Isolation and identification of a Giardia lamblia-specific stool antigen (GSA 65) useful in coprodiagnosis of giardiasis.

A Giardia lamblia-specific antigen (GSA 65) was isolated from stools of G. lamblia-positive patients by crossed- and line-immunoelectrophoresis and counterimmunoelectrophoresis (CIE) in agarose by using rabbit antiserum prepared against G. lamblia cysts. CIE with rabbit anti-GSA 65 monospecific antiserum revealed that GSA 65 was present in aqueous stool eluates of giardiasis patients and in cysts and trophozoites of the parasite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoaffinity-purified antigen followed by Western blotting showed that the molecular weight of this molecule was about 65,000. GSA 65 was detectable by CIE in stool eluates of 36 of 40 giardiasis patients but not in eluates of 10 G. lamblia-negative asymptomatic controls. GSA 65 was detected in stool eluates of 2 of 18 individuals with chronic diarrhea who were negative for parasites by microscopic examination. Cross-specificity studies with other genera of parasitic protozoa performed by using CIE and immunofluorescence indicated that GSA 65 was present only in strains of G. lamblia. Based on these findings, GSA 65 may prove to have an important application in the design of sensitive diagnostic tests for giardiasis.     

Development of an enzyme immunoassay using recombinant expressed antigen to detect hepatitis delta virus antibodies.

Two generic enzyme immunoassays (EIAs) were developed for detection of anti-hepatitis delta virus antibodies (anti-HD) and compared with a commercially available radioimmunoassay. Both generic assays were configured as blocking assays and used hepatitis delta antigen (HDAg) derived from infected chimpanzee liver (EIA-1) or from Escherichia coli transformed with a plasmid containing an insert from within an open reading frame encoding HDAg (EIA-2). Absolute sensitivity was ascertained by endpoint titration, which demonstrated essentially identical endpoints for EIA-1 and EIA-2. The absolute sensitivities of the EIAs were approximately four times greater than that of the radioimmunoassay. Specificity and sensitivity were ascertained by testing a panel of 176 serum specimens by each assay. The specimens were selected to represent a panel composed of sera from individuals with or without markers of viral hepatitis as follows: (i) serologically confirmed by exclusion as posttransfusion non-A, non-B hepatitis; (ii) acute or chronic hepatitis B virus infection, positive for hepatitis B surface antigen; (iii) resolved hepatitis B virus infection, positive for anti-hepatitis B surface antigen; (iv) acute hepatitis A virus infection, positive for anti-hepatitis A virus immunoglobulin M; and (v) normal human sera. All three assays for anti-HD gave similar specificity and sensitivity values. In conclusion, the recombinant expressed HDAg can replace antigen derived from infected liver tissue as a diagnostic reagent used to configure an EIA for detection of anti-HD. Furthermore, the results suggest that the expressed antigen contains the important immunodominant epitope(s).     

Validation of a commercially available SARS-CoV-2 serological immunoassay

ObjectivesTo validate the diagnostic accuracy of a Euroimmun SARS-CoV-2 IgG and IgA immunoassay for COVID-19.MethodsIn this unmatched (1:2) case-control validation study, we used sera of 181 laboratory-confirmed SARS-CoV-2 cases and 326 controls collected before SARS-CoV-2 emergence. Diagnostic accuracy of the immunoassay was assessed against a whole spike protein-based recombinant immunofluorescence assay (rIFA) by receiver operating characteristic (ROC) analyses. Discrepant cases between ELISA and rIFA were further tested by pseudo-neutralization assay.ResultsCOVID-19 patients were more likely to be male and older than controls, and 50.3% were hospitalized. ROC curve analyses indicated that IgG and IgA had high diagnostic accuracies with AUCs of 0.990 (95% Confidence Interval [95%CI]: 0.983-0.996) and 0.978 (95%CI: 0.967-0.989), respectively. IgG assays outperformed IgA assays (p=0.01). Taking an assessed 15% inter-assay imprecision into account, an optimized IgG ratio cut-off > 2.5 displayed a 100% specificity (95%CI: 99-100) and a 100% positive predictive value (95%CI: 96-100). A 0.8 cut-off displayed a 94% sensitivity (95%CI: 88-97) and a 97% negative predictive value (95%CI: 95-99). Substituting the upper threshold for the manufacturer's, improved assay performance, leaving 8.9% of IgG ratios indeterminate between 0.8-2.5.ConclusionsThe Euroimmun assay displays a nearly optimal diagnostic accuracy using IgG against SARS-CoV-2 in patient samples, with no obvious gains from IgA serology. The optimized cut-offs are fit for rule-in and rule-out purposes, allowing determination of whether individuals in our study population have been exposed to SARS-CoV-2 or not. IgG serology should however not be considered as a surrogate of protection at this stage.     

Sensitivity of microscopy versus enzyme immunoassay in the laboratory diagnosis of giardiasis

The substitution of enzyme immunoassay (EIA) techniques for microscopy as a screening tool forGiardia lamblia infection was assessed. Paired stool samples obtained within a ten-day period from 366 patients with persistent diarrhea were examined by microscopy. In addition, two commercially availableGiardia lamblia-specific ElAs were performed. Compared with microscopy, EIA for coproantigen detection was more sensitive, based on examination of either one or two stool samples. Repeated examinations increased the number of cases detected, more so for microscopy than EIA. The negative predictive values of the two EIAs performed on the first stool sample were 98.7% and 97.8%. The results show that EIA for detection of copro-antigens in a single stool sample may be almost as sensitive for identifyingGiardia infection as repeated microscopy on two sequential stool samples.     

Detection of specific IgG and IgM antibodies to Toxoplasma gondii with a commercially available enzyme immunoassay kit system

A total of 138 serum samples submitted for toxoplasma serology have been examined by enzyme immunoassay using kits produced by Labsystems Oy for the detection of specific antibodies of the IgG and IgM class. Results were compared with the dye test, an indirect haemagglutination test, and an indirect immunofluorescence test for specific IgM. The enzyme immunoassay was less sensitive than the dye test, but by running both IgG and IgM enzyme immunoassays, 92.4% sensitivity was achieved. The specificity of the enzyme immunoassay was good, with only one dye test negative serum giving a positive (but weak) IgG enzyme immunoassay reaction. Thirty serum samples from patients with no evidence of exposure to Toxoplasma gondii gave negative results in the IgM enzyme immunoassay. Enzyme immunoassay results were expressed in enzyme immunoassay units, as a percentage value of a standard serum. This convention will be of value in the direct comparison of assay systems and in the application of quality control procedures.     

Evaluation of a commercially available second-generation immunoglobulin G enzyme immunoassay for detection of Helicobacter pylori infection.

We evaluated a commercially available second-generation anti-H. pylori immunoglobulin G enzyme immunoassay (EIA) (Cobas Core Anti-Helicobacter pylori EIA; Roche S. A., Basel, Switzerland) for serodiagnosis of H. pylori infection. The results of the assay were assessed in relation to the results of bacterial culture, urease testing, and histological Giemsa stain of gastric biopsy specimens from 1,134 patients with a variety of symptoms relating to the upper gastrointestinal tract. H. pylori was detected in biopsy specimens from 660 (58.2%) patients: 6 had a normal mucosa, 123 had chronic gastritis only, and 531 were found to have chronic active gastritis by histology; endoscopy showed duodenal and gastric ulcers in 137 and 64 patients of the last two groups, respectively. The test was evaluated with different age and ethnic groups. The prevalence, sensitivity, specificity, and positive and negative predictive values were, respectively, (i) for Belgian patients between 18 and 40 years old, 34, 93, 95, 91, and 96%; (ii) for Belgian patients more than 40 years old, 53, 96, 91, 93, and 95%; and (iii) the Mediterranean patients more than 17 years old, 87, 94, 70, 95, and 64%. All sera showing discordant immunoassay results compared with the results of histology and culture of biopsy specimens, as well as those with borderline immunoassay results, were tested further by immunoblotting. Among the EIA results considered false negative, we demonstrated an absence of seroconversion in 14 of 19 patients tested by immunoblotting. Among the EIA results considered false positive, immunoblotting showed the presence of specific antibodies in 28 of 37 patients tested. Among the borderline results obtained in the first assay with 22 patients' sera, a second assay showed positive results in 10 patients (8 were positive by immunoblotting) and negative reactions in 10 patients (9 were negative by immunoblotting), whereas 2 remained borderline. These data indicate that sera showing borderline immunoassay results must be tested again. In conclusion, this commercially available second-generation EIA, which is easy and quick to perform, was found highly reliable for the serodiagnosis of H. pylori infection.     

Comparison of commercially available enzyme immunoassay with traditional serological tests for detection of antibodies to Coccidioides immitis.

A newly released commercially available enzyme-linked immunosorbent assay (ELISA) was evaluated for its ability to detect immunoglobulin M (IgM) and IgG antibodies against the tube precipitin and complement fixation (CF) antigens of Coccidioides immitis. The ELISA was compared with more traditional diagnostic assays, CF, latex agglutination (LA), and immunodiffusion (ID). When the IgM-specific portion of the ELISA was compared with LA, there was an agreement of 81.8%, a specificity of 75.0%, and a sensitivity of 84.6%. For the determination of the presence of IgG antibodies, the results of the IgG-specific part of the ELISA were compared with the combined results of ID and CF. After resolution of discrepant results, there was an agreement of 95.6%, a specificity of 98.3%, and a sensitivity of 92.6%. When the results of the IgG- and IgM-specific portions of the ELISA combined were compared with the results of the three traditional assays (CF, LA, and ID) there was an agreement of 96.7%, a specificity of 98.5%, and a sensitivity of 94.8%. The ELISA proved to be a reliable assay for the detection of antibodies against the tube precipitin and CF antigens and did not suffer from the objectivity required to interpret the results of the traditional assays and anticomplement interference associated with the traditional assays.     

Rapid diagnosis of tuberculous meningitis with a dot enzyme immunoassay to detect antibody in cerebrospinal fluid

A simple dot enzyme immunoassay (Dot-EIA) was carried out to detect antibody toMycobacterium tuberculosis antigen 5 in cerebrospinal fluid (CSF) specimens from 40 patients with a clinical diagnosis of tuberculous meningitis (TBM). The assay gave a positive reaction in all ten patients with culture proven TBM. In 30 culture negative patients with TBM, the assay was positive at a titre of 1:16 in 18 patients. In 40 patients with non-tuberculous neurological diseases (control group) the assay was negative at a titre of 1:16. The Dot-EIA had an overall sensitivity of 70 % and a specificity of 100 % in the diagnosis of TBM. This assay could be used as a rapid screening test to establish the diagnosis of TBM, particularly in patients in whom bacteriological investigations forMycobacterium tuberculosis in CSF specimens are negative.     

Diagnosis of urogenital gonorrhoea by detecting gonococcal antigen with a solid phase enzyme immunoassay (Gonozyme).

A solid phase enzyme immunoassay (Gonozyme) was used to demonstrate gonococcal antigen in urogenital specimens. Urethral specimens from 101 men and cervical specimens from 150 women were examined, and the diagnostic yields were compared with those obtained by culture. The Gonozyme test was positive in 25 patients, 15 men and 10 women, and negative in 226 patients. Gonococci were isolated by culture in 23 of the patients, 12 men and 11 women. The Gonozyme test gave false-negative results in two men and one woman patient. The sensitivity of the test was 87% for the men and 91% for the women. Correspondingly, the test specificity was 94.3% for the men and 100% for the women, the predictive value of positive test 80% and 100%, and that of negative test 97.7 and 99.3% respectively. The Gonozyme test does not allow antibiotic sensitivity testings but has the advantage of rapidity and is not dependent upon viable organisms. The test is an attractive alternative to culture procedures for screening women patients with symptomatic or asymptomatic gonorrhoea.     

Clinical evaluation of the sensitivity and specificity of a commercially available enzyme immunoassay for detection of rubella virus-specific immunoglobulin M.

A solid-phase capture antigen enzyme immunoassay (Rubazyme-M) was evaluated for sensitivity and specificity on sera from 1,200 blood donors, 51 patients with rubella, 2 infants with congenital rubella, 104 patients with other infections, and 126 patients with immunological abnormalities. The sensitivity was 100% for sera tested between days 3 and 40 after the onset of symptoms of rubella virus infection. Rubella virus-specific immunoglobulin M was detected at birth in sera from congenitally infected infants and persisted for several months. Positive Rubazyme-M responses were observed in some patients in the absence of rubella diagnosis (one blood donor, three other infections, and two immunological abnormalities), providing a test specificity of 99.6%. None of 67 patients with rubella virus-specific immunoglobulin G antibody and high levels of rheumatoid factor were positive in the test.     

Evaluation of four commercially available enzyme immunoassays for laboratory diagnosis of Clostridium difficile-associated diseases.

Four commercial enzyme immunoassays (EIAs) for the detection of Clostridium difficile toxin A have recently been developed and marketed (Premier, Meridian Diagnostics, Cincinnati, Ohio; VIDAS, bioMerierux Vitek, Inc., Hazelwood, Mo.; Tox-A-Test, TechLab, Blacksburg, Va.; and Bartels, Baxter Diagnostics, McGaw Park, Ill.). The performances of these EIAs were compared with those of the tissue culture cytotoxicity assay and a definition of C. difficile-associated disease based on both laboratory and clinical criteria for 329 clinical specimens. Two EIAs (Premier and VIDAS) showed good overall agreement (96 and 95%, respectively) with the cytotoxicity assay. However, they were less sensitive (84 and 71%, respectively) than the Bartels (94%) or Tox-A-Test (93%) EIAs. The Bartels and Tox-A-Test assays were much less specific, resulting in poor positive predictive values (56%) of the two assays when compared with that of the cytotoxicity assay. Tox-A-Test had the added drawback of having a significant number of indeterminate results (6.4%). These data indicate that the four EIAs all have specific shortcomings. When using these EIAs, testing strategies that take these shortcomings into consideration should be developed.     

Detection of histoplasma antigen by a quantitative enzyme immunoassay

The second-generation Histoplasma antigen immunoassay is semiquantitative, expressing results as a comparison to a negative control, which requires repeat testing of the prior specimen with the current specimen to accurately determine a change in antigen. Reporting results in this manner often is confusing to the ordering physician and laboratory. Development of a quantitative assay could improve accuracy, reduce interassay variability, and eliminate the need to test the prior sample with the current sample in the same assay. Calibrators with known concentrations of Histoplasma antigen were used to quantitate antigen in specimens from patients with histoplasmosis and from controls. Samples from cases of disseminated histoplasmosis or other mycoses and controls were tested to evaluate the performance characteristics of the quantitative assay. Paired specimens were evaluated to determine if quantitation eliminated the need to test the current and prior specimens in the same assay to assess a change in antigen. The sensitivity in samples from patients with AIDS and disseminated histoplasmosis was 100% in urine and 92.3% in serum. Cross-reactions occurred in 70% of other endemic mycoses, but not in aspergillosis. Specificity was 99% in controls with community-acquired pneumonia, medical conditions in which histoplasmosis was excluded, or healthy subjects. A change in antigen level categorized as an increase, no change, or decrease based on antigen units determined in the same assay agreed closely with the category of change in nanograms/milliliter determined from testing current and prior specimens in different assays. Sensitivity, specificity, and interassay precision are excellent in the new third-generation quantitative Histoplasma antigen immunoassay.     

Standardization of antigen E in ragweed pollen extracts using a monoclonal antibody-based enzyme immunoassay

A hybridoma-derived monoclonal IgG antibody specific for ragweed AgE was used to develop a competitive binding enzyme immunoassay suitable for quantitation of antigen E levels in ragweed pollen extracts. The assay was capable of detecting as little as 30 ng/ml AgE in crude pollen extracts. The monoclonal antibody was shown to react with AgE present in commercial pooled pollen extracts from a number of ragweed species as well as a laboratory extract from a single species. In contrast to previous conventional xenoantisera, it could distinguish true ragweed (Ambrosia sp) from false ragweed (Franseria sp). The use of monoclonal antibodies in assay systems such as this offers a reproducible and widely applicable method for allergen standardization.     

Detection of alphaviruses in a genus-specific antigen capture enzyme immunoassay using monoclonal antibodies.

A genus-specific antigen capture assay using similar combinations of monoclonal antibodies for capture and detection of 24 alphaviruses belonging to the seven serocomplexes was developed. The sensitivity of the test ranged from 10(3.4) 50% tissue culture infective doses/ml for o'nyong-nyong virus to 10(6.1) 50% tissue culture infective doses/ml for Middelburg virus. The antigen capture test uses a combination of cross-reacting monoclonal antibodies directed against the nucleocapsid protein and envelope glycoprotein E1 of Semliki Forest virus.     

Use of an adaptation of a commercially available PCR assay aimed at diagnosis of chlamydia and gonorrhea to detect Trichomonas vaginalis in urogenital specimens

Trichomonas vaginalis PCR using reagents from a commercially available assay for Chlamydia trachomatis and Neisseria gonorrhoeae was evaluated for detection of infection in women and men attending a sexually transmitted disease clinic. Evaluations included three primer sets, endocervical swabs, vaginal swabs and urine, and various storage conditions. The TVK3/TVK7 primer set was optimal in our hands with sensitivities ranging from 69.5 to 96.8%. In all comparisons, T. vaginalis PCR performed better than routine diagnostics using microscopy for women and culture for men (P > 0.05). The assay performed well for all sample types tested, and vaginal swabs were stable for up to 7 days at ambient temperature. Using samples prepared for, and reagents from, the C. trachomatis-N. gonorrhoeae PCR assay allowed incorporation of T. vaginalis PCR diagnosis into routine clinical testing.     

Use of commercially available rapid chloramphenicol acetyltransferase test to detect resistance in Salmonella species.

Chloramphenicol resistance among Salmonella spp. has important public health and clinical implications, especially in areas of the world where these strains are endemic. The availability of rapid and sensitive screening methods for detection of antibiotic resistance is important. Therefore, we tested 33 strains of Salmonella for chloramphenicol acetyltransferase (CAT) activity using two rapid techniques. Evaluation of a 1-h tube method and a 30-min commercial disk procedure demonstrated that they are as accurate as standardized susceptibility techniques. Both the 1-h tube and 30-min disk methods detected CAT enzymatic activity produced by one CAT gene copy per cell.     

Sandwich enzyme immunoassay of tumor-associated antigen sialosylated Lewisx using beta-D-galactosidase coupled to a monoclonal antibody of IgM isotype

Enzyme conjugates with antibody of IgG type have been used extensively in immunohistochemistry, but conjugates with antibody of IgM type have not been reported. This paper describes the beta-D-galactosidase (Gal) labeling of a monoclonal IgM antibody designated CSLEX1 (for cytotoxic sialosylated Lewisx), which is directed against a tumor-associated antigen sialosylated Lewisx (S-Lex). The antibody was first acylated with a heterobifunctional agent N-(gamma-maleimidobutyryloxy)succinimide (GMBS) to introduce the maleimide groups into the molecule; excess reagent was removed by gel filtration and then the activated antibodies were crosslinked to the thiol groups of Gal. The conjugates were partially purified of free Gal by DEAE-Toyopearl column chromatography with an increasing linear concentration of NaCl. The conjugates thus prepared retained almost full enzyme activity and were demonstrated to be free of CSLEX1 by affinity chromatography using anti-galactosidase antibody bound to Sepharose 4B. The conjugates were used as a label in a sandwich enzyme immunoassay (SEIA) to detect the antigen at concentrations as low as 0.2 U/well. The SEIA was used to measure serum S-Lex levels in both healthy subjects and lung cancer patients and mean concentrations of 70 U/ml and 198.6 U/ml were detected respectively.