A synthetic peptide was prepared based on the antigenic region of Paragonimus westermani pre-procathepsin L, and its applicability for immunodiagnosis for human paragonimiasis (due to Paragonimus heterotremus) was tested using an ELISA to detect IgG4 antibodies in the sera of patients. Sera from other helminthiases, tuberculosis, and healthy volunteers were used as the references. This peptide-based assay system gave sensitivity, specificity, accuracy, and positive and negative predictive values of 100%, 94.6%, 96.2%, 100%, and 88.9%, respectively. Cross reactivity was frequently seen against the sera of fascioliasis (75%) and hookworm infections (50%). Since differential diagnosis between paragonimiasis and fascioliasis can be easily done by clinical presentation and fascioliasis serology, this cross reaction is not a serious problem. Sera from patients with other parasitoses (0-25%) rarely responded to this synthetic antigen. This synthetic peptide antigen seems to be useful for development of a standardized diagnostic system for paragonimiasis.
Adult ; Animals ; Antibodies, Helminth/*blood ; Antigens, Helminth/*diagnostic use ; Clinical Laboratory Techniques/*methods ; Cross Reactions ; Enzyme-Linked Immunosorbent Assay/methods ; Humans ; Immunoglobulin G/*blood ; Paragonimiasis/*diagnosis ; Paragonimus/*immunology ; Parasitology/*methods ; Predictive Value of Tests ; Sensitivity and Specificity

Gnathostoma spinigerum can cause subarachnoid hemorrhage (SAH). The detection of specific antibodies in serum against G. spinigerum antigen is helpful for diagnosis of neurognathostomiasis. There is limited data on the frequency of G. spinigerum infection in non-traumatic SAH. A series of patients diagnosed as non-traumatic SAH at the Srinagarind Hospital, Khon Kaen University, Thailand between January 2011 and January 2013 were studied. CT or MR imaging of the brain was used for diagnosis of SAH. Patients were categorized as aneurysmal subarachnoid hemorrhage (A-SAH) or non-aneurysmal subarachnoid hemorrhage (NA-SAH) according to the results of cerebral angiograms. The presence of specific antibodies in serum against 21- or 24-kDa G. spinigerum antigen was determined using the immunoblot technique. The detection rate of antibodies was compared between the 2 groups. Of the 118 non-traumatic SAH patients for whom cerebral angiogram and immunoblot data were available, 80 (67.8%) patients had A-SAH, whereas 38 (32.2%) had NA-SAH. Overall, 23.7% were positive for specific antibodies against 21- and/or 24-kDa G. spinigerum antigen. No significant differences were found in the positive rate of specific antibodies against G. spinigerum in both groups (P-value=0.350).
Adult ; Aged ; Animals ; Antibodies, Helminth/*blood ; Antigens, Helminth/diagnostic use ; Brain/radiography ; Female ; Gnathostoma/immunology/*isolation & purification ; Gnathostomiasis/*diagnosis/*parasitology ; Humans ; Immunoblotting ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Serum/immunology ; Subarachnoid Hemorrhage/*diagnosis/*etiology ; Thailand ; Tomography, X-Ray Computed

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.
Adult ; Animals ; Antibodies, Helminth/blood ; Antigens, Helminth/*diagnostic use/genetics/isolation & purification ; Central Nervous System Parasitic Infections/*diagnosis/parasitology ; Gnathostoma/enzymology/immunology/*isolation & purification ; Gnathostomiasis/*diagnosis/parasitology ; Healthy Volunteers ; Humans ; Immunoblotting/methods ; Immunoglobulin G/blood ; Matrix Metalloproteinases/*diagnostic use/genetics/isolation & purification ; Parasitology/*methods ; Prospective Studies ; Recombinant Proteins/diagnostic use/genetics/isolation & purification ; Sensitivity and Specificity ; Serologic Tests/methods ; Thailand

The 2 principal species of hookworms infecting humans are Necator americanus and Ancylostoma duodenale. Case studies on zoonotic hookworm infections with Ancylostoma ceylanicum and/or Ancylostoma caninum are known mainly from Asian countries. Of these 2 zoonotic species, only A. ceylanicum can develop to adulthood in humans. In the present study, we report a molecular-based survey of human hookworm infections present in southern and northeastern Thailand. Thirty larval hookworm samples were obtained from fecal agar plate cultures of 10 patients in northeastren Thailand and 20 in southern Thailand. Partial ITS1, 5.8S, and ITS2 regions of the ribosomal DNA genes were amplified using PCR. The amplicons were sequenced, aligned, and compared with other hookworm sequences in GenBank database. The results showed that, in Thailand, N. americanus is more prevalent than Ancylostoma spp. and is found in both study areas. Sporadic cases of A. ceylanicum and A. duodenale infection were seen in northeastern Thailand.
Ancylostoma/classification/genetics/*isolation & purification ; Ancylostomiasis/*epidemiology ; Animals ; Cluster Analysis ; DNA, Ribosomal Spacer/chemistry/genetics ; Feces/parasitology ; Humans ; Molecular Sequence Data ; Necator americanus/classification/genetics/*isolation & purification ; Necatoriasis/*epidemiology ; Phylogeny ; Polymerase Chain Reaction ; Prevalence ; RNA, Ribosomal, 5.8S/genetics ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid ; Thailand/epidemiology

We compared the efficacy and applicability of a modified formalin-ether concentration technique (M-FECT) to the conventional FECT (C-FECT) and the agar plate culture (APC) method for the detection of Strongyloides stercoralis larvae. For this purpose, we used 600 human fecal specimens collected in an endemic area of southern Thailand. In the M-FECT, we used 2 layers of wire meshes, instead of gauze, to avoid the loss by absorption/adhesion of larvae to the gauze during filtration, and we reduced the exposure time of S. stercoralis larvae in stool samples to formalin. By such simple modifications, the efficacy of M-FECT has become comparable to APC and was much better than that of C-FECT for the diagnosis of strongyloidiasis.
Animals ; Ether ; Feces/parasitology ; Formaldehyde ; Humans ; Parasitology/*methods ; Specimen Handling/*methods ; Strongyloides stercoralis/*isolation & purification ; Strongyloidiasis/*diagnosis ; Thailand

A 52-year-old woman presented with lower back pain, progressive symmetrical paraparesis with sensory impairment, and sphincter disturbance. Magnetic resonance imaging (MRI) of the whole spine revealed multiple intradural extramedullary serpiginous-mass lesions in the subarachnoid space continuously from the prepontine to the anterior part of the medulla oblongata levels, C7, T2-T8, and T12 vertebral levels distally until the end of the theca sac and filling-in the right S1 neural foramen. Sparganosis was diagnosed by demonstration of the sparganum in histopathological sections of surgically resected tissues and also by the presence of serum IgG antibodies by ELISA. DNA was extracted from unstained tissue sections, and a partial fragment of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified using a primer set specific for Spirometra spp. cox1. After sequencing of the PCR-amplicon and alignment of the nucleotide sequence data, the causative agent was identified as the larva of Spirometra erinaceieuropaei.
Animals ; Antibodies, Helminth/blood ; Electron Transport Complex IV/genetics ; Enzyme-Linked Immunosorbent Assay ; Female ; Helminth Proteins/genetics ; Histocytochemistry ; Humans ; Immunoglobulin G/blood ; Magnetic Resonance Imaging ; Middle Aged ; Molecular Sequence Data ; Polyradiculopathy/*pathology ; Sequence Analysis, DNA ; Sparganosis/*diagnosis/*pathology ; Spine/radiography ; Spirometra/classification/genetics/*isolation & purification

Eosinophilic meningitis, caused by the nematode Angiostrongylus cantonensis, is prevalent in northeastern Thailand, most commonly in adults. Data regarding clinical manifestations of this condition in children is limited and may be different those in adults. A chart review was done on 19 eosinophilic meningitis patients aged less than 15 years in Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, Thailand. Clinical manifestations and outcomes were reported using descriptive statistics. All patients had presented with severe headache. Most patients were males, had fever, nausea or vomiting, stiffness of the neck, and a history of snail ingestion. Six patients had papilledema or cranial nerve palsies. It was shown that the clinical manifestations of eosinophilic meningitis due to A. cantonensis in children are different from those in adult patients. Fever, nausea, vomiting, hepatomegaly, neck stiffness, and cranial nerve palsies were all more common in children than in adults.
Adolescent ; Adult ; Aged ; Angiostrongylus cantonensis/*isolation & purification ; Animals ; Child ; Child, Preschool ; Eosinophilia/complications/etiology/*pathology ; Female ; Humans ; Male ; Meningitis/complications/etiology/*pathology ; Middle Aged ; Patient Outcome Assessment ; Strongylida Infections/parasitology/*pathology ; Thailand ; Young Adult

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.
Animals ; Asia ; Clonorchis sinensis/*classification/*genetics/isolation & purification ; Feces/parasitology ; Humans ; Multiplex Polymerase Chain Reaction/*methods ; NADH Dehydrogenase/genetics ; Opisthorchis/*classification/*genetics/isolation & purification ; Parasitology/*methods ; Real-Time Polymerase Chain Reaction/*methods ; Sensitivity and Specificity ; Transition Temperature ; Zygote

Paragonimiasis is an important food-borne parasitic zoonosis caused by infection with lung flukes of the genus Paragonimus. Of the 7 members of the genus known in Thailand until recently, only P. heterotremus has been confirmed as causing human disease. An 8th species, P. pseudoheterotremus, has recently been proposed from Thailand, and has been found in humans. Molecular data place this species as a sister species to P. heterotremus, and it is likely that P. pseudoheterotremus is not specifically distinct from P. heterotremus. In this study, we collected metacercariae of both nominal species (identification based on metacercarial morphology) from freshwater crabs from Phetchabun Province in northern Thailand, Saraburi Province in central Thailand, and Surat Thani Province in southern Thailand. In addition, we purchased freshwater crabs imported from Myanmar at Myawaddy Province, western Thailand, close to the Myanmar-Thailand border. The DNAs extracted from excysted metacercariae were PCR-amplified and sequenced for ITS2 and cox1 genes. The ITS2 sequences were nearly identical among all samples (99-100%). Phylogenies inferred from all available partial cox1 sequences contained several clusters. Sequences from Indian P. heterotremus formed a sister group to sequences from P. pseudoheterotremus-type metacercariae. Sequences of P. heterotremus from Thailand, Vietnam, and China formed a separate distinct clade. One metacercaria from Phitsanulok Province was distinct from all others. There is clearly considerable genetic variation in the P. heterotremus complex in Thailand and the form referred to as P. pseudoheterotremus is widely distributed in Thailand and the Thai-Myanmar border region.
Animals ; Cluster Analysis ; DNA, Ribosomal Spacer/chemistry/genetics ; Electron Transport Complex IV/genetics ; *Genetic Variation ; Metacercariae/genetics/isolation & purification ; Molecular Sequence Data ; Myanmar ; Paragonimus/*classification/*genetics/isolation & purification ; Phylogeny ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Sequence Homology ; Shellfish/parasitology ; Thailand

Lung fluke, Paragonimus heterotremus, is a flatworm causing pulmonary paragonimiasis in cats, dogs, and humans in Southeast Asia. We examined the ultrastructure of the testis of adult P. heterotremus with special attention to spermatogenesis and spermiogenesis using scanning and transmission electron microscopy. The full sequence of spermatogenesis and spermiogenesis, from the capsular basal lamina to the luminal surface, was demonstrated. The sequence comprises spermatogonia, spermatocytes with obvious nuclear synaptonemal complexes, spermatids, and eventual spermatozoa. Moreover, full steps of spermatid differentiation were shown which consisted of 1) early stage, 2) differentiation stage representing the flagella, intercentriolar body, basal body, striated rootlets, and electron dense nucleus of thread-like lamellar configuration, and 3) growing spermatid flagella. Detailed ultrastructure of 2 different types of spermatozoa was also shown in this study.
Animals ; Male ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Paragonimus/*physiology/*ultrastructure ; Spermatogenesis ; Spermatozoa/ultrastructure ; Testis/ultrastructure