• View in gallery

    Immunoblot analysis of recombinant Fasciola gigantica cathepsin L1 fused with calmodulin binding peptide. The blotted membrane reacted with pooled negative reference sera (lanes A-D) and pooled positive reference sera (lanes E-H). Lanes A and E, solubilized S1 fraction; lanes B and F, solubilized S2 fraction; lanes C and G, solubilized S3 fraction; lanes D and H, insoluble final remaining pellet. The arrow indicates the 35-kilodalton recombinant F. gigantica cathepsin L1 protease. Values on the left are in kilodaltons (kD).

  • View in gallery

    Cystatin capture enzyme-linked immunosorbent assay using the recombinant cathepsin L1 protease in sera from cases with proven fascioliasis, parasitic diseases other than fascioliasis, cholangiocarcinoma, and healthy controls. Sera groups: A = fascioliasis; B = paragonimiasis; C = opisthorchiasis; D = ascariasis; E = hookworm infection; F = strongyloidiasis; G = capillariasis; H = gnathostomiasis; I = angiostrongyliasis; J = malaria; K = cysticercosis; L = trichinosis; M = other parasitoses; N = cholangiocarcinoma; O = healthy controls as described in Table 1. The horizontal dashed line is the cut off value.

  • 1

    World Health Organization, 1995. Control of foodborne trematode infections. World Health Organ Tech Rep Ser 849 :1–157.

  • 2

    Mas-Coma S, Bargues MD, Esteban JG, 1998. Human fasciolosis. Dalton JP, ed. Fasciolosis. Wallingford, United Kingdom: CABI Publishing, 411–434.

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    Andrews SJ, 1998. The life cycle of Fasciola hepatica. Dalton JP, ed. Fasciolosis. Wallingford, United Kingdom: CABI Publishing, 1–29.

  • 4

    Maleewong W, Wongkham C, Intapan PM, Pipitgool V, 1999. Fasciola gigantica-specific antigens: purification by a continuous-elution method and its evaluation for the diagnosis of human fascioliasis. Am J Trop Med Hyg 61 :648–651.

    • Search Google Scholar
    • Export Citation
  • 5

    Grams R, Vichasri-Grams S, Sobhon P, Upatham ES, Viyanant V, 2001. Molecular cloning and characterization of cathepsin L encoding genes from Fasciola gigantica. Parasitol Int 50 :105–114.

    • Search Google Scholar
    • Export Citation
  • 6

    O’Neill SM, Parkinson M, Strauss W, Angles R, Dalton JP, 1998. Immunodiagnosis of Fasciola hepatica infection (fascioliasis) in a human population in the Bolivian Altiplano using purified cathepsin L cysteine proteinase. Am J Trop Med Hyg 58 :417–423.

    • Search Google Scholar
    • Export Citation
  • 7

    O’Neill SM, Parkinson M, Dowd AJ, Strauss W, Angles R, Dalton JP, 1999. Immunodiagnosis of human fascioliasis using recombinant Fasciola hepatica cathepsin L1 cysteine proteinase. Am J Trop Med Hyg 60 :749–751.

    • Search Google Scholar
    • Export Citation
  • 8

    Strauss W, O’Neill SM, Parkinson M, Angles R, Dalton JP, 1999. Diagnosis of human fascioliasis: detection of anti-cathepsin L antibodies in blood samples collected on filter paper. Am J Trop Med Hyg 60 :746–748.

    • Search Google Scholar
    • Export Citation
  • 9

    Carnevale S, Rodriguez MI, Guarnera EA, Carmona C, Tanos T, Angel SO, 2001. Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase. Diagn Microbiol Infect Dis 41 :43–49.

    • Search Google Scholar
    • Export Citation
  • 10

    Cornelissen JB, Gaasenbeek CP, Borgsteede FH, Holland WG, Harmsen MM, Boersma WJ, 2001. Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease. Int J Parasitol 31 :728–737.

    • Search Google Scholar
    • Export Citation
  • 11

    Rokni MB, Massoud J, O’Neill SM, Parkinson M, Dalton JP, 2002. Diagnosis of human fasciolosis in the Gilan province of northern Iran: application of cathepsin L-ELISA. Diagn Microbiol Infect Dis 44 :175–179.

    • Search Google Scholar
    • Export Citation
  • 12

    Erdman DD, 1981. Clinical comparison of ethyl acetate and di-ethyl ether in the formalin-ether sedimentation technique. J Clin Microbiol 14 :483–485.

    • Search Google Scholar
    • Export Citation
  • 13

    Chomczynski P, Sacchi N, 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162 :156–159.

    • Search Google Scholar
    • Export Citation
  • 14

    Cohen SN, Chang AC, 1977. Revised interpretation of the origin of the pSC101 plasmid. J Bacteriol 132 :734–737.

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    Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 :248–254.

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    Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 :680–685.

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    Ikeda T, 1998. Cystatin capture enzyme-linked immunosorbent assay for immunodiagnosis of human paragonimiasis and fascioliasis. Am J Trop Med Hyg 59 :286–290.

    • Search Google Scholar
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  • 19

    Galen RS, 1980. Predictive value and efficiency of laboratory testing. Pediatr Clin North Am 27 :861–869.

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    Anastasi A, Brown MA, Kembhavi AA, Nicklin MJ, Sayers CA, Sunter DC, Barrett AJ, 1983. Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum. Biochem J 211 :129–138.

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    • Export Citation

 

 

 

 

SERODIAGNOSIS OF HUMAN FASCIOLIASIS BY A CYSTATIN CAPTURE ENZYME-LINKED IMMUNOSORBENT ASSAY WITH RECOMBINANT FASCIOLA GIGANTICA CATHEPSIN L ANTIGEN

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  • 1 Department of Biochemistry, and Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Faculty of Human Health, Tokai Gakuen University, Tenpaku-ku, Nagoya, Aichi, Japan

Cathepsin L1, a cysteine protease secreted by the gastrodermis of juvenile and adult Fasciola gigantica, was expressed in Escherichia coli as a calmodulin binding peptide fusion protein with a molecular mass of approximately 35 kD. The recombinant cathepsin L1 (rCTL1) was tested for its antigenic potential in a cystatin capture enzyme-linked immunosorbent assay (ELISA) to diagnose human fascioliasis. The ELISA plates were sensitized with chicken egg cystatin and incubated with bacterial lysates containing the recombinant protein before the standard ELISA procedures were performed. Analysis of the sera of 13 patients infected with F. gigantica (group 1), 204 patients with other parasitic infections (group 2), 32 cholangiocarcinoma patients (group 3), and 42 healthy controls (group 4) showed that the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of this ELISA using rCTL1 were 100%, 98.92%, 98.97%, 81.25%, and 100%, respectively. These results indicate that this assay has high sensitivity and specificity in the diagnosis of human fascioliasis. In addition, we have produced sufficient amounts of antigen for use in diagnosis.

INTRODUCTION

Fascioliasis is a disease caused by liver flukes of the genus Fasciola, of which F. hepatica and F. gigantica are the most common representatives.1 The disease is recognized as an important infectious condition by the World Health Organization1 and an estimated 17 million peoples are infected worldwide.2 Although F. hepatica has a worldwide distribution but predominates in temperate climates, and F. gigantica is also found in the tropical regions of Asia and Africa.3

The coprodiagnosis of human fascioliasis is often unreliable because the eggs of the parasite are not found during the prepatent period. Even at later times, eggs are only intermittently released. Serologic diagnosis is preferred, particularly since antibodies to Fasciola can be detected as early as two weeks after infection and can facilitate early treatment before irreparable damage to the liver occurs.

A specific 27-kD antigen from F. gigantica (FG27) excretory-secretory products was purified and used as specific and sensitive antigen for the diagnosis of human fascioliasis.4 Recently, we partially sequenced the FG27 antigen and determined the sequence of the 20 amino acids from the N- to the C-terminus (Tantrawatpan C and others, unpublished data). The sequence data proved to be homologous with the deduced amino acid sequence of F. gigantica cathepsin L15 (GenBank accession number AF112566) and cathepsin L1-B5 (GenBank accession number AF239264) at positions 261 to 280, as well as with cathepsin L1-E5 (GenBank accession number AF239267) at positions 154 to 173.

Several serologic tests have been developed with an emphasis on the detection of antibodies to F. hepatica cathepsin L1.6–11 In the present study, we produced the recombinant F. gigantica cathepsin L1 (rCTL1) in Escherichia coli and examined the potential use of rCTL1 as a diagnostic reagent for human fascioliasis based on a cystatin capture enzyme-linked immunosorbent assay (ELISA). The method is relatively simple and does not require chromatographic purification of rCTL1 from a bacterial lysate of a recombinant clone.

MATERIALS AND METHODS

Human sera.

Sera were obtained from serum banks of different sections of the Faculty of Medicine of Khon Kaen University (1992–2003). Each sera was aliquoted and stored at −70°C until used. The study protocol was reviewed and approved by the Human Research Ethics Committee of Khon Kaen University. Informed consent was obtained from adult participants and from parents or legal guardians of minors. Thirteen sera were obtained from parasitologically confirmed cases of infection with F. gigantica. Each of these was selected on the basis of removing F. gigantica adult worms during cholecystectomy and T-tube choledochostomy or other bile duct operations. To evaluate potential cross-reactivity, 204 serum samples from individuals with parasitic diseases other than fascioliasis were included. These samples were obtained from parasitologically confirmed cases of paragonimiasis, opisthorchiasis, ascariasis, hookworm infection, strongyloidiasis, capillariasis philippinensis, gnathostomiasis, angiostrongyliasis, malaria, cysticercosis, and trichinosis. Other parasitosis sera were obtained from cases who had mixed infections with parasites (Table 1). Thirty-two sera were obtained from patients who were residents of area endemic for Opisthorchis viverrini infection whose pathologic features were compatible with a diagnosis of bile duct cancer or cholangiocarcinoma (nine were proven cases of opisthorchiasis-associated cholangiocarcinoma). Negative control sera were obtained from 42 healthy adults and stool examinations were performed at the time of the blood collections using the formalin-ether concentration method.12 No evidence of intestinal parasitic infections was found. Human sera were pooled as positive and negative reference sera by combining equal volumes of proven fascioliasis and healthy control sera, respectively.

Purification of mRNA and rapid amplification of cDNA polymerase chain reaction (RACE PCR).

Total RNA of adult F. gigantica was extracted from frozen worms in RNAlater™ (Promega, Madison, WI) by acid guanidinium thiocyanate phenol chloroform extraction.13 The mRNA was purified from an adult fluke using an oligo (dT)-latex kit (Takara, Shiga, Japan). The synthesis of single and double- stranded cDNA from isolated mRNA was carried out using the Marathon™ cDNATamplification kit (Clontech, Palo Alto, CA) according to the manufacturer’s instructions. After adapter ligation with the Marathon cDNA adapter, the 5′-and 3′-RACE PCRs were carried out using gene specific primers (GSPs) and the adapter primer (AP1; 5′-CCATCCTAATACGACTCACTATAGGGC-3′). The GSPs were designed based on the conserved DNA sequences obtained from F. gigantica cathepsin L15 (GenBank accession number AF112566), cathepsin L1-B5 (GenBank accession number AF239264), and cathepsin L1-E5 (GenBank accession number AF239267) mature enzyme. The GSP1 (anti-sense primer) for the 5′-RACE PCR was 5′-TCACG-GAAATCGTGCCACCATCGGGAGACT-3′ and the GSP2 (sense primer) for the 3′-RACE PCR was 5′-CCCGACAAAATTGACTGGCGTGAATCTGGT-3′.

Cloning of cDNA and DNA sequencing.

The 5′-RACE PCR product was selected based on the sequence of the full-length cathepsin L1 and cloned into pGEM®-T Easy vector (Promega). Electrotransformation of E. coli JM 109 high efficiency competent cells was performed according to the protocol as previously described.14 White bacterial colonies were randomly selected and plasmids containing inserts were purified and sequenced. The nucleotide sequence of the gene was sequenced in both directions by the dideoxynucleotide chain termination method using Dye Terminator Cycle Sequencing kits (Applied Biosystems, Foster City, CA) and an ABI DNA sequencer 373A (Applied Biosystems). The BLAST network service was used to search the DNA and protein databases of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). The alignment of the sequences was carried out using the Multilin program (http:// prodes.toulouse.inra.fr/multalin/multalin.html). The plasmids containing inserts were digested with Eco RI. The inserts were purified and subcloned into pCAL-n-FLAG expression vectors (Stratagene, La Jolla, CA) that were previously digested with Eco RI. The resulting plasmids were transformed into E. coli BL21 gold (DE3; Stratagene). The transformed cells were then examined and the existence of an insert was confirmed by a PCR and sequencing.

Expression and purification of the expressed protein.

The expression of calmodulin binding peptide fused with cathepsin L-like in transformed cells was induced with 1 mM isopropyl-β-D-thiogalactopyranoside for three hours at 30°C. The cells were then harvested and the cell pellet was resuspended in cooled 0.01 M phosphate-buffered saline (PBS), pH 7.4, containing 0.1% Triton-X and 1% sarcosine. The cells were then sonicated and the resulting suspension was centrifuged at 15,000 × g for 10 minutes at 4°C. Recombinant protein expressed as inclusion bodies was obtained from the pellet. The pellet was then washed several times with 0.01 M PBS, pH 7.4, and resuspended in solubilizing solution (50 mM Tris, pH 8.0, 50 mM NaCl, 5 mM EDTA, 10 mM dithiothreitol, 8 M urea) for 30 minutes. After centrifugation at 15,000 × g for 30 minutes, the first supernatant fraction (S1) was collected. The remaining pellet was repeatedly solubilized and centrifuged two times. The second (S2) and third (S3) supernatant fractions as well as the final remaining pellet were also collected. The S1, S2, S3, and pellet were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The S2 and S3 were selected for use as antigen for the cystatin capture ELISA.

Protein determination.

The protein concentration was determined by the method of Bradford15 with bovine serum albumin (BSA) used as the standard.

Analytical SDS-PAGE and immunoblotting.

The tested samples were analyzed by SDS-PAGE using the Mini-Protean II Cell (Bio-Rad Laboratories, Hercules, CA), under reducing conditions on a 10–18% polyacrylamide gradient gel prepared by the method of Laemmli.16 After electrophoresis, the resolved polypeptides were electrophoretically transferred to a nitrocellulose membrane for immunoblotting.17

For immunoblotting, the blotted membrane was blocked with a blocking solution (100 mM PBS, pH 7.5, 0.1% Tween 20, 1% skim milk) for 30 minutes at room temperature, followed by incubation with each serum samples (diluted 1:100 in blocking solution) for two hours. After washing with blocking buffer, the membrane was incubated with 1:1,000 dilution of secondary, peroxidase-conjugated goat anti-human immunoglobulin G (Cappel; ICN Pharmaceuticals, Inc., Aurora, OH). The blots were developed with a diaminobenzidine solution.

Preparation of recombinant protein antigen for the cystatin capture ELISA.

The S2 and S3 fractions containing rCTL1 were pooled, dialyzed, lyophilized, and resuspended in 1% SDS at a final concentration of 2 mg/mL. The resulting suspension was stored at 4°C until used.

Cystatin capture ELISA.

The method was performed as previously described18 with some modifications. Each well of the ELISA plate was sensitized with 1 μg of chicken egg cystatin (Sigma, St. Louis, MO) in 0.1 mL of 0.1 M carbonate buffer, pH 9.6, overnight at 4°C. The wells were washed five times with 10 mM PBS, pH 7.4, 0.05% Tween 20 (PBS/T) and blocked with 2% BSA in PBS/T for one hour at room temperature. After washing with PBS/T, 3 μg of recombinant protein antigen diluted with 1% BSA in PBS/T was added to the well and the plate was incubated overnight at 4°C. After another washing step with PBS/T, the wells were incubated for one hour at 37°C with 0.1 mL of human sera diluted 1:200 with 1% BSA in PBS/T. After washing with PBS/T, peroxidase-conjugated anti-human IgG (Cappel; ICN Pharmaceuticals, Inc.) diluted 1:40,000 with 1% BSA in PBS/T was used as secondary antibody. The wells were then washed with PBS/T and incubated with the 0.1 mL of o-phenylenediamine dihydrochloride substrate for 30 minutes. The reaction was stopped with 0.05 mL of 8 N H2SO4 and the absorbance was measured at 492 nm using a microplate ELISA reader (Tecan, Salzburg, Austria).

Statistical analysis.

Statistical analysis was performed with the Student’s t-test using Sigma Stat (San Rafael, CA) statistical software. The diagnostic sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were calculated and expressed using the method of Galen.19 These values were calculated and expressed as follows: sensitivity was the [number of true positives/(number of true positives + number of false negatives) × 100]; specificity was the [number of true negatives/(number of false positives + number of true negatives) × 100]; accuracy was the [number of true positives + number of true negatives/(number of true positives + number of false negatives + number of false positives + number of true negatives) × 100]; positive predictive value was the [number of true positives/(number of false positives + number of true positives) × 100]; negative predictive value was the [number of true negatives/(number of false negatives + number of true negatives) × 100]; true negative was the number of control samples (other parasitosis, cholangiocarcinoma, and healthy controls) that were negative by the assay; true positive was the number of proven fascioliasis samples that were positive by the assay; false positive was the number of control samples that were positive by the assay; false negative was the number of proven fascioliasis samples that were negative by the assay.

RESULTS

Sequence analysis of the cathepsin L1 gene.

Following the RACE PCR, we obtained the full-length cathepsin L1 gene of the F. gigantica adult worm. An analysis of the deduced amino acid sequence of the selected subcloned PCR product showed a homology level of 99.0% compared with the F. gigantica cathepsin L1 sequence (GenBank accession number AF112566) with only two differences at position 123 from leucine to valine and position 212 from valine to alanine. The expression of the cathepsin L1 gene inserted into the pCAL-n-FLAG expression vector in E. coli resulted in an expressed fusion protein with an addition of a 46-amino acid fusion on the N-terminus, which contained a calmodulin binding protein tag (MKRRWKKNFIAVSAANRFKKISSSGALLVPR-GSDYKDDDDKGRGSE) and an enterokinase cleavage site. The recombinant protein was expressed in the inclusion body. Cells were then lysed under denaturating conditions using urea as the denaturant. The SDS-PAGE and immunoblot analysis of the expressed protein showed a protein with a molecular mass of approximately 35 kD. The expressed protein reacted strongly with the pooled positive reference sera, but not with the pooled negative reference sera (Figure 1).

Diagnostic values of the cystatin capture ELISA.

The pooled S2 and S3 fractions containing rCTL1 were used as antigen in the cystatin capture ELISA for the detection of IgG antibody in the sera of patients with proven fascioliasis. The results were compared with those obtained using sera from patients with parasitic diseases other than fascioliasis, those with cholangiocarcinoma, and from healthy controls (Figure 2). The mean ± SD and ranges of optical densities (ODs) at 492 nm of the four sera groups are shown in Table 1. There was a significant difference in mean OD values between cases of proven fascioliasis (group 1) and cases of parasitic diseases other than fascioliasis (group 2), cholangiocarcinoma (group 3), and healthy controls (group 4) (P < 0.001). If the absorbance value of 0.464 (which was equivalent to the absorbance value of the mean plus 3 SD of the healthy control group) was used as the cut-off limit between positivity and negativity for fascioliasis, the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were 100%, 98.92%, 98.97%, 81.25%, and 100%, respectively. However, two cases of paragonimiasis and one case of gnathostomiasis showed false-positive results.

DISCUSSION

Serologic tests for the diagnosis of fascioliasis have been developed as standard assays using authentic F. hepatica cathepsin L16–8,11 or rCTL1 as marker antigens.7,9,10 In the present study, the cDNA-encoded mature cathepsin L1 was expressed in E. coli. The expressed protein aggregated in the cytoplasm of E. coli and was solubilized only with strong denaturants such as urea. Therefore, investigation of the precise role of the proteinase was not performed. However, we have developed a diagnostic cystatin capture ELISA to detect antibodies to F. gigantica in humans that is based on the use of an rCTL1 protein as the marker antigen.

This study used chicken cystatin, which has a very low Ki value and high specificity for cysteine proteinase,20 as a capture reagent for rCTL1 in the ELISA. This method could detect the antibodies to proteinases without prior purification of rCTL1 from the bacterial lysate. This represents a big improvement in time, labor, and costs because purification of the recombinant protein requires specialized equipment and procedures, and it is troublesome to prepare rCTL1 because it requires several purification steps.

The cystatin capture ELISA is a sensitive and specific tool for the serodiagnosis of human fascioliasis. The procedure is simple and easy for use. The sensitivity was the same as for the ELISA using authentic FG27 antigen for the detection of human fascioliasis, while the specificity of rCTL1 was slightly lower (1.08%) than the one observed with the authentic FG27 antigen. Cross-reactions with sera from two cases of paragonimiasis and one case of gnathostomiasis were demonstrated, which did not react with authentic FG27 antigen.4 This could either be due to a subclinical infection with Fasciola spp. or because the rCTL1 shares some epitope with paragonimiasis and gnathostomiasis. However, the another important element of this study was the ability to demonstrate that, contrary to FG27, the rCTL1 antigen could be produced in large amounts to produce a standardized serological test for human fascioliasis.

Table 1

Absorbance values of various sera in a cystatin capture enzyme-linked immunosorbent assay using the Fasciola gigantica recombinant cathepsin L1 protease

Type of serumGroup*RangeMean ± SDNo. positive/total (%)
* 1 = proven fascioliasis; 2 = parasitic diseases other than fascioliasis; 3 = cholangiocarcinoma; 4 = healthy controls.
† Total of 9 cases, 7 with Plasmodium falciparum, 1 with P. vivax, and 1 mixed infection with P. falciparum and P. vivax.
‡ Total of 33 cases, 5 patients infected with Echinostoma spp., 4 patients infected with Taenia spp., 4 patients infected with Trichuris trichiura, 3 patients infected with Entamoeba histolytica, 2 patients infected with Giardia lamblia, 4 patients infected with T. trichiura and Ascaris lumbricoides, 3 patients infected with T. trichiura and hookworm, 4 patients infected with T. trichiura, A. lumbricoides, and hookworm, 1 patient infected T. trichiura, Strongyloides stercoralis, and hookworm, and 3 patients infected with T. trichiura, A. lumbricoides, S. stercoralis, and hookworm.
Fascioliasis10.491–1.1610.744 ± 0.18813/13 (100)
Paragonimiasis20.252–0.7730.359 ± 0.1262/25 (8)
Opisthorchiasis20.147–0.3900.274 ± 0.0670/25 (0)
Ascariasis20.201–0.3640.264 ± 0.0510/14 (0)
Hookworm infection20.067–0.4470.215 ± 0.1080/12 (0)
Strongyloidiasis20.073–0.4110.178 ± 0.0760/30 (0)
Capillariasis20.106–0.4390.207 ± 0.1260/12 (0)
Gnathostomiasis20.154–0.4740.248 ± 0.0841/13 (7.69)
Angiostrongyliasis20.126–0.3000.207 ± 0.0610/9 (0)
Malaria†20.151–0.4180.325 ± 0.0840/9 (0)
Cysticercosis20.207–0.2740.245 ± 0.0260/6 (0)
Trichinosis20.067–0.4570.192 ± 0.1190/16 (0)
Other parasitoses‡20.079–0.3890.230 ± 0.0900/33 (0)
Cholangiocarcinoma30.147–0.4630.303 ± 0.1010/32 (0)
Healthy controls40.131–0.4210.260 ± 0.0680/42 (0)
Figure 1.
Figure 1.

Immunoblot analysis of recombinant Fasciola gigantica cathepsin L1 fused with calmodulin binding peptide. The blotted membrane reacted with pooled negative reference sera (lanes A-D) and pooled positive reference sera (lanes E-H). Lanes A and E, solubilized S1 fraction; lanes B and F, solubilized S2 fraction; lanes C and G, solubilized S3 fraction; lanes D and H, insoluble final remaining pellet. The arrow indicates the 35-kilodalton recombinant F. gigantica cathepsin L1 protease. Values on the left are in kilodaltons (kD).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.82

Figure 2.
Figure 2.

Cystatin capture enzyme-linked immunosorbent assay using the recombinant cathepsin L1 protease in sera from cases with proven fascioliasis, parasitic diseases other than fascioliasis, cholangiocarcinoma, and healthy controls. Sera groups: A = fascioliasis; B = paragonimiasis; C = opisthorchiasis; D = ascariasis; E = hookworm infection; F = strongyloidiasis; G = capillariasis; H = gnathostomiasis; I = angiostrongyliasis; J = malaria; K = cysticercosis; L = trichinosis; M = other parasitoses; N = cholangiocarcinoma; O = healthy controls as described in Table 1. The horizontal dashed line is the cut off value.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.82

Authors’ addresses: Chairat Tantrawatpan, Chaisiri Wongkham, and Sopit Wongkham, Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. Wanchai Maleewong and Pewpan M. Intapan, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. Kunio Nakashima, Faculty of Human Health, Tokai Gakuen University, Nakahira 2-901, Tenpaku-ku, Nagoya, Aichi, Japan.

Acknowledgments: We thank Dr. Nimit Morakote (Faculty of Medicine, Chiang Mai University, Chang Mai, Thailand) for providing trichinosis sera and Dr. Mark Roselieb for improving the English language presentation of the manuscript.

Financial support: This research was supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (grant no. PHD/0155/2542 to Chairat Tantrawatpan) and Wanchai Malee-wong and a grant from Khon Kaen University.

REFERENCES

  • 1

    World Health Organization, 1995. Control of foodborne trematode infections. World Health Organ Tech Rep Ser 849 :1–157.

  • 2

    Mas-Coma S, Bargues MD, Esteban JG, 1998. Human fasciolosis. Dalton JP, ed. Fasciolosis. Wallingford, United Kingdom: CABI Publishing, 411–434.

  • 3

    Andrews SJ, 1998. The life cycle of Fasciola hepatica. Dalton JP, ed. Fasciolosis. Wallingford, United Kingdom: CABI Publishing, 1–29.

  • 4

    Maleewong W, Wongkham C, Intapan PM, Pipitgool V, 1999. Fasciola gigantica-specific antigens: purification by a continuous-elution method and its evaluation for the diagnosis of human fascioliasis. Am J Trop Med Hyg 61 :648–651.

    • Search Google Scholar
    • Export Citation
  • 5

    Grams R, Vichasri-Grams S, Sobhon P, Upatham ES, Viyanant V, 2001. Molecular cloning and characterization of cathepsin L encoding genes from Fasciola gigantica. Parasitol Int 50 :105–114.

    • Search Google Scholar
    • Export Citation
  • 6

    O’Neill SM, Parkinson M, Strauss W, Angles R, Dalton JP, 1998. Immunodiagnosis of Fasciola hepatica infection (fascioliasis) in a human population in the Bolivian Altiplano using purified cathepsin L cysteine proteinase. Am J Trop Med Hyg 58 :417–423.

    • Search Google Scholar
    • Export Citation
  • 7

    O’Neill SM, Parkinson M, Dowd AJ, Strauss W, Angles R, Dalton JP, 1999. Immunodiagnosis of human fascioliasis using recombinant Fasciola hepatica cathepsin L1 cysteine proteinase. Am J Trop Med Hyg 60 :749–751.

    • Search Google Scholar
    • Export Citation
  • 8

    Strauss W, O’Neill SM, Parkinson M, Angles R, Dalton JP, 1999. Diagnosis of human fascioliasis: detection of anti-cathepsin L antibodies in blood samples collected on filter paper. Am J Trop Med Hyg 60 :746–748.

    • Search Google Scholar
    • Export Citation
  • 9

    Carnevale S, Rodriguez MI, Guarnera EA, Carmona C, Tanos T, Angel SO, 2001. Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase. Diagn Microbiol Infect Dis 41 :43–49.

    • Search Google Scholar
    • Export Citation
  • 10

    Cornelissen JB, Gaasenbeek CP, Borgsteede FH, Holland WG, Harmsen MM, Boersma WJ, 2001. Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease. Int J Parasitol 31 :728–737.

    • Search Google Scholar
    • Export Citation
  • 11

    Rokni MB, Massoud J, O’Neill SM, Parkinson M, Dalton JP, 2002. Diagnosis of human fasciolosis in the Gilan province of northern Iran: application of cathepsin L-ELISA. Diagn Microbiol Infect Dis 44 :175–179.

    • Search Google Scholar
    • Export Citation
  • 12

    Erdman DD, 1981. Clinical comparison of ethyl acetate and di-ethyl ether in the formalin-ether sedimentation technique. J Clin Microbiol 14 :483–485.

    • Search Google Scholar
    • Export Citation
  • 13

    Chomczynski P, Sacchi N, 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162 :156–159.

    • Search Google Scholar
    • Export Citation
  • 14

    Cohen SN, Chang AC, 1977. Revised interpretation of the origin of the pSC101 plasmid. J Bacteriol 132 :734–737.

  • 15

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Author Notes

Reprint requests: Wanchai Maleewong, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand, Telephone: 66-43-348387, Fax: 66-43-202475, E-mail: wanch_ma@ kku.ac.th.
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