Seung Yull Cho

Foodborne Outbreaks of Human Toxoplasmosis

Two outbreaks of acute toxoplasmosis involving 8 adult patients in Korea were linked to eating uncooked pork. In the first outbreak, 3 patients developed unilateral chorioretinitis within 3 months of eating a meal consisting of raw spleen and liver of a wild pig. In the second outbreak, 5 of 11 soldiers who ate a meal consisting of raw liver of a domestic pig developed lymphadenopathy. All 8 patients had high levels of IgG Toxoplasma gondii antibodies (> or = 1:1024) in the Sabin-Feldman dye test, modified agglutination test incorporating mercaptoethanol, and latex agglutination test. T. gondii IgM antibodies persisted in these patients for several months. Most patients had a favorable response to anti-T. gondii chemotherapy with pyrimethamine and sulfanomides.

A Recombinant 10-kDa Protein of Taenia solium Metacestodes Specific to Active Neurocysticercosis

Neurocysticercosis (NCC) is an important cause of neurological disease worldwide. A 10-kDa antigen of Taenia solium metacestodes (TsMs) has been shown to be specific for immunodiagnosis of NCC. Screening of a TsM complementary DNA (cDNA) library isolated a cDNA encoding this protein. The cloned cDNA contained a 258-bp complete open-reading frame that encodes an 86-amino acid polypeptide with a calculated molecular weight of 9582 Da. It showed 73% homology with a 10-kDa antigen of T. crassiceps. The recombinant protein was expressed bacterially as a fusion protein at a high level. In immunoblot with recombinant protein, 97% (184/190) of sera from patients with active NCC showed strong reactivity, whereas 14% (4/29) of those from patients with chronic calcified NCC reacted weakly. In 180 sera from other patients with parasitic infections and from normal controls, it showed 98% specificity. A single recombinant TsM antigen has a high potential for serological differentiation of active NCC.

Excystment of Paragonimus westermani metacercariae by endogenous cysteine protease

To infect definitive or paratenic hosts, metacercariae of Paragonimus westermani should excyst in the host intestine. Optimum conditions for the excystment have been known to be pH 8-9 and a temperature of 40 C. Under these conditions, excystment of P. westermani metacercariae was accelerated in the presence of 1 mM dithiothreitol (DTT). The DTT acceleration was antagonized dose-dependently by cysteine protease inhibitors of L-trans-epoxysuccinylleucylamido(4-guanidino)butane (E-64, 2-20 microM) or leupeptin (0.1-1 mM), suggesting that certain cysteine proteases of the metacercaria are involved in excystment. Protease activities were detected in excretory-secretory products (ESP) of newly excysted metacercariae. Two distinct proteases were purified by DEAE anion-exchange chromatography of the ESP. While a 27-kDa protease exhibited endodipeptidolytic activity at pH 5-8.5 and remained stable at neutral pH for 3 days, the 28-kDa enzyme was stable at pH 5-7.5, with lower activity at pH 8.5. Both proteases hydrolyzed collagen, fibronectin, and myosin within 1 hr at pH 8. These results suggest that cysteine proteases secreted by P. westermani metacercariae modulate excystment.

Cleavage of immunoglobulin G by excretory–secretory cathepsin S-like protease of Spirometra mansoni plerocercoid

When immunoglobulin G (IgG) was incubated with Spirometra mansoni plerocercoid (sparganum), it was cleaved into Fab and Fc fragments. Fab/c fragments were also hydrolysed. The digestion was accelerated by dithiothreitol (DTT), indicating that cleavage of IgG heavy chain was due to a cysteine protease secreted into the medium. The responsible enzyme, of M(r) 27 (+/- 0.8) kDa, was purified by a series of thiopropyl affinity, Sephacryl S-300 HR and DEAE-anion exchange chromatographies, either from worm extracts or from excretory-secretory products (ESP). The purified, thiol-dependent protease showed an optimal activity at pH 5.7 with 0.1 M sodium acetate but was active over the pH range 4.5-8.0. Its activity was inhibited completely by 10(-5) M L-trans-epoxysuccinylleucylamido(4-guanidino) butane (E-64) and 1 mM iodoacetamide (IAA), but by only 53% using the specific cathepsin L inhibitor, Z-Phe-Phe-CHN2 (5 x 10(-5) M). Partial NH2-terminal amino acid sequence was Leu-Pro-Asp-Ser-Val-Asn-Trp-Arg-Glu-Gly-Ala-Val-Thr-Ala-Val which showed 80% homology to human cathepsin S. Immunoblot analysis showed that sera from infected patients exhibited IgE antibody reaction. It is proposed that cleavage of immunoglobulin by an excreted-secreted, cathepsin S-like, allergenic protease is a mechanism of immune evasion used by the sparganum.

Critical roles for excretory–secretory cysteine proteases during tissue invasion of Paragonimus westermani newly excysted metacercariae

Paragonimus westermani is a trematode parasite, which causes pulmonary and/or extrapulmonary granulomatous disease in humans. Successful invasion of the host tissue is critical for the survival of this tissue‐invasive parasite. The enzymatic hydrolysis of host proteins is clearly a prerequisite of this process. In this study, we have investigated the functional roles of the excretory–secretory cysteine proteases of P. westermani newly excysted metacercariae (PwNEM) in tissue invasion. The 27 and 28 kDa enzymes (PwMc27 and PwMc28) purified from PwNEM excretory–secretory products (ESP), preferentially degraded fibrillar proteins, but not globular proteins. PwMc28 significantly facilitated the invasion of PwNEM into mouse peritoneum, whereas a diffusible cysteine protease inhibitor, trans‐epoxysuccinyl‐l‐leuciloamido‐(4‐guanidino) butane (E‐64) inhibited this process dose‐dependently. Two distinct isoforms of PwMc28 (PwMc28a and PwMc28b), which exhibited two amino acid differences in their mature domains, were identified by tandem mass spectrometry and sequence analysis. Both enzymes were localized at the tegument on the anterior border and on the oral sucker, which suggests excretion–secretion via exocytosis or via the excretory canal network. The mRNA transcripts of PwMc28a and b were expressed abundantly during the active invasion/migration through the host’s tissues, suggesting their relevant function to tissue invasion/migration in the definitive host.

Hepatic visceral larva migrans of Toxocara canis: CT and sonographic findings.

OBJECTIVE The aim of this study was to describe the CT and sonographic findings of hepatic visceral larva migrans of Toxocara canis. MATERIALS AND METHODS Fifty-four patients (44 men, 10 women; age range, 30-80 years; mean age, 53 years) with serologically confirmed visceral larva migrans of Toxocara canis underwent evaluation of the liver with CT (n = 25), sonography (n = 48), or both. Two radiologists used consensus for retrospective evaluation of CT and sonographic findings. Correlation between the presence and severity of hepatic abnormalities on images and the degree of peripheral eosinophilia was assessed. RESULTS Seventeen (68%) of 25 patients who underwent CT had single or multiple ill-defined, oval or elongated, small, low-attenuating lesions in the liver. Eighteen (38%) of 48 patients who underwent sonography had single or multiple small, poorly defined, oval or elongated, hypoechoic scattered focal lesions in the liver. In the 19 patients who underwent both CT and sonography, the two techniques had no significant difference in rate of detection of hepatic lesions (p = 0.375, McNemar test). The lesion numbers on CT and sonography showed excellent linear correlation (r = 0.844, p = 0.001) by Pearsons correlation test. An independent samples t test showed that eosinophil count and percentage in the peripheral blood were significantly higher in patients with hepatic lesions on CT and sonography than in patients without lesions. CONCLUSION CT and sonographic findings of hepatic visceral larva migrans of T. canis are multiple, ill-defined, oval or elongated, small, nodular lesions scattered in the liver parenchyma. The presence of hepatic lesions on images was associated with higher peripheral eosinophil count and percentage.

Cerebral sparganosis : analysis of 34 cases with emphasis on CT features

SummaryCerebral sparganosis is a rare parasitic CNS disease, producing chronic active granulomatous inflammation. We retrospectively reviewed the clinical data, CT scans and histopathologic specimens in 34 patients with cerebral sparganosis. The majority of the patients (89%) were rural inhabitants; 75% had a history of ingestion of frogs and/or snakes. The major presenting symptoms were seizure (84%), hemiparesis (59%) and headache (56%) of chronic course. On CT scans, the disease most frequently involved the cerebral hemispheres, particularly frontoparietal lobes, with occasional extension to the external and internal capsules and basal ganglia. The cerebellum was rarely involved. Bilateral involvement was seen in 26%. The main CT findings consisted of white matter hypodensity with adjacent ventricular dilatation (88%), irregular or nodular enhancing lesion (88%), and small punctate calcifications (76%). In combination, the CT triad above appears to be specific for this disease, and was noted in 62% of cases. Of 16 follow-up CT scans, 5 (38%) showed a change in the location of the enhancing nodule. With a single CT scan, it does not appear to be possible to determine whether the worm is alive or dead, information important for deciding whether to intervene surgically. Change in the location of the enhancing nodule and/or worsening of the other CT findings on sequential CT scans would suggest that the worm is alive and that the patient is a candidate for surgery.

Effect of Control Strategies on Prevalence, Incidence and Re-infection of Clonorchiasis in Endemic Areas of China

Background A pilot clonorchiasis control project was implemented to evaluate the efficacies of various chemotherapy strategies on prevalence, incidence and re-infection in Heilongjiang Province, China. Methods and Findings Seven intervention groups (14,139 residents, about 2000 in each group) in heavily or moderately endemic areas were subjected to repeated praziquantel administration from 2001 to 2004. In the selective chemotherapy groups, residents were examined for fecal eggs, and those who tested positive were treated with three doses of 25 mg/kg praziquantel at 5-hour-intervals in one day. However, all residents were treated in the mass chemotherapy groups. In heavily endemic areas, two mass treatments of all residents in 2001 and 2003 reduced the prevalence from 69.5% to 18.8%, while four annual mass treatments reduced the prevalence from 48.0% in 2001 to 8.4% in 2004. Selective annual treatments for egg-positive subjects reduced the egg-positive rates from 54.9% in 2001 to 15.0% in 2004 or from 73.2% in 2001 to 12.3% in 2004. Selective treatments every 6 months significantly reduced the prevalence from 59.5% in 2001 to 7.5% in 2004. All of the repeated treatments reduced EPG (eggs per gram of feces) significantly. The annual mass treatment and selective treatment every 6 months produced lower prevalence and re-infection rates and higher egg reduction rate than annual selective treatments did. In the moderate endemic areas, egg positive rates were 24.8% and 29.7% in 2001 but were 1.9% and 1.3% after 2 or 3 selective treatments. The prevalence, incidence, re-infection rates in a moderately endemic area were significantly lower than those of heavy endemic areas. Conclusions Repeated mass treatment or selective treatment with praziquantel every 6 to 12 months is highly effective for clonorchiasis control in heavily endemic areas. In contrast, one or two selective treatments with health education is effective in moderately endemic areas.

Evaluation of Clonorchis sinensis Recombinant 7-Kilodalton Antigen for Serodiagnosis of Clonorchiasis

ABSTRACT The diagnostic applicability of the Clonorchis sinensis recombinant 7-kDa protein was evaluated. In enzyme-linked immunosorbent assays and immunoblots, the protein showed high sensitivities (81.3 and 71.9%, respectively) and specificities (92.6 and 89.7%, respectively) for sera obtained from various helminthic infections. Some paragonimiasis sera showed cross-reactions. The antigen might be valuable in the serodiagnosis of human clonorchiasis.

A cathepsin F of adult Clonorchis sinensis and its phylogenetic conservation in trematodes.

A novel 28 kDa cysteine protease (Cs28CF) secreted by the hepatobiliary trematode, Clonorchis sinensis was identified. The protease was purified from the excretory-secretory products (ESP) of the adult worm using DEAE-ion exchange and Arginine-Sepharose 4B chromatography. It showed a high activity between pH 6.5 and 7.5 in a dithiothreitol (DTT)-dependent manner. Inhibitors specific to cysteine proteases down-regulated the activity. Addition of Cs28CF to monkey cholangiocyte cultures resulted in approximately 95% cell death after 7 days. The full-length cDNA (1078 bp) encoded a single peptide of 328 amino acids (aa) with an N-terminal hydrophobic sequence, an ERFNAQ motif in the propeptide and a mature domain. Expression of mRNA transcripts of Cs28CF was observed in both the metacercaria and adult stages. Bacterially expressed recombinant protein exhibited a specific antibody reaction with clonorchiasis sera. Deduced aa exhibited 52-76% sequence identity with the cathepsin F analogues from other organisms. A novel E/DXGTA motif was recognized in the propeptide region. Phylogenetic analysis of 63 papain family members revealed that the trematode cysteine proteases formed 2 major clades of cathepsins F and L. The trematode cysteine proteases classified as cathepsin F shared higher homology among themselves than those classified as cathepsin L. Cathepsin F is phylogenetically conserved in the trematode parasites as well as in mammals.