kit. The specific B. hominis primers (BH1: GCT TAT CTG GTT GAT CCT GCC AGT and BH2: TGA TCC TTC CGC AGG TTC ACC TAC A) successfully produced the PCR product of about 1,770 bp with all the 7 Blastocystis isolates tested. The restriction fragment length polymorphism (RFLP) patterns yielded by 13 out of 25 restriction endonucleases showed that the 7 isolates could be grouped into 4 subgroups: subgroup-1 consisted of isolate C; subgroup-2 of isolates H4 and H7; subgroup-3 of isolates KP1, Y51 and M12; and subgroup-4 of isolate 27805. The differences between subgroups manifested as clear-cut RFLP patterns. A common band of 230 bp was revealed by Eco R1 in all the Blastocystis isolates tested. The band of about 180 bp was revealed by Alu I, differentiated symptomatic from asymptomatic isolates of this parasite, and might indicate the pathogenicity of this parasite.

The Climate Range Expansion of Aedes albopictus (Diptera: Culicidae) in Asia Inferred From the Distribution of Albopictus Subgroup Species of Aedes (Stegomyia).

We compared climatic distribution ranges between Aedes albopictus (Skuse) (Diptera: Culicidae) and the five wild (nondomesticated) species of Albopictus Subgroup of Scutellaris Group of Aedes (Stegomyia) in southern Asia. Distribution sites of the wild species concentrate in seasonal forest and savannah climate zones in India, Indochina, and southern China. The distribution of Ae. albopictus is broader than the wild species under 1) tropical rain-forest climate, 2) steppe and temperate savannah climate, and 3) continental climate with large seasonal temperature variation (hot summer and cold winter) at temperate lowlands (northernmost sites 40°N in Ae. albopictus vs 32°N in the wild species). However, the distribution of Ae. albopictus is more limited at tropical and subtropical highlands where the climate is cool but less continental (small seasonal variation, mild summer, and winter). We discuss a possibility that the broader climate ranges of Ae. albopictus are ecological or eco-evolutionary consequences of adaptation to human habitats. We also propose a general scenario for the origin, dispersal, and adaptation of Ae. albopictus in Asia as a hypothesis for future research.

Molecular identification of Gnathostoma spinigerum (Nematoda: Gnathostomatidae) as causative agent of human gnathostomiasis in Thailand.

A 43-year-old male residing in Prachin Buri Province, Thailand, was admitted to the Siriraj University Hospital of Mahidol University, Thailand, in July 2014 with right eyelid swelling and serpiginous lesion for three weeks. A nematode specimen was accidentally recovered from his upper right eyelid area. The body of the worm was cylindrical and measured 11.0 × 1.4 mm. The head bulb had eight circles of transverse spines. Anterior half of the body was covered with rows of comb-like pointed spines. The tail part was rounded dorsally and flattened ventrally and no copulatory spicule was observed at the posterior end. It was morphologically identified as an immature female worm of gnathostome species. Sequence analysis for partial cytochrome c oxidase subunit I (COI) gene revealed this recovered nematode as Gnathostoma spinigerum.

Geographical distribution and incidence of Angiostrongylus lungworms (Nematoda: Angiostrongylidae) and their rodent hosts in Thailand.

The rat lungworm Angiostrongylus cantonensis, a zoonotic parasite, is known to be responsible for eosinophilic meningitis and meningoencephalitis in humans in many countries worldwide. Another congener A. malaysiensis is a potential pathogen. Rodents as natural definitive host of the parasites are abundant and globally widespread. In this study, the prevalence of Angiostrongylus infection in wild rats was investigated in twenty-four provinces of Thailand during the period December 2011 to June 2014. Of the 669 wild rats sampled, 46 (6.88%) were infected with Angiostrongylus lungworms. The rodents harbouring A. cantonensis worms included Bandicota indica, Bandicota savilei, Rattus exulans, Rattus norvegicus, Rattus rattus complex and Rattus tiomanicus, and those harbouring A. malaysiensis were B. savilei, Rattus losea, R. norvegicus and R. rattus complex. No parasite was recovered from Maxomys surifer (n=11), Mus musculus (n=1), Niviventer fulvescens (n=2), Rattus argentiventer (n=4), Rattus nitidus (n=3) and Sundamys muelleri (n=3). In positive rats, the incidence of infection with Angiostrongylus lungworms was variable among host species and provinces. There were also considerable variation in the proportion of male and female worms among rodent hosts and localities. Two hundred and thirty-five of the collected worms were male and 282 were female. The mean worm burden in the positive rats was 11.24 and ranged from 1 to 61. 81.82% (423/517) of the adult worms were morphologically identified as A. cantonensis, and 18.18% (94/517) were A malaysiensis. One R. rattus from Prachuap Khiri Khan had mixed infection of A. cantonensis and A. malaysiensis (10 worms of each species). The overall number of male (202) and female (221) A. cantonensis worms was not significantly different (χ2 = 0.86, 0.50 > P > 0.30). However, the overall number of male (33) and female (61) A. malaysiensis worms was significantly different (χ2 = 8.34, P < 0.01). The present study added one new definitive host (R. tiomanicus) for A. cantonensis and two new definitive hosts (B. savilei and R. losea) for A. malaysiensis in Thailand. Our data update and contribute significantly to existing knowledge of the geographical distribution of A. cantonensis in wild rats in Thailand and confirm the occurrence of A. malaysiensis throughout the country.

Double cancer of the stomach and oesophagus with situs ambiguus with polysplenia: the importance of preoperative evaluation.

A rare case of double cancer with situs ambiguus with polysplenia is presented. A 58-year-old patient was initially diagnosed with an early gastric cancer. On evaluation, the computed tomography of the abdomen demonstrated situs ambiguus with polysplenia. We performed a subtotal gastrectomy with the stomach being reconstructed in a Billroth-II fashion. Three months after the operation, he again visited our department complaining nausea and dysphagia. Examinations confirmed the other oesophageal malignancy with advanced stage. Because of unfamiliarity to situs anomaly and rarity of double cancer, we missed the other coexistent cancer. This is the first case presentation of a double carcinoma occurring in a patient with situs ambiguus with polysplenia. The literature is reviewed and the importance of preoperative evaluation is discussed.

Genetic variation of NADH dehydrogenase subunit 1 (nad1) mitochondrial gene sequence in adult Necator americanus hookworms recovered from a female patient in Thailand

@#Two female and one male adult hookworms were recovered from a female patient in Thailand. Based on gross and microscopic morphology, the three hookworms are members of Necator americanus. Phylogenetic reconstruction based on partial NADH dehydrogenase subunit 1 (nad1) mitochondrial gene sequences shows that these hookworms belong to the same genetic lineage as N. americanus adult worm from Zhejiang, China. The male and female hookworms were genetically distinct, belonging to two different nad1-haplotypes. This is the first report targeting the nad1 gene on the identification and genetic characterization of the human hookworms originated from infected patient. The nad1 gene marker is useful for species and higher taxa differentiation of hookworms.

Evaluation of dot immunogold filtration assay (DIGFA) for rapid serodiagnosis of eosinophilic meningitis due to Angio-strongylus cantonensis (Nematoda: Metastrongyloidea).

Angiostrongylus cantonensis is the most frequent cause of eosinophilic meningitis in humans in Thailand and worldwide. Because of difficulty of recovering the Angiostrongylus larvae from infected patients, detection of parasite-specific antibodies is used to support clinical diagnosis. This study tested serum samples from eosinophilic meningitis patients and individuals at risk of infection with A. cantonensis to evaluate a recently developed simple and rapid dot-immunogold filtration assay (DIGFA) for detection of specific antibodies against A. cantonensis. Purified 31-kDa glycoprotein of A. cantonensis and protein A colloidal gold conjugate were employed to detect the 31-kDa anti-A. cantonensis antibody in patients sera from the parasite endemic areas of northeast Thailand. The results were compared with those obtained by dot-blot enzyme-linked immunosorbent assay (ELISA) with 31-kDa A. cantonensis antigen. The overall positivity rate of DIGFA and dot-blot ELISA for A. cantonensis infection in 98 clinically diagnosed cases from three highly endemic districts in Khon Kaen province were 39.79% and 37.75%, respectively. Among 86 sera of subjects at risk of infection with A. cantonensis, 24.41% were positive by DIGFA and 23.25% by dot-blot ELISA. There were good correlation between the visual grading of DIGFA and dot-blot ELISA in both groups of defined sera. DIGFA is as sensitive and specific as dot-blot ELISA for confirming eosinophilic meningitis due to A. cantonensis infection, with advantages of simplicity, rapidity and without the use of specific and expensive equipment, and can be used in field settings.

Diffuse cystitis glandularis. Associated with adenocarcinomatous change.

Although cystitis glandularis has been considered a premalignant lesion, the instance of cystitis glandularis progression to adenocarcinoma or cystitis glandularis associated with adenocarcinomatous change is rare. This article includes 4 cases of neurogenic bladder with urinary diversion for different periods of time. In the first case with ureterocutaneostomy for twenty-five years diffuse cystitis glandularis with multifocal adneocarcinomatous change developed. The second case with suprapubic cystostomy for twenty-two years had diffuse cystitis glandularis of gastrointestinal type without evidence of malignancy. The other 2 cases with suprapubic cystostomy for merely ten years showed only mild to moderate cystitis glandularis and chronic cystitis with squamous metaplasia, respectively. The extent of cystitis glandularis appeared to correlate with the duration of urinary stasis. Ureterocutaneostomy rendered constant infection of the urinary bladder of the first case because of inadequate drainage. Thus, we assume that the intensity of the infection with a toxic product and virulence of organism may be responsible for the development of adenocarcinoma in this patient. Cystitis glandularis, especially diffuse type, can undergo malignant degeneration under constant irritation, but it is a long-term process.

Aquatic arthropod communities in Nepenthes pitchers: the role of niche differentiation, aggregation, predation and competition in community organization.

The structure and organization of aquatic arthropod communities in Nepenthes ampullaria pitchers were studied at two sites (M in Malacca and K in Kuching) in Malaysia. The communities consisted mainly of aquatic dipteran larvae. Community M was dominated by a filter feeder, Tripteroides tenax, which reached a high density despite a strongly aggregated distribution. Community K had five trophic groups: carrion feeders, filter feeders, detritus feeders, nipping predators and hooking predators, each including multiple species. The summed density of filter feeders in Community K remained much below the level attained by filter feeders in Community M. Niche differentiation within each trophic group with regard to pitcher age and feeding behaviour was not sufficient to allow species coexistence through niche separation alone. Aggregated distributions directly reduced interspecific encounters. Nevertheless, species belonging to the same trophic group commonly shared the same pitcher, because of high occurrence probabilities of dominant species and positive associations between some taxa (due mainly to similar occupancies by pitcher age). Predator coexistence in Community K may have been facilitated by self-limitation of the large predators through intraspecific cannibalism strengthened by aggregation. Prey coexistence, on the other hand, may have relied more on population suppression by predation, especially the selective removal of old instar Tripteroides.

Synlophe of Batrachonema synaptospicula (Nematoda:Amphibiophilidae) collected from Malaysian frogs.

The synlophe of Batrachonema synaptospicula Yuen, 1965 collected from Rana limnocharis Boie, 1835 of peninsular Malaysia was found to be identical morphologically to that in the specimens from Rana narina Stejneger, 1901 of Okinawa, and R. limnocharis of Taiwan. In the midbody, 20-22 ridges are present, and the ridges increase gradually in size and are oriented from right to left in the dorsal and left ventral fields, whereas the right ventral ridges are small and almost perpendicular to the body wall. The orientation of ridges from right to left is considered to be a key characteristic of the genus Batrachonema. Because Amphibiophilus ranae Wang et al., 1978 and Amphibiophilus sp. from R. limnocharis of south China are regarded to be conspecific with B. synaptospicula, this nematode is surmised to be distributed widely in southeast and east Asia.

Schistosoma malayensis n. sp.: a Schistosoma japonicum-complex schistosome from Peninsular Malaysia.

Schistosoma malayensis n. sp., a member of the Schistosoma japonicum complex is described from Rattus muelleri in Peninsular Malaysia and 2 strains are characterized. The only morphological differences noted among adults from natural hosts were that S. malayensis are in general smaller than S. mekongi and S. japonicum. But these differences may be the result of host-induced variations and therefore are of little taxonomic value. To minimize the effects of host-induced variations, adult worms recovered from laboratory mice with similar worm burdens at 50-56 days postinfection were compared. These comparisons revealed only minor morphometric differences among these 3 species. Schistosoma malayensis eggs from naturally and experimentally infected hosts are most similar to those of S. mekongi, with eggs of both species being, in general, smaller than those of S. japonicum. The egg index for S. malayensis is usually higher than for S. japonicum and lower than for S. mekongi. Differences were noted in the developmental rates in mice for 2 isolates of S. malayensis, S. mekongi, and S. japonicum (Philippine strain), but relatively large differences observed between isolates of S. malayensis indicate that, in this case, the developmental rate is not a useful taxonomic character. Schistosoma malayensis is erected principally on the basis of differences, reported elsewhere, in the life histories and in the electrophoretic migration patterns of isoenzymes of adult worms as compared to S. mekongi and S. japonicum. These comparisons indicate that S. malayensis is more closely related to S. mekongi than to S. japonicum.