Qualitative evaluation of selective tests for detection of Neospora hughesi antibodies in serum and cerebrospinal fluid of experimentally infected horses

Neospora hughesi is a newly recognized protozoan pathogen in horses that causes a myeloencephalitis similar to Sarcocystis neurona. There are no validated serologic tests using the gold standard sera that are currently available to detect specific N. hughesi antibodies and, thus, no tests available to detect antemortem exposure or estimate seroprevalence in the horse. The objectives of the present study were to establish a bank of gold standard equine sera through experimental infections with N. hughesi and to assess several serologic tests for the detection of related protozoan antibodies. Seven horses were inoculated with N. hughesi tachyzoites, and 7 horses received uninfected cell culture material. The horses were monitored, and blood and cerebrospinal fluid were collected repeatedly over a 4-mo period. With the sera, 4 different serologic techniques were evaluated. including a whole-parasite lysate enzyme-linked immunosorbent assay (ELISA), a recombinant protein ELISA, a modified direct agglutination test, and an indirect fluorescent antibody test. Qualitative and quantitative evaluation of the results showed that the N. hughesi indirect fluorescent antibody test (IFAT) consistently discriminated between experimentally infected and noninfected horses, using a cutoff of 1:640. Sera from 3 naturally infected horses had titers >1:640. Cerebrospinal fluid in all but I infected horse had very low N. hughesi IFAT titers (<1:160), starting at postinoculation day 30.     

Prevalence of antibodies to Neospora sp. in horses from Alabama and characterisation of an isolate recovered from a naturally infected horse [corrected

An IFAT was used to determine the prevalence of Neospora-specific IgG antibodies in serum from Alabama horses. Serum samples (n = 536) were from asymptomatic horses routinely submitted for equine infectious anaemia virus infection testing. We also subjected a 13-year-old horse with CNS disease to necropsy examination for isolation and in vitro cultivation of protozoal organisms. In antemortem tests, this horse was positive for antibodies to Neospora sp. in the IFAT and western immunoblot. Results of the prevalence survey indicated that IgG antibodies to Neospora were present in 62 (11.5%) of the 536 serum samples. Endpoint titres for the positive samples were 1:50 (35/6.5%), 1:100 (19/3.5%), 1:200 (7/1.3%) and 1:1600 (1/0.2%). Tachyzoites were first seen in cultured bovine turbinate cells 32 days after inoculation with spinal cord homogenates from the horse with CNS disease. Tachyzoites reacted with known N. caninum-positive serum from horses, cows, dogs and mice, but did not react with murine anti-Toxoplasma gondii or equine anti-Sarcocystis neurona serum. Ultrastructural features of tachyzoites and results of comparison of tachyzoite immunodominant proteins revealed that they were identical to those of N. hughesi, a species described recently from a naturally infected horse. The isolate recovered from the naturally infected horse in the present study (designated NA1) is thought to be an isolate of N. hughesi, although confirmation of this awaits additional molecular characterisation. These results provide some additional evidence that N. hughesi is a valid species and that Neospora infections in horses may occur in widely separated geographic regions of the United States.     

Differentiation of Neospora hughesi from Neospora caninum based on their immunodominant surface antigen, SAG1 and SRS2

Neospora hughesi is a newly recognised parasite that is closely related to Neospora caninum, and is a cause of equine protozoal myeloencephalitis. We have characterised two N. hughesi immunodominant tachyzoite antigens which exhibit antigenic and molecular differences from the homologous tachyzoite antigens on N. caninum. These antigens on N. hughesi are referred to as NhSAG1 and NhSRS2, using the same mnemonics as used for the N. caninum antigens (NcSAG1 and NcSRS2), and are homologous to Toxoplasma gondii surface antigen 1 (SAG1) and SAG1-related sequence 2 (SRS2). The NcSAG1 and NcSRS2 were antigenically conserved in six different N. caninum isolates from cattle and dogs. The two equine-derived Neospora isolates, one designated as N. hughesi, were similar to each other but different from N. caninum. There was 6% difference in amino acid identity between NcSAG1 and NhSAG1, whereas there was a 9% difference when NcSRS2 and NhSRS2 were compared. The polymorphism of these genes and their corresponding proteins provide additional markers which can be used to distinguish N. caninum from N. hughesi.     

Characterization of the Oregon isolate of Neospora hughesi from a horse

Neospora hughesi was isolated in cell cultures inoculated with homogenate of spinal cord from a horse in Oregon. Tachyzoites of this Oregon isolate of N. hughesi were maintained continuously by cell culture passage and tachyzoites were infective to immunosuppressed mice. Gamma interferon gene knockout (KO) mice injected with tachyzoites developed fatal myocarditis and numerous tachyzoites were seen in lesions. Gerbils (Meriones unguiculatus) inoculated with tachyzoites developed antibodies (> or = 1:500) as indicated by the Neospora caninum agglutination test but did not develop clinical signs, and Neospora organisms were not demonstrable in their tissues. Tissue cysts were not found in gerbils, nude mice, KO mice, immunosuppressed outbred Swiss Webster mice, or BALB/c mice injected with the Oregon isolate of N. hughesi. Ultrastructurally, tachyzoites of the Oregon isolate from the myocardium of infected KO mice and from cell culture were similar to N. caninum tachyzoites. Western blot analysis using NcSAG1 and NcSRS2 polyclonal and monoclonal antibodies and characterization of the internal transcribed spacer 1 sequences from the equine isolates and different isolates of N. caninum from dogs and cattle indicated that the Oregon isolate of N. hughesi is distinct from N. caninum isolates from cattle and dogs.     

Molecular comparison of the dense granule proteins GRA6 and GRA7 of Neospora hughesi and Neospora caninum

Neospora hughesi is a recently described apicomplexan parasite that has been associated with several cases of equine protozoal myeloencephalitis. The biology of this new parasite is just beginning to be defined. Towards this understanding, we report important differences between the nucleotide and deduced amino acid sequences of the dense granule proteins GRA6 and GRA7 of N. hughesi and Neospora caninum. This information can be used to differentiate the two species and contribute to further understanding of the prevalence and biology of N. hughesi. The newly defined proteins of N. hughesi are referred to as NhGRA6 and NhGRA7 in keeping with the protocol for naming homologous proteins of the Apicomplexa. Genes of the two dense granule proteins of N. hughesi (isolate Nh-A1) and four different isolates of N. caninum were isolated via PCR and their DNA sequences were determined. Computer analysis indicated that the two gene sequences were identical among all four N. caninum isolates. However, the gene for NhGRA6 was found to be 96 nucleotides longer at the 3' end than that of NcGRA6, resulting in a protein product that is 32 amino acids larger than NcGRA6. Two tandem repeat sequences were identified at the 3' end of the NhGRA6 gene. These repeat sequences contributed to the lengthening of the carboxy terminus of NhGRA6 in comparison with that of NcGRA6. The larger size of NhGRA6 was further confirmed by Western blot analysis in which NcGRA6 monospecific antibodies recognised a protein of approximately 42 kDa in N. hughesi whole tachyzoite preparation but a protein of 37 kDa in N. caninum whole tachyzoite preparation. Analysis of GRA7 gene sequences indicated a 6 and 14.8% difference at nucleotide and amino acid sequence level, respectively, between NcGRA7 and NhGRA7. Despite the same number of residues in the deduced amino acid sequences of all the GRA7 proteins, Western blot analysis indicated a difference in the migration pattern of NhGRA7 in comparison with NcGRA7. Results of our study indicate that diagnostic tests based on differences in dense granule sequences and antigenicity may have potential to differentiate between N. hughesi and N. caninum. Such diagnostic tests would be valuable tools to aid in our understanding of the epidemiology of these parasites. Additionally, dense granule proteins are immunogenic and they may have potential as use in recombinant vaccines against neosporosis.     

A random amplified polymorphic DNA polymerase chain reaction technique that differentiates between Neospora species

Neospora caninum is a recently described coccidial parasite that was first isolated from a dog in 1988 and has subsequently been shown to infect a wide range of mammals. Neospora hughesi, a new species of this genus, has recently been isolated from the spinal cord of horses showing clinical signs of equine protozoal myeloencephalitis. The random amplified polymorphic DNA polymerase chain reaction technique is capable of differentiating between N. caninum and N. hughesi.     

Endogenous transplacental transmission of Neospora hughesi in naturally infected horses

Over a 2-yr study period, we investigated possible endogenous transplacental transmission of Neospora hughesi in 74 mare and foal pairs following the diagnosis of neuronal neosporosis in a weanling foal. Presuckle and postsuckle serum of each foal, serum and colostrum of each periparturient mare, and serum of each mare and foal pair, collected at 3-mo intervals thereafter, were tested for N. hughesi using an indirect fluorescent antibody test (IFAT). Furthermore, whole blood and colostrum samples and placentae were tested for the presence of N. hughesi by real-time PCR. The mares' seroprevalence at foaling based on IFAT (titer ≥ 160) was 52 and 6% in 2006 and 2007, respectively. Colostral antibodies against N. hughesi were detected in 96 and 11% of the mares in the 2-yr study. With the exception of 3 foals, all remaining foals were born seronegative to N. hughesi. Passive transfer of colostral antibodies to N. hughesi was documented in 15 foals. Three foals born from 2 different mares had presuckle antibodies at a titer ranging from 2,560 to 20,480. All 3 foals were born healthy. Two foals were born to the same dam that also gave birth to the weanling diagnosed with neuronal neosporosis in 2005. The third foal was born to a second mare with no previous foaling history at the farm. Seroconversion was documented in 10 foals and 9 mares over the 2-yr study. All blood and colostrum samples tested PCR negative for N. hughesi. Only 1 placenta collected in 2007 from the mare with the 2 congenitally infected foals tested PCR positive for N. hughesi. In conclusion, N. hughesi persisted in this population via endogenous transplacental infection.     

Canine and bovine Neospora caninum control sera examined for cross-reactivity using Neospora caninum and Neospora hughesi indirect fluorescent antibody tests

Neospora caninum is a well known protozoan parasite of domestic and wild animals. Neospora hughesi is a closely related protozoan with an unknown life cycle, host range, and infection prevalence. Many serologic surveys of N. caninum have been performed without consideration of potential cross-reactions with N. hughesi, which could confound results. The aim of this study was to investigate whether postexposure sera from animals experimentally infected with N. caninum exhibit significant reactivity differences when tested using N. caninum and N. hughesi Immunofluorescent Antibody Tests (IFAT). Pre- and postinfection serum samples from 10 dogs, 20 calves, and 17 cows were tested by dual IFATs. All pre-exposure samples for N. caninum tested seronegative for both organisms. All postexposure samples that were seropositive for N. caninum were also positive for N. hughesi, although N. hughesi antibody titers were usually 1 dilution lower (P < 0.02). Serologic surveys for N. caninum may be confounded by cross-reacting titers with N. hughesi, but true positive N. caninum antibody titers are greater than, or equal to, cross-reacting N. hughesi antibody titers.     

Risk of transplacental transmission of Sarcocystis neurona and Neospora hughesi in California horses

The study objective was to assess the risk of transplacental transmission of Sarcocystis neurona and Neospora hughesi in foals from 4 California farms during 3 foaling seasons. Serum of presuckle foals and serum and colostrum of periparturient mares were tested using indirect fluorescent antibody tests for S. neurona and N. hughesi. Serum antibody titers were < or =10 in 366 presuckle foals tested. There was no serologic or histologic evidence of either parasite in aborted fetuses or placentas examined. Positivity for S. neurona and N. hughesi in mares increased with age. Mares < or =9 yr that originated from Kentucky were 3.8 and 1.4 times more likely to be positive for S. neurona and N. hughesi, respectively, than mares from California. The strength of association between positivity to either parasite and state of birth decreased as age increased. Mares positive for S. neurona and N. hughesi were 2.2 and 1.7 times more likely, respectively, to have a previous abortion than negative mares, adjusted for age and state of birth. The annual mortality rate for mares was 4%. The annual incidence rate of equine protozoal myeloencephalitis was 0.2%. In conclusion, there was no detectable risk of transplacental transmission of S. neurona and N. hughesi. Prevalence of antibodies against both parasites in mares increased with age.     

Expression of Kaposi＇s Sarcoma-associated Herpesvirus ORFK8.1 and Its Preliminary Diagnostic Application

The ORFK8.1 of Kaposi's sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf K8.1 was induced by isopropyl-b-D-thiogalactopyranoside (IPTG). The fusion protein was purified by chromatyography. The expressed protein and its purified product were identified by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE). SDS-PAGE showed that a protein of 26 kDa was visualized as expected. A western blot assay was established to analyze the immunogenicity of purified recombinant 0RFK8.1 protein. The optimal condition of the recombinant ORFK8.1 ELISA assay was confirmed: the concentration of antigen was 5 ug/mL, the dilution of serum was 1:200. We used the ELISA method to investigate the recombinant ORF K8.1 protein's specificity, the data showed that the specificity of ORF K8.1 to detect KSHV was 100%. At the same time, 560 sera samples from Hubei province were detected by using ORFK8.1 ELISA to investigate KSHV seroprevalence in this region. The KSHV seroprevalence in Hubei province is shown to be 6.80%.     

Enzyme-linked immunosorbent assay (ELISA) using recombinant Fasciola cathepsin L1 for the diagnosis of human fasciolosis caused by Fasciola hepatica/gigantica hybrid type

Recombinant Fasciola cathepsin L-1 (rCatL1) was evaluated in enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of human fasciolosis in Japan. Quality characteristics of the test were accessed by receiver operating characteristic (ROC) analysis, with sera from fasciolosis patients (n = 10), patients with no evidence of parasitic infections (n = 29), and patients with other helminth infections (n = 119). Both the sensitivity and specificity of the test achieved 100% with the control samples. To test the performance of the assay in an authentic situation, 311 serum samples, which had been sent to our laboratory for the diagnosis of parasitic infections from January 2018 to February 2019, were re-assessed using the rCatL1 ELISA. In this case, the sensitivity of the rCatL1 ELISA was 100%, giving positive results to all fasciolosis sera (n = 7), and the specificity was 99.0%, in which three of the 304 non-fasciolosis samples were judged positive. Careful re-examination of the laboratory data and medical imaging of these three patients revealed that one of the patients, who had been diagnosed as having larva migrans syndrome, was judged to be infected with Fasciola, in addition to ascarid nematodes. Thus the true specificity of the assay in the authentic reached 99.3% (302/304). As the rCatL1 ELISA exhibited a highly significant positive likelihood ratio (152.0) and negative likelihood ratio (0.0), calculated from the 311 sample data, this rCatL1 ELISA can be used for routine screening and definitive diagnosis test for fasciolosis in reference laboratories.     

Comparison of the Serion IgM ELISA and Microscopic Agglutination Test for diagnosis of Leptospira spp. infections in sera from different geographical origins and estimation of Leptospira seroprevalence in the Wiwa indigenous population from Colombia

Leptospirosis is among the most important zoonotic diseases in (sub-)tropical countries. The research objective was to evaluate the accuracy of the Serion IgM ELISA EST125M against the Microscopic Agglutination Test (MAT = imperfect reference test); to assess its ability to diagnose acute leptospirosis infections and to detect previous exposure to leptospires in an endemic setting. In addition, to estimate the overall Leptospira spp. seroprevalence in the Wiwa indigenous population in North-East Colombia. We analysed serum samples from confirmed leptospirosis patients from the Netherlands (N = 14), blood donor sera from Switzerland (N = 20), and sera from a cross-sectional study in Colombia (N = 321). All leptospirosis ELISA-positive, and a random of negative samples from Colombia were tested by the MAT for confirmation. The ELISA performed with a sensitivity of 100% (95% CI 77% - 100%) and a specificity of 100% (95% CI 83% - 100%) based on MAT confirmed Leptospira spp. positive and negative samples. In the cross-sectional study in Colombia, the ELISA performed with a sensitivity of 100% (95% CI 2–100%) and a specificity of 21% (95% CI 15–28%). Assuming a 5% Leptospira spp. seroprevalence in this population, the positive predictive value was 6% and the negative predictive value 100%. The Leptospira spp. seroprevalence in the Wiwas tested by the ELISA was 39%; however, by MAT only 0.3%. The ELISA is suitable to diagnose leptospirosis in acutely ill patients in Europe several days after onset of disease. For cross-sectional studies it is not recommended due to its low specificity. Despite the evidence of a high leptospirosis prevalence in other study areas and populations in Colombia, the Wiwa do not seem to be highly exposed to Leptospira spp.. Nevertheless, leptospirosis should be considered and tested in patients presenting with febrile illness.     

水泡性口炎病毒核蛋白基因的表达及初步应用

将水泡性口炎病毒编码群特异性抗原的N基因片段克隆至pMD18-T克隆载体质粒中,构建N基因克隆重组质粒,进行核苷酸序列分析。然后亚克隆插入pBAD/Thio TOPO表达载体,经PCR限制性内切酶分析、测序鉴定,筛选获得N基因正向插入、有正确读码框的阳性克隆,成功构建了水泡性口炎病毒N基因重组表达载体。经L-Arabinose诱导表达,可稳定、高效地表达N蛋白抗原。SDS-PAGE、Western blotting及间接ELISA试验结果表明,表达蛋白为融合蛋白,质量约63.5 kD,其表达产量约占菌体总蛋白的16％,相当于92mg/L。融合蛋白中含有水泡性口炎病毒群特异性的核蛋白抗原,应用表达的VSV核蛋白抗原建立了酶联免疫吸附试验,通过对186份山羊、豚鼠实验动物人工感染VSV的血清样品和参考血清样品的检测,并与微量血清中和试验进行了比较,结果表明：以表达的VSV核蛋白为包被抗原的酶联免疫吸附试验是一种特异性强、敏感性高、快速、简单、安全的检测方法,抗原制备成本低。     

Detection of antibodies to Neospora caninum in cattle by enzyme-linked immunosorbent assay with truncated NcSRS2 expressed in Escherichia coli

The surface antigen 1-related sequence 2 of Neospora caninum (NcSRS2) is considered as an immunodominant antigen. In this study, the gene encoding truncated NcSRS2 (NcSRS2t) lacking an N-terminal signal peptide and C-terminal hydrophobic regions was expressed in Escherichia coli, and its diagnostic potential in an enzyme-linked immunosorbent assay (ELISA) was evaluated. ELISA could discriminate clearly between known N. caninum-positive and -negative sera from cattle. Field serum samples collected from cattle in Brazil were examined for the diagnosis of N. caninum infection using ELISA. Of the 197 samples analyzed, 64 (32.5%) samples were positive for antibodies to N. caninum. Of the 64 ELISA-positive samples, 58 (90.6%) were confirmed as positive by Western blot analysis with whole-parasite antigens. These results suggest that ELISA with recombinant NcSRS2t is an effective method for diagnosis of N. caninum infection in cattle.     

Seroprevalence of antibodies against Anisakis simplex larvae among health-examined residents in three hospitals of southern parts of Korea

The present study was performed to estimate the seroprevalence of larval Anisakis simplex infection among the residents health-examined in 3 hospitals in southern parts of Korea. A total of 498 serum samples (1 serum per person) were collected in 3 hospitals in Busan Metropolitan city, Masan city, and Geoje city in Gyeongsangnam-do (Province) and were examined by IgE-ELISA and IgE-western blotting with larval A. simplex crude extract and excretory-secretory products (ESP). The prevalence of antibody positivity was 5.0% and 6.6% with ELISA against crude extracts and ESP, respectively. It was also revealed that infection occurred throughout all age groups and higher in females than in males. A specific protein band of 130 kDa was detected from 10 patients with western blot analysis against crude extract and ESP among those who showed positive results by ELISA. Our study showed for the first time the seroprevalence of anisakiasis in Korea. The allergen of 130 kDa can be a candidate for serologic diagnosis of anisakiasis.     

Evaluation of the usefulness of commercial enzyme-linked immunosorbent assays for diagnosis of Mycoplasma pneumoniae infections

The usefulness of the ELISA distributed by BioChem ImmunoSystems, Medial Polska, Biomedica/Virotech and prepared in our laboratory (ELISA FH-K) for diagnosis of the M. pneumoniae infections was estimated. Eighty six serum samples obtained from 86 patients with respiratory tract infections were simultaneously tested by ELISA-IgM/-IgG and by complement fixation test which was accepted as a reference test. The highest sensitivity in relation to the CFT was displayed by the ELISA BioChem ImmunoSystems and Medial Polska (100%), slightly lower sensitivity by the ELISA Biomedica/Virotech--96.5% and ELISA FH-K--90.9% when determining mycoplasmal antibodies of IgM. The lowest sensitivity was displayed by the ELISA Biomedica/Virotech when determining antibodies of the IgG class (54.9%). The specificity of ELISA in relation to the CFT was generally higher when detecting mycoplasmal antibodies of the IgM class then of IgG class. The study demonstrated that all 4 ELISA may be used in routine serodiagnosis of M. pneumoniae infection. For the improve of sensitivity of ELISA it's recommended to measure simultaneously the level of mycoplasmal antibodies of IgM and IgG.     

Characterization of secreted recombinant <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> surface antigen 2 (SAG2) heterologously expressed by the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The surface antigen 2 (SAG2) gene of the protozoan parasite, Toxoplasma gondii, was cloned and extracellularly expressed in the yeast Pichia pastoris. The effectiveness of the secreted recombinant SAG2 (rSAG2-S) as a serodiagnosis reagent was assessed by western blots and ELISA. In the western blot assay, rSAG2-S reacted with all Toxoplasma-antibody positive human serum samples but not with Toxoplasma-negative samples. In the ELISA, rSAG2-S yielded sensitivity rates ranging from 80% (IgG negative, IgM positive) to 100% (IgG positive, IgM negative). In vivo experiments showed that serum from mice immunized with rSAG2-S reacted specifically with the native SAG2 of T. gondii. These mice were protected when challenged with live cells of T. gondii.     

Increasing peptide identifications and decreasing search times for ETD spectra by pre-processing and calculation of parent precursor charge

Background

Early diagnosis and treatment of Mycobacterium tuberculosis infection can prevent most deaths resulting from this pathogen; however, multidrug-resistant strains present serious threats to global tuberculosis control and prevention efforts. In this study, we identified antigens that could be used for the serodiagnosis of drug-resistant M. tuberculosis strains, using a proteomics-based analysis.

Results

Serum from patients infected with drug-resistant or drug-susceptible M. tuberculosis strains and healthy controls was subjected to two-dimensional gel electrophoresis using a western blot approach. This procedure identified nine immunoreactive proteins, which were subjected to MALDI-TOF-MS analysis. Six recombinant proteins, namely rRv2031c, rRv0444c, rRv2145c, rRv3692, rRv0859c, and rRv3040, were expressed and used to determine the immuno-reactivity of 100 serum samples. Antibody reactivity against rRv2031c, rRv3692, and rRv0444c was consistently observed. Among them, the best sensitivity and specificity of rRv3692 were 37% and 95% respectively. Furthermore, when rRv2031c and rRv3692 or rRv2031c, rRv3692, and rRv0444c were combined in 2:1 or equal amounts, the assay sensitivity and specificity were improved to 56.7% and 100% respectively.

Conclusions

These results suggest that Rv2031c, Rv3692, and Rv0444c are possible candidate biomarkers for effective use in the serodiagnosis of drug-resistant tuberculosis infections, and a combined formula of these antigens should be considered when designing a subunit assay kit.     

Comparative Assessment of ELISAs Using Recombinant Saposin-Like Protein 2 and recombinant Cathepsin L-1 from Fasciola hepatica for the Serodiagnosis of Human Fasciolosis

Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2) and cathepsin L-1 (recCL1), were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG) conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory–secretory products (FhES) from adult stage liver flukes was assessed by receiver operator characteristic (ROC) analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n = 20), patients with other parasitic infections (n = 87) and patients with malignancies (n = 121). The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy) employing the threshold (cut-off) to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls.     

Diagnostic value of an enzyme‐linked immunosorbent assay using the recombinant CT694 species‐specific protein of Chlamydia trachomatis

Aim: To study the performance of the CT694 protein in relation to the microimmunofluorescence (MIF) and the pELISA tests for the serodiagnosis of Chlamydia trachomatis infections. Methods and Results: The CT694 protein was produced as recombinant protein and was used as antigen in ELISA test for the detection of C. trachomatis IgG antibodies. The performance of the developed ELISA test was compared to the MIF test at two cut‐off values of 16 and 64, and to the specific pELISA test using a panel of 342 sera. These sera were from children MIF C. trachomatis and Chlamydophila pneumoniae negative, patients MIF C. pneumoniae positive, patients MIF C. trachomatis positive, patients suspected to have chlamydial infections diagnosed by the Cobas Amplicor test, healthy blood donors and prostitutes. Our results indicate that the developed ELISA test has performed better compared with the MIF and the pELISA tests. The highest performance was obtained when comparing the developed ELISA test in relation to the pELISA, yielding an overall sensitivity and specificity of 85% and 87% respectively. Conclusions: The CT694 ELISA showed the best performance when compared to the species‐specific pELISA test and may be used for the serodiagnosis of C. trachomatis infections. Significance and Impact of the Study: The CT694 ELISA test responds to the criteria of both sensitivity and specificity according to the MIF and pELISA tests and may be used for serodiagnosis of C. trachomatis infections.