Pewpan M. Intapan

The Opisthorchis viverrini genome provides insights into life in the bile duct

Opisthorchiasis is a neglected, tropical disease caused by the carcinogenic Asian liver fluke, Opisthorchis viverrini. This hepatobiliary disease is linked to malignant cancer (cholangiocarcinoma, CCA) and affects millions of people in Asia. No vaccine is available, and only one drug (praziquantel) is used against the parasite. Little is known about O. viverrini biology and the diseases that it causes. Here we characterize the draft genome (634.5 Mb) and transcriptomes of O. viverrini, elucidate how this fluke survives in the hostile environment within the bile duct and show that metabolic pathways in the parasite are highly adapted to a lipid-rich diet from bile and/or cholangiocytes. We also provide additional evidence that O. viverrini and other flukes secrete proteins that directly modulate host cell proliferation. Our molecular resources now underpin profound explorations of opisthorchiasis/CCA and the design of new interventions.

Comparison of the Quantitative Formalin Ethyl Acetate Concentration Technique and Agar Plate Culture for Diagnosis of Human Strongyloidiasis

ABSTRACT The quantitative formalin ethyl acetate concentration technique (QFEC) was compared to agar plate culture (APC) for the detection of Strongyloides stercoralis larvae. QFEC could substitute for APC only when the parasite load was higher than 50 larvae per g of stool. This study serves as a good reminder to those conducting stool exams about the sensitivity and specificity limitations of both techniques.

Detection of Opisthorchis viverrini in experimentally infected bithynid snails and cyprinoid fishes by a PCR-based method.

A PCR procedure for the detection of Opisthorchis viverrini in experimentally infected bithynid snails and cyprinoid fishes was developed. This procedure was based on primers designed from a pOV-A6 specific probe sequence giving a 330 base-pair product. The detection was accomplished in host tissue homogenates to which a single cercaria or metacercaria was introduced. PCR can detect as little as a single cercaria artificially inoculated in a snail or a single metacercaria artificially inoculated in a fish sample. The method gave a 100% positivity rate for all infected snails or fishes. The method did not yield a 330 base-pair amplified product with other digenean fluke DNAs such as Haplorchis taichui, Centrocestus spp., Echinostoma malayanum, Fasciola gigantica, animal schistosomes, Paragonimus heterotremus or Haplorchoides spp. The assay has great potential for application in epidemiological surveys of both snail and fish intermediate hosts as well as for investigation of foodborne parasites in freshwater fishes.

Immunoblot Diagnostic Test for Neurognathostomiasis

Neurognathostomiasis is a rare but severe form of human gnathostomiasis. Diagnosis of neurognathostomiasis is made presumably by using clinical manifestations. Serologic tests for neurognathostomiasis are not widely available and limited. We studied 12 patients with diagnoses of neurognathostomiasis at Srinagarind Hospital, Khon Kaen University, Thailand. There were three types of neurognathostomiasis (five patients with intracerebral hemorrhage, one patient with subarachnoid hemorrhage, and six patients with myelitis). All patients were tested for antibodies against Gnathostoma spinigerum by an immunoblotting technique. The sensitivity and specificity of the 21-kD and 24-kD diagnostic bands were 83.3% and 100%, and 91.7% and 100%, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for the 21-kD and 24-kD diagnostic bands were all 100%. Both diagnostic bands are a helpful diagnostic tool for neuro gnathostomiasis and show good diagnostic properties.

Clinical factors predictive of encephalitis caused by Angiostrongylus cantonensis.

Angiostrongylus cantonensis is mainly caused eosinophilic meningitis in humans, whereas a minority of patients develop encephalitic angiostrongyliasis (EA). EA is an extremely fatal condition, and the clinical factors predictive of EA have never been reported. A comparison study was conducted in a hospital situated in an endemic area of Thailand. We enrolled 14 and 80 angiostrongyliasis patients who developed encephalitis and meningitis, respectively. Logistic regression analysis was used to assess the clinical variables predictive of encephalitis. Age (adjusted odds ratio [OR], 1.22; 95% confidence interval [CI], 1.05-1.42), duration of headache (adjusted OR, 1.26; 95% CI, 1.03-1.55), and fever > 38.0 degrees C (adjusted OR, 37.05; 95% CI, 1.59-862.35) were identified as statistically significant factors for EA prediction. Elderly patients with angiostrongyliasis experiencing fever and prolonged headaches were at the highest risk of developing EA.

Cerebrospinal fluid cytokine responses in human eosinophilic meningitis associated with angiostrongyliasis

The levels of interleukin 5 (IL5), IL10, and IL13 in the cerebrospinal fluid (CSF) were markedly higher in 30 patients with eosinophilic meningitis associated with angiostrongyliasis (EOMA) than in the controls (P<0.001). IL2, IL4, interferon gamma (IFNgamma), and tumor necrosis factor alpha (TNFalpha) levels were not significantly different (P>0.05). IL5, IL10, and TNFalpha levels correlated with eosinophil levels (P=0.023, P=0.018, and P=0.005, respectively) while IL2, IL4, IL13, and IFNgamma did not (P>0.05). Our data suggest that local T-helper-2 (TH2) cytokine responses are predominant in the CSF of patients with EOMA. Data on T lymphocyte-parasite interactions are important for the design of effective vaccines and immunotherapies. The measurement of T-helper-1 (TH1)/TH2 cytokines in the CSF may also have some potential for the diagnosis of parasite associated meningitis.

Factors Affecting Recovery of Strongyloides stercoralis Larvae: an Approach to a Newly Modified Formalin-Ether Concentration Technique for Diagnosis of Strongyloidiasis

ABSTRACT To improve the diagnosis efficiency of human strongyloidiasis by using formalin-ether concentration technique (FECT), the effects of various factors on the recovery rates of Strongyloides stercoralis larvae were comparatively evaluated. Fresh stool and a short time exposure of larvae to formalin yielded significantly higher numbers of larvae than preserved stool and 10-min exposure. Likewise, straining through wire mesh yielded a significantly higher number of larvae recovered than straining through gauze did. In addition, centrifugation for 5 min for separation of larvae from debris yielded a significantly greater number of larvae recovered than centrifugation for 2 min did. The efficacies of the five versions of FECT with different factors-FECT 1, FECT 2, FECT 3, FECT 4, and FECT 5-were then compared. It was found that FECT 5 was 1.8, 2.0, 1.9, and 1.4 times more effective than FECT 1, FECT 2, FECT 3, and FECT 4, respectively. FECT 5 is a modified FECT method, whose modifications included using fresh stool without a preservative substance; a short-time rather than 10-min formalin exposure; and the use of wire mesh instead of gauze.

Sequential imaging studies of cerebral gnathostomiasis with subdural hemorrhage as its complication

Cerebral gnathostomiasis is a severe form of human infection caused by Gnathostoma spinigerum. We report sequential brain imaging in serologically proven cerebral gnathostomiasis. The clinical and radiographic correlation showed the probable route taken by the parasite during its migration through the central nervous system. We offer additional insight into pathogenesis of cerebral gnathostomiasis and its rare complication, subdural hemorrhage, a finding not previously reported in the literature.

Detection of Paragonimus heterotremus Eggs in Experimentally Infected Cats by a Polymerase Chain Reaction–Based Method

A polymerase chain reaction (PCR) procedure for the detection of Paragonimus heterotremus eggs in stool samples was developed and compared with Stolls egg count method. The primers were designed on the basis of a previously constructed pPH-13–specific DNA probe, which produced an approximate 0.5-kb amplified product. This PCR method could detect as few as 5 eggs in 0.6 g of artificially inoculated feces of a healthy control cat or as little as 1 × 10−4 ng of P. heterotremus genomic DNA. The assay had 100% sensitivity in all infected cats. The method did not yield an approximate 0.5-kb product with DNA from other parasites such as Gnathostoma spinigerum, Trichinella spiralis, Fasciola gigantica, Echinostoma malayanum, Opisthorchis viverrini, Dirofilaria immitis, and Taenia saginata; exceptions were Paragonimus siamensis and Paragonimus westermani. In addition, no genomic DNA from Escherichia coli, Burkholderia pseudomallei, Acinetobacter anitratus, Mycobacterium tuberculosis, Staphylococcus aureus, β-Streptococcus grA, and Proteus mirabilis or from the vertebrate and invertebrate hosts of P. heterotremus was amplified in the PCR assay. This assay has great potential for application in clinical epidemiological studies.

Evaluation of Immunoglobulin G Subclass Antibodies against Recombinant Fasciola gigantica Cathepsin L1 in an Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Human Fasciolosis

ABSTRACT A cystatin capture enzyme-linked immunosorbent assay (ELISA) using recombinant Fasciola gigantica cathepsin L1 antigen was developed to detect specific immunoglobulin G (IgG) subclass antibodies (IgG1, IgG2, IgG3, and IgG4) and was evaluated for its diagnostic potential for human fasciolosis. In an analysis of the sera of 13 patients infected with F. gigantica, 209 patients with other parasitic infections, 32 cholangiocarcinoma patients, and 42 healthy controls, the IgG4-ELISA gave the highest diagnostic values. The sensitivity, specificity, accuracy, and positive and negative predictive values of this method based on the detection of IgG4 antibody were 100%, 99.3%, 99.3%, 86.7%, and 100%, respectively. The results revealed that restricting the ELISA to the detection of specific IgG4 antibody enhanced the specificity and accuracy for the serodiagnosis of human fasciolosis.