Fasciolosis, caused by Fasciola hepatica and F. gigantica, is an impediment to the livestock industry’s expansion and has a massively negative socio-economic impact due to its widespread prevalence in livestock. It is a waterborne zoonosis affecting human populations in the countries where rural economies are associated with livestock rearing. Conventional diagnosis of Fasciola infection is done by detecting parasite eggs in the faeces of infected animals or by immunological methods. Accurate and quick immunodiagnosis of Fasciola infection in animals and humans is based on the detection of antibodies and specific antigens expressed in the prepatent stage of the parasite. Both molecular and serodiagnostic tests developed thus far have enhanced the reliability of Fasciola diagnosis in both man and animals but are not widely available in resource-poor nations. A pen-side diagnostic test based on a lateral flow assay or a DNA test like loop-mediated isothermal amplification (LAMP) would be simple, fast, and cost-effective, enabling clinicians to treat animals in a targeted manner and avoid the development of drug resistance to the limited flukicides. This review focuses on the recent advances made in the diagnosis of this parasite infection in animals and humans.

To evaluate the diagnostic accuracy of three types of antigenic preparations from Strongyloides venezuelensis infective larvae for detection of serum IgG anti-Strongyloides antibodies by enzyme-linked immunosorbent assay (ELISA). Soluble somatic fractions (SSF) and membrane somatic fractions (MSF) and excretory−secretory (E/S) products from S. venezuelensis infective larvae were evaluated against 71 sera from individuals with strongyloidiasis, 105 sera from healthy individuals, and 84 sera from individuals with other helminth infections. Using an ELISA cut-off for 100% sensitivity, E/S products were 97.88% specific followed by MSF (93.12%) and then by SSF (85.2%). The occurrence of cross-reactivity with other helminths was 4.76% (4/84) with E/S products, 8.33% (7/84) with MSF, and 17.86% (15/84) with SSF. For a cut-off for 100% specificity, E/S products showed a sensitivity of 88.73% whereas MSF and SSF showed sensitivities of 59.15% and 53.52%, respectively. In conclusion, E/S products were the best antigenic option for the serodiagnosis of human strongyloidiasis.

Cerebral angiostrongyliasis is a central nervous system disease caused by Angiostrongylus cantonensis and can produce eosinophilic meningitis or meningoencephalitis in humans. Sero-immunological techniques, such as the enzyme-linked immunosorbent assay (ELISA) and immunoblotting, are most commonly used for the diagnosis of human angiostrongyliasis. However, diagnosis in remote areas remains problematic because sophisticated equipment and specialized skills are required. To overcome, we have developed the immunochromatographic test (ICT) kit for rapid serological diagnosis of angiostrongyliasis through the detection of anti-A. cantonensis-specific antibodies in human serum. A recombinant A. cantonensis galectin-2 (rAcGal2) from young adult female worms was used as an antigen for the ICT kit development. Diagnostic values were evaluated and compared using the ELISA. The sensitivity, specificity and positive and negative predictive values of the ICT kit were 87.0, 96.5, 94.6 and 91.4%, respectively, and those of the ELISA were 91.0, 97.2, 95.8 and 94.0%, respectively. The concordance of the ICT kit was 93.9%. We, thus, determined that the ICT kit is sensitive and specific and provides reliable diagnostic results. It is rapid and simple to perform and can be utilized for both point-of-care diagnosis in the bedside laboratory and epidemiological surveys in endemic regions where access to diagnostic equipment is limited.

There is no suitable vaccine against human visceral leishmaniasis (VL) and available drugs are toxic and/or present high cost. In this context, diagnostic tools should be improved for clinical management and epidemiological evaluation of disease. However, the variable sensitivity and/or specificity of the used antigens are limitations, showing the necessity to identify new molecules to be tested in a more sensitive and specific serology. In the present study, an immunoproteomics approach was performed in Leishmania infantum promastigotes and amastigotes employing sera samples from VL patients. Aiming to avoid undesired cross-reactivity in the serological assays, sera from Chagas disease patients and healthy subjects living in the endemic region of disease were also used in immunoblottings. The most reactive spots for VL samples were selected, and 29 and 21 proteins were identified in the promastigote and amastigote extracts, respectively. Two of them, endonuclease III and GTP-binding protein, were cloned, expressed, purified and tested in ELISA experiments against a large serological panel, and results showed high sensitivity and specificity values for the diagnosis of disease. In conclusion, the identified proteins could be considered in future studies as candidate antigens for the serodiagnosis of human VL.

We have previously shown that the C-terminal region of the intermediate subunit of Entamoeba histolytica galactose- and N-acetyl-D-galactosamine-inhibitable lectin (C-Igl) is a useful antigen for serodiagnosis of amebiasis. An immunochromatographic kit was developed using fluorescent silica nanoparticles coated with C-Igl prepared in Escherichia coli. Samples for examination were added to the freeze-dried particles and then applied to the immunochromatographic device, in which a test line on the membrane was also coated with C-Igl. Fluorescent intensity was measured using a hand-held reader. In an evaluation of the kit using a human monoclonal antibody, the minimum amount of C-Igl specific antibody showing positive results was 100 pg. In the evaluation of serum samples with different antibody titers in indirect immunofluorescent antibody tests in the kit, 20 µL of serum was sufficient to obtain positive results at 30 min. Serum samples from symptomatic patients with amebic colitis and amebic liver abscess and those from asymptomatic E. histolytica-cyst carriers showed positive results in the kit. Based on evaluation using sera from healthy controls and patients with other infectious diseases, the sensitivity and specificity of the kit were 100 and 97.6%, respectively. Therefore, we conclude that the newly developed kit is useful for rapid serodiagnosis of amebiasis.

Due to the lack of an effective prophylactic intervention and diagnosis, human liver fluke Clonorchis sinensis continues to afflict a large human population, causing a chronic inflammatory bile duct disease. With an aim to identify target antigens for sensitive serodiagnosis, adenylate kinase 3 of C. sinensis (CsAK3) was successfully expressed in soluble form in Escherichia coli by fusion to an RNA-interacting domain derived from human Lys-tRNA synthetase and purified by Ni2+-affinity chromatography. Anti-CsAK3 serum was raised by immunization of mice, and Western blotting confirmed that CsAK3 was expressed in adult-stage C. sinensis. Histochemical analysis showed that CsAK3 was localized to the subtegumental tissue of C. sinensis and was excreted into the bile duct of the host. When tested against sera from various parasite-infected patients by enzyme-linked immunosorbent assay, the recombinant CsAK3 elicited a specific response to C. sinensis-infected sera. The results suggest that CsAK3, either alone or in combination with other antigens, could be used for improving the clinical diagnosis of clonorchiasis.

Scabies is a parasitic disease caused by the ectoparasite Sarcoptes scabiei, affecting different mammalian species, including rabbits, worldwide. In the present study, we cloned and expressed a novel inorganic pyrophosphatase, Ssc-PYP-1, from S. scabiei var. cuniculi. Immunofluorescence staining showed that native Ssc-PYP-1 was localized in the tegument around the mouthparts and the entire legs, as well as in the cuticle of the mites. Interestingly, obvious staining was also observed on the fecal pellets of mites and in the integument of the mites. Based on its good immunoreactivity, an indirect enzyme-linked immunosorbent assay (ELISA) using recombinant Ssc-PYP-1 (rSsc-PYP-1) as the capture antigen was developed to diagnose sarcoptic mange in naturally infected rabbits; the assay had a sensitivity of 92·0% and specificity of 93·6%. Finally, using the rSsc-PYP-1-ELISA, the Ssc-PYP-1 antibody from 10 experimentally infected rabbits could be detected from 1 week post-infection. This is the first report of S. scabiei inorganic pyrophosphatase and the protein could serve as a potential serodiagnostic candidate for sarcoptic mange in rabbits.

Euthanasia of infected dogs is one of the measures adopted in Brazil to control visceral leishmaniasis (VL) in endemic areas. To detect infected dogs, animals are screened with the rapid test DPP® Visceral Canine Leishmaniasis for detection of antibodies against K26/K39 fusion antigens of amastigotes (DPP). DPP-positives are confirmed with an immunoenzymatic assay probing soluble antigens of promastigotes (ELISA), while DPP-negatives are considered free of infection. Here, 975 dogs from an endemic region were surveyed by using DPP, ELISA and real-time PCR (qPCR) for the diagnosis of VL. When DPP-negative dogs were tested by qPCR applied in blood and lymph node aspirates, 174/887 (19·6%) were positive in at least one sample. In a second sampling using 115 cases, the DPP-negative dogs were tested by qPCR in blood, lymph node and conjunctival swab samples, and 36/79 (45·6%) were positive in at least one sample. Low-to-moderate pairwise agreement was observed between all possible pair of tests. In conclusion, the official diagnosis of VL in dogs in Brazilian endemic areas failed to accuse an expressive number of infected animals and the impact of the low accuracy of serological tests in the success of euthanasia-based measure for VL control need to be assessed.

Ovine babesiosis is one of the most important tick-borne haemoparasitic diseases of small ruminants. The ovine parasite Babesia sp. Xinjiang is widespread in China. In this study, recombinant full-length XJrRAP-1aα2 (rhoptry-associated protein 1aα2) and C-terminal XJrRAP-1aα2 CT of Babesia sp. Xinjiang were expressed and used to evaluate their diagnostic potential for Babesia sp. Xinjiang infections by indirect enzyme-linked immunosorbent assay (ELISA). Purified XJrRAP-1aα2 was tested for reactivity with sera from animals experimentally infected with Babesia sp. Xinjiang and other haemoparasites using Western blotting and ELISA. The results showed no cross-reactivities between XJrRAP-1aα2 CT and sera from animals infected by other pathogens. High level of antibodies against RAP-1a usually lasted 10 weeks post-infection (wpi). A total of 3690 serum samples from small ruminants in 23 provinces located in 59 different regions of China were tested by ELISA. The results indicated that the average positive rate was 30·43%, and the infections were found in all of the investigated provinces. This is the first report on the expression and potential use of a recombinant XJrRAP-1aα2 CT antigen for the development of serological assays for the diagnosis of ovine babesiosis, caused by Babesia sp. Xinjiang.

The aim of this study was to fractionate and partially characterize the antigenic extract of filariform larvae of Strongyloides venezuelensis in ion-exchange resin diethylaminoethyl sepharose (DEAE), to obtain antigenic fractions potentially applicable in immunoassays. Somatic antigen (SA) and its fractions DEAE S1 and DEAE S2 - which interacted with the resin - were evaluated by 1-dimensional electrophoresis to obtain protein profiles. SA and its fractions were tested in serum samples for IgG detection by ELISA. Serum samples (n = 155) were analysed: 50 from strongyloidiasis patients (G1), 55 from patients with other parasitic infections (G2) and 50 from healthy volunteers. Sensitivity (Se), specificity (Sp), area under curve (AUC) and likelihood ratios (LR) were calculated. The DEAE S2 fraction provided a high diagnostic value for IgG detection (Se 92·0%, Sp 91·4%, AUC 0·981, LR+ 10·75, LR − 0·09). In conclusion, the DEAE S2 fraction would probably be a source of immunodominant polypeptides for IgG detection in human strongyloidiasis serodiagnosis.

Sera of Sudanese patients with active infections of Schistosoma mansoni or S. haematobium were tested in immunoblots for their reactivity with Mr31/32 000 proteins of adult S. mansoni (Sm31/32). All sera from patients with intestinal (n=123) and all but one from those with urinary schistosomiasis (n=35) had antibodies against Sm31/32. These and additional data suggest that both specificity and sensitivity of Sm31/32 to detect schistosome infections are close to 100%. Antibodies against these proteins developed also in monkeys after experimental infection with S. haematobium. Sm31/32 antigens reacted in immunoblots as a doublet with most S. haematobium-patient sera and as a broad band with many S. mansoni-sera suggesting that at least two components are present in the molecular weight region of Mr31/32 000. The data demonstrate the potential use of Sm31/32 from adult worms to diagnose patients with intestinal or urinary schistosomiasis in endemic areas.

The sensitivity and specificity of double immunodiffusion (DID), indirect haemagglutination test (IHA), immunoelectrophoresis (IEP), counterimmunoelectrophoresis (CIEP) and enzyme-linked immunosorbent assay (ELISA) were evaluated and compared using saline extracted of Taenia solium larval scolex and its Sephadex G-200 fractionated 1st and 2nd peak as antigens. Various immunodiagnostic tests gave different results with different antigens. Highest sensitivity (92·5%) was obtained with ELISA using 1st peak antigen and lowest sensitivity was obtained with DID. However, 88·4% and 84·6% sensitivity was obtained with IHA and CIEP respectively using scolex antigen. CIEP gave better results as compared to IEP. Crude antigen gave high sensitivity but less specificity. It was concluded that CIEP can be used as a field test for the anti-mortem diagnosis and ELISA can be employed for laboratory confirmation of T. solium cysticercosis in pigs using frationated 1st peak antigen.

A specific enzyme-linked immunosorbent assay, based on a 48 kDa metallo-proteinase isolated from the frontal glands of a porocephalid pentastomid (Porocephalus crotali) in rat intermediate hosts, is described. SDS–PAGE analysis of whole nymphal homogenate, followed by Western blotting and probing with immune rat serum, revealed that only 2 protein bands label consistently; these are the 48 kDa metallo-proteinase and a 150 kDa metallo-proteinase from the body fluid, but only the former labels strongly. Furthermore this response is detected earlier and persists much longer. These characteristics uniquely qualify the 48 kDa metallo-proteinase as a diagnostic antigen. We review the literature concerning the related porocephalid Linguatula serrata from the nasal cavities of domestic dogs, which can zoonotically infect man principally through the ingestion of eggs. We conclude that the high prevalences in dogs in certain areas, most notably the Middle East, must inevitably signal significant human infections. Our ELISA can be readily adapted for the serodiagnosis of linguatulosis and this should establish whether our prediction is true.

The enzyme-linked immunosorbent assay (ELISA) was used to survey human sera for antibodies to Encephalitozoon cuniculi using spores obtained from in vitro cultures as antigen. Sera were obtained from patients with tropical diseases, neurological and renal disorders, patients who were HIV positive and those who had been tested for HIV but found to be negative. Sera from inhabitants of the village of Jali, The Gambia and from healthy blood donors were also examined. Numerous sera from all groups except the blood donors gave positive ELISA reactions at dilutions of 1:400. On titration, those with titres of 1:400 were reclassified as negative. Antibody titres of 1:800 and above were considered to be indicative of past or present infections with E. cuniculi. Many of these ELISA seropositives were also positive by IFAT or PAP. When examined by Western blotting of SDS–PAGE protein profiles of E. cuniculi spores, sera from many patients who had a tropical association reacted with the characteristic profiles shown by known positive mouse and rabbit sera. Others in the tropical group showed antibody binding to some but not all of the immunodominant polypeptides and yet others were negative in spite of their reactivity in the ELISA, IFAT or PAP test. Less agreement between ELISA and Western blotting results was obtained with the other groups of patients, although reactivity with one or more of the major polypeptide bands was sometimes seen. Serum from one blood donor, examined by ELISA and Western blotting, was positive. Differences in the methods of antigen preparation and of epitopes recognized by individuals may account for different reactivities in the tests. It is concluded that infections of E. cuniculi are common in the tropics and that reactivations of these infections might be a hazard to AIDS patients.

A low molecular weight (15 kDa) surface antigen of the cattle filarial nematode, Setaria digitata, was earlier shown to be specifically recognized by the antibodies from human bancroftian filarial (Mf positive) patients' sera (Theodore & Kaliraj, 1990). The filarial specific antibodies bound to a 15 kDa peptide in preparative Western blots were eluted and employed in screening of candidate antigens expressed in the genomic library of Wuchereria bancrofti at the IgG4 subclass antibody level. A recombinant clone (λ WbG7) reacting strongly with filarial sera but poorly with sera from patients infected with non-filarial helminths was selected for further studies. The 2 kb DNA insert of the clone λ WbG7 was recloned into a pMAL vector and the recombinant clone pGT7 thus obtained was over-expressed and affinity purified. The purified 105 kDa fusion protein of the clone pGT7 was specific and was not recognized by the non-filarial sera at the IgG4 level. All microfilaraemic individuals were positive by IgG4 assay. However, similar attempts to diagnose by filarial-specific IgE assays failed to recognize microfilaraemic individuals. Moreover, by filarial-specific IgG4 assays, the endemic normals were distinctly divided into two groups, showing higher and lower recognition for this antigen indicating current and past/no infection. Among the filarial-IgG4 (assay)-positive ‘endemic normals’, 14% showed ‘microfilariae’ during repeated peripheral night blood examination, confirming the validity of the recombinant antigen, pGT7 based assay.

The purpose of this study was to prepare recombinant K26 antigen from Leishmania infantum and evaluate its performance by enzyme-linked immunosorbent assay (ELISA) test for serodiagnosis of visceral leishmaniasis (VL) in endemic regions of Iran. The results were compared with those obtained by direct agglutination test (DAT) and whole cell ELISA using crude parasite antigen. Of 93 sera from patients with confirmed VL, 90 sera were positive with rK26 ELISA (sensitivity=96·8%), whereas 85 sera were positive with DAT (sensitivity=91·4%) and 89 sera were positive with whole cell ELISA (sensitivity=95·7%). Of 130 subjects who either had other infectious diseases (n=30) or were healthy (n=100), rK26 ELISA were negative in all cases (specificity=100%), whereas DAT were negative in 116 cases (specificity=89·2%) and whole cell ELISA was negative in 114 cases (specificity=87·7%). The results of this study indicate that the rK26 ELISA is more sensitive and specific than conventional methods and could be used for reliable diagnosis of VL caused by Leishmania infantum.

To improve the diagnosis of human fascioliasis caused by Fasciola gigantica, we developed a peptide-based enzyme-linked immunosorbent assay (peptide-based ELISA) based on the detection of specific IgG4 subclass antibody. Two identified B-cell epitopes of F. gigantica cathepsin L1 were synthesized as single synthetic peptides, acetyl-DKIDWRESGYVTELKDQGNC-carboxamide (peptide L) and acetyl-DKIDWRESGYVTEVKDQGNC-carboxamide (peptide V), and their diagnostic potential was evaluated. The sera of 25 patients infected with F. gigantica, 212 patients with other parasitic infections, 32 cholangiocarcinoma patients and 57 healthy controls were analysed. The sensitivity, specificity, accuracy, and positive and negative predictive values of this assay were the same with both peptides at 100%, 99·7%, 99·7%, 96·2% and 100%, respectively. These highly sensitive and specific peptide-based ELISAs for the detection of specific IgG4 antibody could be useful for laboratory diagnosis of human fascioliasis in future large-scale surveys throughout Southeast Asia where this disease is prevalent.

Since an indirect fluorescence immunoassay (IFA) for the detection of specific antibodies against Babesia divergens in human sera is not commercially available, an in-house prepared B. divergens IFA for the examination of bovine sera was established for serological studies in humans. To determine whether the described IFA is appropriate for such studies, 2 B. divergens antigens (of human or bovine origin) were tested using serum samples obtained from febrile human patients with a history of ‘tick bite’. Sera from other species of animals infected with B. divergens, Babesia EU1, B. microti or B. canis were also included for comparative purposes. All serum samples were also tested using a commercially available IFA for the detection of antibodies against B. microti, and the results compared with those obtained using blood smear and molecular techniques. This study showed that the evaluation and standardization of a B. divergens IFA for testing human sera is critical and that different B. divergens antigens provide different end-point titres of antibodies, leading to false negative or positive results. Serological cross-reactivity between B. divergens and Babesia EU1 needs to be taken into account when interpreting IFA results.

Both CEF6, a cation-exchange fraction of soluble Schistosoma mansoni egg antigens (SEA), composed of the 2 antigens, alpha-1 and omega-1, and haemocyanin from the keyhole limpet, Megathura crenulata, have shown potential for immunodiagnosis of human schistosomiasis. Possible cross-reactivity between antigens in SEA and keyhole limpet haemocyanin (KLH) was explored by Western immunoblotting and enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with KLH, SEA, CEF6, alpha-1, omega-1, or egg antigen k5. Both immunoassays revealed a high degree of serological cross-reactivity between the schistosome egg antigens and KLH, much of it due to sodium periodate- sensitive epitopes. Cross-reactivity with schistosome antigens with proven diagnostic efficacy may thus, in part, explain the usefulness of KLH for the diagnosis of human schistosomiasis mansoni.

The kinetics of serum antibody response against infection with Echinococcus multilocularis eggs was evaluated in AKR mice. The animals were infected by oral inoculation with 300 parasite eggs, and necropsied at 1, 2, 4, 6, 9, 12 and 16 weeks post-infection (p.i.), respectively. The parasite formed the laminated layer at 4 weeks p.i., the brood capsule with a massive proliferation of germinal cells at 9 weeks p.i. and protoscoleces at 16 weeks p.i. Serum antibody responses of the mice to antigen preparations from metacestodes of different stages and protoscoleces were evaluated by ELISA, immunoblotting and immunohistochemistry. In ELISA, the antibody responses began to increase at 4 weeks and became more apparent at 9 weeks p.i. and thereafter. Immunoblots using sera collected at 16 weeks p.i. showed some common bands among the 3 different antigen preparations. In addition to this, the germinal cells and brood capsules of mature metacestodes were stained strongly in an immunohistochemical study. From above, it is suggested that some antigen molecules are expressed in the parasite through these stages and stimulated host antibody responses.