Background: Paragonimus heterotremus is the main causative agent of paragonimiasis in Thailand. In Western blot diagnostic assays for paragonimiasis, the 35 kDa band present in crude P. heterotremus somatic extracts represents one of the known diagnostic bands. This study aimed to use a P. heterotremus cDNA library to create a recombinant version of this antigen for use in immunodiagnosis of paragonimiasis. Methods: To accomplish this aim a cDNA expression library was constructed from adult worm mRNA and immuno- screened using antibodies from mice that had been immunized with the 35 kDa antigen. Screening resulted in the identification of an immunoreactive protein encoded by clone CE3, which contained an inserted sequence composed of 1292 base pairs. This clone was selected for use in the construction of a recombinant P. heterotremus protein because of its similarity to proactivator polypeptide. For recombinant protein expression, the CE3 gene sequence was inserted into the plasmid vector pRset and the resulting product had the expected molecular weight of 35 kDa. An IgG-ELISA based on the CE3 recombinant protein was evaluated by using sera from healthy individuals, from patients with paragonimiasis and other parasitic infections. This ELISA was performed by using human sera diluted at 1:2000, an optimized antigen concentration of 1 μg/ml, and anti-human IgG diluted at 1:4000. Results: The cut-off optical density value was set as the mean + 2 standard deviations (0.54), which resulted in the test having a sensitivity of 88.89% and a specificity of 95.51%. The recombinant antigen could react with antibodies from P. heterotremus, P. pseudoheterotremus and P. westermani infections. Cross-reactivity occurred with a few cases of Blastocystis hominis infection (2/3), Bancroftian filariasis (1/10), opisthorchiasis (3/10), strongyloidiasis (4/10) and neurocysticercosis (1/11). Conclusions: Given the high test sensitivity and specificity, reflected in the low level of heterologous infection cross- reactivity (11/215 serum samples), observed in the IgG-ELISA, this 35 kDa antigen may be useful for the detection of paragonimiasis. Keywords: Paragonimus heterotremus, cDNA library, Recombinant protein, Paragonimiasis, Immunodiagnosis, IgG-ELISA * Correspondence: firstname.lastname@example.org Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pothong et al. Parasites & Vectors (2018) 11:322 Page 2 of 10 Background assay) based on this protein. Results of testing against a Paragonimus species, also known as lung flukes, are the panel of human sera indicate that the recombinant protein causative agents of pulmonary paragonimiasis in humans. generated by this study may be useful in the serodiagnosis In Asia, the occurrence of paragonimiasis is 90%, with of human paragonimiasis. about 20 million individuals infected [1, 2]. Seven species of the genus Paragonimus have been recorded in Methods Thailand: P. bangkokensis, P. harinasutai, P. heterotremus, Worm collection P. pseudoheterotremus, P. siamensis, P. westermani,and P. The metacercariae were recovered from freshwater crabs macrorchis.Ofthese, P. heterotremus, P. pseudoheterotre- (Larnaudia larnaudii), which were collected from parago- mus and probably P. westermani are reported in human nimiasis endemic areas of Nakon Nayok Province, central cases [3–5]. People become infected with Paragonimus Thailand. Gerbils were infected by feeding five fresh meta- parasites when they frequently eat improperly cooked cercariae to each animal using a stomach tube connected freshwater crabs containing Paragonimus metacercariae to a syringe and maintained for at least 35 days, at which collected from mountainous streams. time P. heterotremus eggs were observed in the feces using Several suspected cases of paragonimiasis have been a simple smear technique. Gerbils were then euthanized clinically identified without the detection of Paragoni- and the adult worms isolated from the lungs and sur- mus eggs or worms in the sputum, feces, or tissues. Not- rounding lung tissues. Isolated worms were washed 3 ably, the ectopic foci of this worm in various host tissues times in 0.85% sterile NaCl and used for preparing crude can result in cerebral, cutaneous and other clinical forms antigen and for isolation of total worm RNA. of paragonimiasis that can be difficult to diagnosis. Therefore, immunodiagnosis is an important supplement Construction and immunoscreening of the cDNA library to the parasitological methods used in the detection of To construct the cDNA, total RNA was extracted from Paragonimus infections [6, 7]. Specifically, immunoblot- fresh P. heterotremus adult worms by using the single- ting tests using P. heterotremus crude extracts of adult step method of total RNA extraction with TRIzol® re- worms show high sensitivity and specificity to paragoni- agent (Invitrogen-Life Technologies, Carlsbad, CA, miasis heterotremus with diagnostic bands correspond- USA). Messenger RNA (mRNA) was isolated using a ing to 32.5, 33, and 35 kDa native antigens  and can solid-phase oligo-dT matrix (Oligotex mRNA mini kit; differentiate between P. heterotremus and P. westermani Qiagen, Hilden, Germany) according to the manufac- Korean strain . Another P. heterotremus component, turer’s instructions. Two micrograms of mRNA template a 31.5 kDa partially purified antigen, also has been used was used in the Universal RiboClone® cDNA Synthesis in the diagnosis of human paragonimiasis . Molecu- System (Promega, Madison, WI, USA) and further li- lar techniques have been applied as a tool for producing gated into a pJET 1.2/blunt Cloning Vector (CloneJET recombinant protein antigens to replace the native anti- PCR Cloning Kit, Thermo Fisher Scientific, Rockford, IL, gens produced by other parasitic worm species from ani- USA). The ligation mixture was used directly to trans- mal hosts in similar tests . Obtaining Paragonimus form Escherichia coli XL1-Blue (Stratagene, Agilent adult worms for native antigen preparation is a very Technologies, Santa Clara, CA, USA) competent cells, time-consuming and costly process which involves crab followed by plating on LB agar plates supplemented with collection, recovery of metacercariae from crabs, infect- ampicillin for Ampicillin-selection. To identify the ing and maintaining mammalian definitive hosts, antigen-producing clones by immunoscreening, the confirming infections by eggs detection, and finally pooled negative (pre-immunized sera) and positive sera obtaining worms from host lungs and processing them from mice that had been immunized with the crude for diagnostic testing. Therefore, the aim of the present eluted 32.5, 33 and 35 kDa antigens of P. heterotremus study was to apply a molecular approach to generate P. were used to identify clones carrying the 32.5, 33 and 35 heterotremus DNA recombinant antigens for use in im- kDa antigens. Positive colonies were identified by align- munodiagnosis of human paragonimiasis. To this end, ing the membrane with the agar plates. For DNA se- we constructed a complementary DNA (cDNA) library quencing, the PCR was performed by using pJET 1.2 from adult P. heterotremus worms and used a molecular forward and pJET 1.2 reverse sequencing primers. The cloning approach to identify and express recombinant resulting gene sequences were searched against the proteins that exhibited selective immunoreactivity with NCBI database. antibodies recognizing the 32.5, 33 and 35 kDa bands comprising P. heterotremus native antigens. Herein we Target sequence amplification report the DNA cloning, identification and expression of a The protein targets for DNA cloning were selected recombinant protein for the 35 kDa antigen and the con- based on the results of immunoscreening tests, aimed to struction of an IgG-ELISA (enzyme-linked immunosorbent extend the target sequences. The L_2F was selected as Pothong et al. Parasites & Vectors (2018) 11:322 Page 3 of 10 target clone. The designed primers were: Ph3 forward Immunoreactivity of purified recombinant proteins to sera primer 5'-CTC TAG AAG ATC TCC TAC-3' and Ph3 of immunized mice reverse primer 5'-AGC CGA ACG ACC GAG CGC-3'. Three mice were each injected via the intraperitoneal After PCR amplification, the PCR product was further route (IP) with 1 μg of the 35 kDa recombinant protein, purified. The purified DNA was cloned, designated mixed 1:1 with Inject® Alum (Thermo Fisher Scientific). cloneCE3,byusing aStrataClone PCRCloning kit Mice were given IP booster injections with the same (Stratagene, Agilent Technologies), according to the man- dose twice more at 2-week intervals. Blood samples were ufacturer’s instructions. collected at 1 week after the last injection, and the resulting sera were tested against each recombinant pro- tein antigen by Western blot analyses. Construction of a recombinant protein clone Plasmid creation Evaluation of recombinant proteins for serodiagnosis of Primers for clone CE3 were designed based on the paragonimiasis heterotremus DNA sequencing results. These include: CE3 forward Human sera primer, 5'-CGA ACG TCG AGC GCT TAC TGT GAC The collection of patients’ sera used as the diagnostic A-3', which contained XhoI restriction enzyme sites, test panel in the present study was stored specimens that and CE3 reverse primer, 5'-AGC CGA GAA TTC GAG had been used in the previous serological surveys. All CGC CTT GCA AAA G-3', which contained EcoRI re- samples had been maintained at -80 °C in the Depart- striction enzyme sites. To amplify the target protein, ment of Helminthology, Faculty of Tropical Medicine, PCR was performed as described above. Both the Mahidol University for several years, and contained no pRsetB vector (Invitrogen Ltd., Paisly, UK) and the patient identifying information (see Ethical Approval purified DNA were digested with XhoIand EcoRI and Consent section). Details of the diseases and corre- restriction enzymes using the Fermentas FastDigest sponding diagnostic methods used on the groups of Restriction Enzymes kit (Thermo Fisher Scientific). Fi- serum samples (Group A: paragonimiases, Group B: nally, ligation was performed, and the obtained plasmid other parasitic infections and Group C: healthy controls) was transformed into E. coli host cells (strain DH5α or for the evaluation of the recombinant antigens are strain DE3BL21pLysS). shown in Table 1. Protein expression and purification of the recombinant Enzyme-linked immunosorbent assay (ELISA) protein An indirect ELISA was created based on the recombin- A single colony of CE3 protein-expressing host cells was ant 35 kDa protein antigen and was evaluated using inoculated in a tube that contained LB broth with ampicil- groups of sera from patients infected with paragoni- lin and chloramphenicol and further incubated overnight miases (n = 36), heterologous infections (n = 215) and at 37 °C with shaking at 180 rpm (New Brunswick Scien- healthy controls (n = 30). The ELISAs were performed tific Innova® 40, Edison, NJ, USA). The cultured cells were as described by Dekumyoy et al.  with minor modifi- then inoculated into ampicillin-chloramphenicol LB broth cations. Single serum dilutions, 1:2000, of individual and shaken at 37 °C for 3 h. IPTG (1 mM isopropyl-l- positive and negative sera against 1 μg/ml purified re- thio-β-D-galactopyranoside) was added to induce protein combinant antigen were performed to establish the opti- expression and shaken at 180 rpm for an additional 4 h at mal conditions for the tests. In addition, 1:4000-diluted 37 °C. The cultured cells were centrifuged for the pellet horseradish peroxidase-conjugated rabbit anti-human and stored at -70 °C until required. IgG (Southern Biotech, Birmingham, USA) and color- Following recombinant protein induction, host cells metric substrate solution [ABTS, 2, 2-azino-di-(3-ethyl- were pelleted and then extracted by suspending cells in benzthiazoline sulfonate); Sigma, Oakville, Ontario, BugBuster™ (Novagen™, Merck, Darmstadt, Germany). Canada] were used. The reactions were stopped by add- The extract mixture was centrifuged for collecting the ing 50 μl of 1% SDS solution. Antigen-antibody binding supernatant containing solubilized proteins. The putative reactions were measured by reading the OD (optical recombinant His6 (Histidine 6) fusion protein was then density) using an ELISA Reader (Tecan, Männedorf, purified using a Ni-NTA (nickel-nitrilotriacetic acid) Switzerland). chromatography column according to the manufac- turer’s instructions (Ni-NTA ProBond, Invitrogen, USA). Data analysis Eluted fractions were collected and checked by SDS- The ELISA cut-off point was chosen by using receiver PAGE analysis for the desired protein. Eluted fractions operating characteristic (ROC) curve analysis, sensitivity that possessed a single protein band were pooled, sub- versus 1-specificity or false positive. The SPSS version 18 jected to dialysis, and lyophilization. was used as tool for ROC curve analysis. All data were Pothong et al. Parasites & Vectors (2018) 11:322 Page 4 of 10 Table 1 Diseases, and corresponding diagnostic methods, Table 1 Diseases, and corresponding diagnostic methods, detected in the serum samples detected in the serum samples (Continued) Group Diseases No. of Diagnostic methods Group Diseases No. of Diagnostic methods serum serum samples samples Fascioliasis 3 Egg detection, immunoblot A Paragonimiasis 29 Worm and egg detection, heterotremus serum samples collected for Minute intestinal 10 Worm detection over 25 years, patients resided flukes infections in 2 endemic provinces of P. Creeping eruption 3 Symptoms, negative for heterotremus in the central strongyloidiasis and Thailand where metacercariae gnathostomiasis from crabs were identified by PCR a few years ago Entamebiasis 4 Cyst detection Paragonimiasis 3 Eggs in feces and PCR (1 case), Giardiasis 3 Cyst detection pseudoheterotremus immunoblot detection and PCR for detection of Blastocyctis hominis 3 Cyst detection metacercariae from crabs (2 infection cases). Patients resided along Falciparum malaria 5 Blood stage detection Thai-Myanmar border Vivax malaria 5 Blood stage detection Paragonimiasis 4 Serum samples were westermani supported by Korean Tuberculosis 5 Acid fast stained researcher Mycobacterium tuberculosis Total 36 Total 215 B Gnathostomiasis 10 Worm and immunoblot C Healthy serum 30 Negative ELISA using ten kinds detection of antigens and fecal examinations Strongyloidiasis 10 Larva detection Total 30 Hookworm infection 10 Egg detection Key: Group A, paragonimiases; group B, heterologous infections; group C, Trichinellosis 10 Larva and immunoblot healthy controls detection Capillariasis 3 Egg, larva, adult worm further analyzed by using the Mann-Whitney U-test. detection The calculation was carried out on SPSS version 18 and a Toxocariasis 10 Immunoblot P-value < 0.05 was considered as significant. The accur- Angiostrongyliasis 10 Worm detection, immunoblot acies of the diagnostic test, including sensitivity, specifi- city, and predictive values, were determined using the Ascariasis 6 Egg and worm detection method of Parikh et al.  and SPSS version 18 and Trichuriasis 9 Egg and worm detection MedCal (free statistical calculators, diagnostic test evalu- Trichostrongyliasis 10 Egg detection ation calculator) were used at 95% CI. The ELISAs were Bancroftian filariasis 10 Microfilariae detection analyzed by calculating the means and standard deviations Enterobiasis 4 Egg and worm detection (SD). A cut-off value at 0.543 was done by ROC curve Brugian filariasis 10 Microfilariae detection analysis and the mean + 2SD. The AUC was 0.975. Brugian filariasis 10 ELISA using recombinant antigen Results Isolation of total RNA, purification of mRNA, and Dirofilariasis 2 Worm detection construction of a P. heterotremus cDNA library Neurocysticercosis 11 Cyst detection, immunoblot A double-stranded P. heterotremus cDNA reaction was Sparganosis 6 Sparganum detection run on a 1.2% agarose gel along with a 1 kb DNA size Taeniasis 13 Egg or segments (T. solium or marker. The sizes of the cDNA bands ranged from 0.5 T. saginata) to 12 kb. After the cDNA was purified by using Promega Echinococcosis 3 Protoscolices detection Wizard® DNA Clean-UP System (Promega, Madison, Hymenolepiasis 5 Egg detection WI, USA), the double-stranded cDNA was successfully nana ligated into the pJET 1.2/blunt cloning vector and used Hymenolepiasis 1 Egg detection to transform in E. coli XLI Blue on LB agar. In order to diminuta check the size of inserts, 14 colonies were randomly Opisthorchiasis 10 Worm detection picked and PCR was performed. The sizes of insert Echinostomiasis 1 Egg detection and PCR cDNAs, as determined by performing colony PCR, malayanum ranged from approximately 500 to 1000 bp (Additional file 1: Figure S1). The products of the positive clones Pothong et al. Parasites & Vectors (2018) 11:322 Page 5 of 10 that were larger than 500 bp were further assessed by calculated by the NCBI-Blastx program show that it colonies amplification and sequencing. shares identity with the proactivator polypeptides of C. sinensis and Schistosoma haematobium at 48 and 29%, re- Sequence analysis spectively (Table 3). An overview of the CE3 clone using We determined the DNA sequences of each of the 11 DNAMan software consists of 1292 bp with an open read- plasmid clones described above, the insert sizes of which ing frame at 49–1047 bp (Additional file 3:FigureS3), ranged from 500 to 1000 bp. Homology searches against under the accession number KX180136, and the estimated NCBI databases (https://www.ncbi.nlm.nih.gov/) revealed molecular weight of the corresponding protein is 35 kDa some clones matched were a trematode group i.e. clone (Fig. 1). IDs, P89-1, P51-R, P55-R, P3_1F. The identities of these matches are listed in Table 2, and the percentage of iden- IPTG-induced protein expression and purification of tity of these eleven clones ranged from 31 to 100% when recombinant proteins searched against the trematodes, bacteria and protozoans We examined the effect of IPTG-induction on recombin- in the NCBI database. ant protein expression. The IPTG-induction was started from 0 h (non-IPTG-induced) to 3 h (IPTG-induced); PCR confirmatory test and DNA cloning proteins were observed every hour. The protein expres- After the initial cloning described above, the inserted sion was increased following timing. Whole bacterial ly- cDNA that was > 500 bp in size was run on a 1.2% agar- sates from non-IPTG-induced and IPTG-induced CE3 ose gel. The band containing the inserted cDNA was clones at 3 h were analyzed by SDS–PAGE in a 12% poly- then cut and purified for further second cloning to ex- acrylamide gel and stained with Coomassie brilliant blue. tend sequences. Eleven plasmid clones were identified by After IPTG induction for 3 h, there was an increase in the immuno-screening of the cDNA library. The inserted amount of an expressed 35 kDa protein (Fig. 1), indicating cDNA from clone L_2F (designated CE3) was run on a that the fusion protein from clone CE3 was successfully 1.2% agarose gel, and based on comparison with the expressed in E. coli upon IPTG induction. Kapa Universal ladder, was approximately 900 bp long After extraction of bacterial cells with BugBuster™ and (Additional file 2: Figure S2). The CE3 clone was selected centrifugation and washes with lysis buffer (8M urea in as representative of the antigenic clones since it possessed LEW buffer), the crude recombinant protein extract was the longest sequence that contained no stop codons. subjected to passage through a nickel-agarose affinity column, followed by elution with buffer containing vari- Sequence analysis ous concentrations of imidazole ranging from 100 to After the DNA sequences were checked for open reading 500 mM. A SDS-PAGE analysis of the purified protein frames and stop codons, clone number 3 (CE3) was fractions eluted at imidazole concentrations of 25, 50, 100, chosen for recombinant protein production since CE3 was 200 and 500 mM showed the expected enrichment of the given the similar identity of sequences to other clones and 35 kDa band in the eluted fractions. The isolated fractions had a long sequence without stop codon between the se- recovered from the 100, 200 and 500 mM imidazole elu- quences. Specific primers were used to amplify the DNA tions were then pooled and dialyzed against 0.1× PBS via PCR. A sequence analysis of the recombinant protein (phosphate buffered saline-Tween20 buffer, pH 7.2) for from clone CE3 and the corresponding alignment scores further use. Table 2 The sequence analysis results after being searched against NCBI databases Sample No. Clone ID Name Percentage of identity 1 P89-1 Fatty acid-binding protein type 3 (Clonorchis sinensis)70 2 P51-F Microcystin synthetase (uncultured Microcystis sp.) 73 3 P51-R Dynein beta Chain ciliary (C. sinensis)97 4 P55-R Hypothetical proteinSMP_137720 (Schistosoma mansoni)31 5 P3_1F SJCH6C65074 protein (Schistosoma japonicum)78 6 P7_2UF Histone lysine methyltransferases (Toxoplasma gondii)43 7 L_2F Hypothetical protein (Echinococcus multilocularis)92 8 L_3R Axonemal beta dynein heavy chain, putative (T. gondii ME49)37 9 L_2F2 Hypothetical protein (Plasmodium chabaudi chabaudi)99 10 L_8F Protein Bm11556, partial (Brugia malayi) 100 11 L3R Hypothetical protein Emj 000005500 (E. multilocularis)92 Pothong et al. Parasites & Vectors (2018) 11:322 Page 6 of 10 protein concentrations. As a result, in subsequent assays, the primary antibodies (human sera) were diluted to 1: 2000, and 1 μg/ml of recombinant protein was used to coat wells of ELISA plates. Based on the ELISA results from 30 negative serum samples, the cut-off value was selected from the mean ± 2 SD at OD = 0.54. Evaluating the IgG- ELISA with this cut-off value produced sensitivity, specifi- city, and positive and negative predictive values at 88.89, 95.51, 74.42 and 98.32%, respectively (Table 4) (calculated by MedCal with 95% CI). Based on this OD cut-off value of 0.54, 32 of 36 serum samples from patients with con- firmed paragonimiasis gave positive assay results. The 4 false negative sera, although below the 0.54 OD threshold, were not far from the cut-off value. Regarding to paragoni- miasis, this recombinant antigen strongly react with serum Fig. 1 Assessment of recombinant fusion protein production in antibodies from 3 cases of paragonimiasis pseudoheterotre- CE3-clone transformed E. coli cells. The proteins from these cells were mus and 2 cases of paragonimiasis westermani in the high separated on a 12% gel by SDS-PAGE and stained with Coomassie ODs-ranges, 0.856–1.145 and 0.799–0.853, respectively. blue. Lane M: molecular weight markers; Lane 1: non-IPTG-induced CE3 The antigen weakly reacted with antibodies from tubercu- cells; Lane 2: IPTG-induced CE3 cells. Arrow indicates the prominent band at 35 kDa losis. Importantly, this recombinant antigenic protein re- sulted in true negative readings for sera representing 27 of 32 different parasitic diseases, as well as for all negative Mouse immunization with the 35 kDa recombinant controls. Cross-reactivity occurred for five diseases (11/ protein 215 serum samples), including Blastocystis hominis infec- After isolation, the 35 kDa purified recombinant protein tion (2/3), Bancroftian filariasis (1/10), opisthorchiasis (3/ was used to immunize mice for specific antibody produc- 10), strongyloidiasis (4/10), and neurocysticercosis (1/11). tion. ELISAs were performed against the purified recom- We observed that one of four false positives for stron- binant protein. The mouse antibody against the 35 kDa gyloidiasis gave a quite high OD-value, above the cut- recombinant protein had titer over 1:1600. Additionally, off value. The OD-values of false positives from Blasto- using Western blot analysis, the 35 kDa recombinant pro- cystis hominis infection, Bancroftian filariasis, neurocys- tein from the CE3 clone also was recognized by the anti- ticercosis and opisthorchiasis were close to the cut-off bodies in the sera of immunized mice, but not by sera value (Fig. 3). from normal, unimmunized mice (Fig. 2). Statistical analysis Serodiagnostic evaluation of the 35 kDa recombinant All data were analyzed by SPSS version 18 and were found protein by ELISA to be non-parametric. A value of P < 0.0001 was considered An indirect ELISA was developed using the 35 kDa re- as significant (Wilcoxon signed-rank test: Z = 30365.000, combinant protein and sera from healthy controls or Mann-Whitney U-test: U = 230, Z = -9.306, P < 0.0001). from patients with human paragonimiasis or other para- The statistical results indicated that there was a significant sitic infections (Table 1) were used to assess the specificity difference between paragonimiasis samples and non- and sensitivity of this assay as an immunodiagnostic tool. paragnimiasis samples, heterologous infected groups and The ELISA conditions were optimized by fixing the con- healthy control. Moreover, the median scores of the para- centration of the secondary antibody at a 1:4000 dilution gonimiasis and non-paragonimiasis groups were 257.78 and then optimizing the primary antibody and recombinant and 123.94, respectively. Table 3 Alignment score of the putative amino acid sequences of the 35 kDa antigen as calculated by the NCBI-BLASX program from clone CE3 Accession No. Sequence description Identity (%) Score (Bits) E-value GAA56617.1 Proactivator polypeptide (Clonorchis sinensis) 48 297 4E-92 XP_012799469.1 Proactivator polypeptide (Schistosoma haematobium) 29 197 3E-34 AAW27625.1 SJCHGC01869 protein (Schistosoma japonicum) 28 237 1E-31 CCD59012.1 Saposin-containing protein (Schistosoma mansoni) 27 245 1E-31 Pothong et al. Parasites & Vectors (2018) 11:322 Page 7 of 10 and positive and negative predictive values of 88.89, 95.51, 74.42 and 98.32%, respectively, and resulted in false po- sitives for only four helminthic infections and one pro- tozoan infection. Specificity of the test using this recombinant antigen is quite high, which was evaluated against 32 different diseases as indicated above. It should be noted by including serum samples from patients in- fected with the three human Paragonimus spp: P. hetero- tremus, P. pseudoheterotremus and P. westermani,we found that the ELISA was not able to discriminate be- tween species-specific infections. However, given the simi- larities in infection transmission, clinical disease and treatment options for these lung fluke infections, having a broad-based diagnostic for human paragonimiasis, regard- less of the species, provides a valuable tool for infection screening and/or diagnoses. The expressed recombinant P. heterotremus protein (CE3), when incorporated into the ELISA immunoassay, not only exhibited the expected high specificity to antibodies from mice immunized with the recombinant 35 kDa protein, but also showed positive reactive and high specificity to antibodies from 32 of 36 human paragonimiasis serum samples. As noted, the other Fig. 2 Reactivity of mouse anti-CE3 clone against the CE3 recombinant four paragonimiasis sera produced the OD-values close to protein. The reactivity of serum samples from mice immunized with the cut-off point. Antibody cross-reactivity is largely re- the recombinant protein from CE3 clone against CE3 recombinant protein was assessed by Western blot analysis. His-tagged CE3 lated to the presence of helminth protein epitopes that are recombinant protein was blotted with anti-his tag antibodies shared in common with similar proteins from other para- (Lanes 1, 2), normal (non-immunized) mouse sera (Lanes 3, 4), or site species resulting in a false positive test outcome, or anti-CE3 clone antibody (Lanes 5, 6). Lane M: protein molecular the individual donor may have been previously infected weight standards with lung flukes. However, only a few cases of other parasitic diseases, opisthorchiasis, strongyloidiasis, Ban- Discussion croftian filariasis, neurocysticercosis and B. hominis in- In the present study, a novel recombinant protein antigen fection cross-reacted with the proactivator polypeptide was produced from a cloned cDNA of P. heterotremus recombinant antigen. Considering location of these para- worms, designated CE3. This insert cDNA sequence ex- sites in patients, as is generally known, B. hominis is an in- hibited closest homology to a proactivator polypeptide of testinal protozoan and mostly asymptomatic, Opisthorchis the liver fluke, Clonorchis, and the encoded a protein anti- viverrini is located in the bile duct and Wuchereria ban- gen that specifically reacted to antibodies directed against crofti is located in the lymphatic. Symptoms typically seen the specific 35 kDa diagnostic band seen in native adult in infections by all three of these parasites are quite differ- worm antigen extracts. The IgG-ELISA used to evaluate ent from lung fluke infections, and therefore, can be more this recombinant protein yielded sensitivity, specificity, easily diagnosed based on clinical presentations. However, Table 4 Cut-off values, sensitivity, specificity, and positive and negative predictive values of the P. heterotremus 35 kDa recombinant fusion protein ELISA. The ELISA was performed using 1 μgof P. heterotremus 35 kDa recombinant fusion protein and 30 negative serum samples diluted at 1:2000. Test results were determined by the OD values of all serum samples under the cut-off value of 0.54 Cut-off value OD Sensitivity Specificity Predictive values (%) at (%) (%) Positive Negative nm Mean + SD 0.44 80 80 80 80 Mean + 2 SD 0.54 100 100 100 100 Mean + 3 SD 0.64 0 100 0 50 Evaluation of test under the cut-off value in this study Mean + 2 SD 0.54 88.89 95.51 74.42 98.32 Pothong et al. Parasites & Vectors (2018) 11:322 Page 8 of 10 Fig. 3 Scatter pattern of ELISA absorbance values was developed by using purified recombinant antigen, and the specificity of this assay was assessed using serum samples from paragonimiasis patients, healthy human controls (negative control) and patients with various parasitic and bacterial diseases (cut-off at a mean + 2 SD = 0.54) S. stercoralis larvae pass the lungs during their develop- produced a partially purified adult worm antigen of ment before moving to the intestine to become adult P. heterotremus using Sephadex column chromatography worms, but most OD-values were lower than the cut-off and subsequently used this antigen preparation in the value. The serum sample exhibiting cross-reactivity above serodiagnosis of human paragonimiasis heterotremus. the cut-off may be a result from a co-infection with Para- The IgG–ELISA constructed using this antigen showed gonimus because it has been previously shown that 100% sensitivity and 100% specificity (seven diseases). A antibodies produced in naturally-acquired opisthorchiasis, partially purified antigen has been prepared from adult strongyloidiasis, and neurocysticercosis do not react with worms of P. heterotremus using an isoelectric focusing cell 35 kDa antigen of adult P. heterotremus worm. We noted (Rotofor, Bio-Rad, Hercules, California, USA). This par- in the study of Yoonuan et al.  that antibody cross- tially purified antigen shows a specific antigen at 31.5 kDa, reactivity of opisthorchiasis, ascariasis and strongyloidiasis which gave ELISA values, 100% sensitivity and 99% speci- also occurs in IgG-ELISA using 35 kDa cathepsin L lack- ficity (12 diseases). Only one of fascioliasis showed a false ing signal peptide (rPpsCatL, recombinant P. pseudoheter- positive . Besides, enzymes in the excretory-secretory otremus cathepsin L). However, although both the products of lung flukes can also be useful in the serodiag- proactivator polypeptide (current study) and the cathepsin nosis of paragonimiasis westermani, as shown for cysteine L proteins share a similar 35-kDa molecular mass, both protease antigens in an IgG-ELISA . In another study appear antigenically distinct based on differences in their was a chicken cystatin capture ELISA for detection of immunoreactivity profiles. Also our antigen did not cross- antibodies to P. westermani. In this assay, the cystatin react with ascariasis patient sera, further distinguishing it binds with fluke cysteine proteinases in excretory- from the recombinant cathepsin L. secretory products, and then is exposed to sera from It is known that serodiagnosis can play an important paragonimiasis patients containing antibodies to the role as a supplementary method of human paragonimia- cycteine proteases. The paragonimiasis cystatin cap- sis diagnosis. An ELISA is a complementary method for ture ELISA showed high reactivity , although na- diagnosis of cerebral paragonimiasis, chronic cerebral tive cysteine proteinases are highly conserved and paragonimiasis, pulmonary and ectopic pulmonary para- possess common epitopes that are shared among gonimiasis [14, 15]. In 1991, Indrawati et al.  many Paragonimus species including P. westermani, Pothong et al. Parasites & Vectors (2018) 11:322 Page 9 of 10 P. miyazakii and P. ohirai , thereby limiting their contrast to diagnoses, symptoms and history of intermedi- immunodiagnostic potential. ate host consumption are helpful for physicians to predict In addition to recombinant technology allowing the early stages of disease. However, for clinically unpredicted production of an unlimited amount of antigen, its appli- or suspected cases of paragonimiasis, which may result cation in generating a DNA recombinant protein of P. from immature or mature Paragonimus worms located westermani eggs resulted in a significant increase in the outside the lungs, serodiagnosis can be of great assistance sensitivity and specificity of an ELISA for paragonimiasis as an adjunct diagnostic tool. Therefore, the incorporation diagnosis to 90.2 and 100%, respectively, and also useful of the recombinant proactivator polypeptide as a sero- for an epidemiological study of paragonimiasis . A logical test antigen shows promise for the detection of recently developed IgG-ELISA using the recombinant human paragonimiasis. cysteine protease antigen (21 kDa) derived from cDNA of the Paragonimus skrjabini juvenile stage was reported Additional files to have a sensitivity of 95.5% and no cross-reaction with antibodies from the other human diseases in the study Additional file 1: Figure S1. Sizes of the inserted cDNA in randomly by Yu et al. , i.e. echinococcosis granulosus, taeniasis selected clones from the P. heterotremus cDNA library. PCR products resulting from amplification with the pJET forward primer and pJET reverse solium, schistosomiasis japonicum and trichinellosis primer were separated on a 1.2% agarose gel. The sizes of inserted cDNA spiralis . Another recombinant antigen is the 35 kDa fragments from random clones (Lanes 1–14) were determined by cathepsin L lacking signal peptide (rPpsCatL), which is comparison with the Kapa universal ladder (Lane M). (TIF 1102 kb) produced from the cDNA of P. pseudoheterotremus Additional file 2: Figure S2. The sizes of CE3 PCR products. PCR was performed on CE3 and the resulting products were run on a 1.2% worms and belongs to the cysteine protease group. This agarose gel. Lane M: marker; Lane 1: CE3 clone. (TIF 780 kb) rPpsCatL was used in IgG-ELISA for paragonimiasis het- Additional file 3: Figure S3. Overview of clone CE3. DNAMan software erotremus, resulting in 100% sensitivity and 95.6% speci- was used to produce a schematic overview of clone CE3. (TIF 405 kb) ficity (27 diseases). Cross-reactivity occurred from ten cases of strongyloidiasis, toxocariasis, Brugian filariasis, Abbreviations ascariasis, opisthorchiasis and fascioliasis . Despite ABTS: 2,2-azino-di-(3-ethyl-benzthiazoline sulfonate); AUC: area under the the fact that the recombinant antigens used in that study ROC curve; CI: confidence interval; ELISA: enzyme-linked immunosorbent assay; His6: histidine 6; IgG: immunoglobulin G; IP: intraperitoneal route; (rPpsCatL) and the current study (proactivator polypep- IPTG: isopropyl-l-thio-β-D-galactopyranoside; Ni-NTA: nickel-nitrilotriacetic tide) possessed the same molecular weight, the sensitivity acid; OD: optical density; PBS: phosphate-buffered saline; ROC: receiver and specificity results for their corresponding IgG-ELISAs operating characteristic; rPpsCatL: recombinant Paragonimus pseudoheterotremus cathepsin L; SDS-PAGE: sodium dodecyl sulphate- suggest that they differ in antigenic reactivity to anti- polyacrylamide gel electrophoresis bodies. However, both of these recombinant antigens were cross-reactive with antibodies from strongyloidiasis and Acknowledgments opisthorchiasis. Further studies of the P. heterotremus We would like to thank Dr Tippayarat Yoonuan, Mr Nirundorn Homsuwan proactivator recombinant antigen could lead to improve- and the staff of the Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Thailand, and the staff of the Department of ments in its diagnostic specificity and sensitivity. For ex- Pathobiological Sciences, School of Veterinary Medicine, University of ample, identification and immune testing of individual Wisconsin, Madison, USA, for providing laboratory facilities and assistance in antibody-reactive epitopes through epitope mapping of laboratory techniques. the protein could lead to highly-specific single-epitope Funding diagnostic testing formats. Currently we are exploring this Financial support from the Thailand Research Fund through the Royal Golden and other, serodiagnostic tests for the detection of ectopic Jubilee PhD Programme (Grant No. PHD/0269/2549) is acknowledged. human paragonimiasis infections. Availability of data and materials Conclusions The sequence of interested target clone CE3 was submitted to the GenBank database under the accession number KX180136. The data used in the In the present study, a novel recombinant protein antigen present study are available from the corresponding author upon request. was produced from the cDNA of P. heterotremus worms. This antigen specifically responded to antibody directed Authors’ contributions against the specific diagnostic band of native 35 kDa anti- KP (PhD student) performed all experiments, provided data and results and gen. The insert of cDNA is suggested to be homologous contributed to manuscript preparation. CK supervised the animal experiments. TK supervised on molecular aspects of the study experiments. to a proactivator polypeptide. The IgG-ELISA was used to DW supervised the molecular part of the experiments involving clinical evaluate this recombinant protein and this resulted in samples in Thailand. TPY supervised KP in the development of molecular sensitivity, specificity, and positive and negative predictive part of the experiments at the University of Wisconsin and advised manuscript preparation. PD provided financial support from the Royal values of 88.89, 95.51, 74.42 and 98.32%, respectively. False Golden Jubilee PhD Program through TRF and supervised all aspects of the positives were found from only a few cases of four thesis experiments in Thailand and advised manuscript preparation. All helminthic infections and one protozoan infection. In authors read and approved the final manuscript. Pothong et al. Parasites & Vectors (2018) 11:322 Page 10 of 10 Ethics approval and consent to participate 13. Yoonuan T, Nuamtanong S, Dekumyoy P, Phuphisut O, Adisakwattana All animal protocols were approved by the Faculty of Tropical Medicine- P. Molecular and immunological characterization of cathepsin L-like Animal Care and Use Committee (FTM-ACUC) of Mahidol University (Project cysteine protease of Paragonimus pseudoheterotremus. Parasitol Res. No. FTM-ACUC.2011/008). The human sera used in the present study were 2016;115:4457–70. stored specimens that had been used in previous serological surveys/diagnostic 14. Choo JD,Suh BS, Lee HS,Lee JS, Song CJ,Shin DW,Lee YH.Chronic testing. All patient identifiers had been removed, and no patient consent was cerebral paragonimiasis combined with aneurysmal subarachnoid required for this study. All procedures were approved by the Human Ethics hemorrhage. Am J Trop Med Hyg. 2003;69:466–9. Committee of the Faculty of Tropical Medicine, Mahidol University (Project No. 15. Chen Z, Zhu G, Lin J, Wu N, Feng H. Acute cerebral paragonimiasis presenting TMEC 11-040). as hemorrhagic stroke in a child. Pediatr Neurol. 2008;39:133–6. 16. Ikeda T, Oikawa Y, Nishiyama T. Enzyme-linked immunosorbent assay using cysteine proteinase antigens for immunodiagnosis of human paragonimiasis. Competing interests Am J Trop Med Hyg. 1996;55:434–7. The authors declare that they have no competing interests. 17. Ikeda T. Cystatin capture enzyme-linked immunosorbent assay for immunodiagnosis of human paragonimiasis and fascioliasis. Am J Trop Med Hyg. 1998;59:286–90. Publisher’sNote 18. Lee JS, Lee J, Kim SH, Yong TS. Molecular cloning and characterization of a Springer Nature remains neutral with regard to jurisdictional claims in major egg antigen in Paragonimus westermani and its use in ELISA for the published maps and institutional affiliations. immunodiagnosis of paragonimiasis. Parasitol Res. 2007;100:677–81. 19. Yu S, Zhang X, Chen W, Zheng H, Ai G, Ye N, Wang Y. Development of an Author details 1 immunodiagnosis method using recombinant PsCP for detection of Department of Helminthology, Faculty of Tropical Medicine, Mahidol 2 Paragonimus skrjabini infection in human. Parasitol Res. 2017;116:377–85. University, Bangkok 10400, Thailand. Mahidol-Bangkok School of Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand. Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand. Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53706, USA. Received: 6 November 2017 Accepted: 29 April 2018 References 1. World Health Organization. Control of foodborne trematode infections: report of a WHO study group. Geneva: World Health Organization; 1995. 2. Vijayan VK. Tropical parasitic lung diseases. Indian J Chest Dis Allied Sci. 2008;50:49–66. 3. Vajrasthira S, Paragonimiasis in Thailand. In: Harinasuta C, editor. Proceedings of the Fourth Southeast Asian Seminar on Parasitology and Tropical Medicine, Schistosomiasis and Other Snail-transmitted Helminthiases. Bangkok: Thai Watana Panich Press; 1969. p. 299–304. 4. Sugiyama H, Morishima Y, Rangsiruji A, Binchai S, Ketudat P, Kawanaka M. Application of multiplex PCR for species discrimination using individual metacercariae of Paragonimus occurring in Thailand. Southeast Asian J Trop Med Public Health. 2006;37(Suppl. 3):48–52. 5. Waikagul J. A new species of Paragonimus (Trematoda: Troglotrematidae) from a cat infected with metacercariae from mountain crabs Larnaudia larnaudii. J Parasitol. 2007;93:1496–500. 6. Indrawati I, Chaicumpa W, Setasuban P, Ruangkunaporn Y. Studies on immunodiagnosis of human paragonimiasis and specific antigen of Paragonimus heterotremus. Int J Parasitol. 1991;21:395–401. 7. Kong Y, Ito A, Yang HJ, Chung YB, Kasuya S, Kobayashi M, et al. Immunoglobulin G (IgG) subclass and IgE responses in human paragonimiases caused by three different species. Clin Diagn Lab Immunol. 1998;5:474–8. 8. Dekumyoy P, Setasuban P, Waikagul J, Yaemput S, Sa-nguankiat S. Human lung fluke (Paragonimus heterotremus): differentiation of antigenic proteins of adult worms by enzyme–linked immunoelectrotransfer blot technique. Southeast Asian J Trop Med Public Health. 1995;26:434–8. 9. Dekumyoy P, Waikagul J, Eom KS. Human lung fluke Paragonimus heterotremus: differential diagnosis between Paragonimus heterotremus and Paragonimus westermani infections by EITB. Trop Med Int Health. 1998;3:52–6. 10. Wongkham C, Maleewong W, Intapan P, Morakote N, Chaicumpa W. Partially purified antigens of Paragonimus heterotremus for serodiagnosis of human paragonimiasis. Southeast Asian J Trop Med Public Health. 1994;25:176–80. 11. Ito A, Xiao N, Liance M, Sato MO, Sako Y, Mamuti W, et al. 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