Electron microscopy in the diagnosis of giardiasis

Biopsy specimens of intestinal mucosa from two patients with malabsorption syndrome were examined with routine light and electron microscopy. Both showed intact intestinal mucosa, but by electron microscopy, the presence of Giardia lamblia in the intervillar spaces was identified. Electron microscopy is useful for the identification of this parasite, since the stool examinations in up to 50% may be negative. The parasites appear in light microscopy as cellular debris and can be easily overlooked, while by electron microscopy the typical morphologic features of the parasite are diagnostic.     

Comparative evaluation of enzyme immunoassay and culture for the laboratory diagnosis of gonorrhea

A commercially available, solid-phase enzyme immunoassay (EIA), called Gonozyme, was compared to Martin-Lewis medium in Jembec plates for the laboratory diagnosis of gonorrhea. A total of 577 clinical specimens (419 urethral and 158 endocervical) were collected from a high-risk, walk-in patient population attending a sexually transmitted disease clinic. The results showed that EIA was comparable to a conventional cultural procedure for identifying infected and noninfected males. In addition, the system may be used reliably for performing test of cure on urethral samples obtained from this male population. Gonozyme was also comparable to culture in identifying females who had gonococcal infection. However, because of the high incidence of false positive test results, most likely attributable to antigen persistence in endocervical secretions, EIA is not recommended for performing test of cure in the female. Overall, the Gonozyme system is an easily performed, rapid, and reliable system that provides for a noncultural alternative for the laboratory diagnosis of gonorrhea.     

Microparticle agglutination versus antibody-capture enzyme immunoassay for diagnosis of community-acquiredMycoplasma pneumoniae pneumonia

Community-acquiredMycoplasma pneumoniae pneumonia is a common disease which is usually diagnosed by serological methods. The objective of the present study was to understand the diagnostic significance and test characteristics of two different serological tests used to identify currentMycoplasma pneumoniae infection. Three hundred sixty-six patients who suffered from community-acquired pneumonia served as the study population. Six hundred ninety-four (328 paired and 38 unpaired) sera were examined for the presence of antibodies toMycoplasma pneumoniae with commercial kits based on two serological methods, microparticle agglutination and antibody-capture EIA. Agreement between the two kits was 85.2 % when individual sera were compared (Kappa=0.62) and 88.5 % when patients were compared (Kappa=0.69). The positive predictive value and the specificity for the identification of currentMycoplasma pneumoniae infection using a single acute-phase serum were 49.3 % and 86.9 %, respectively, for the microparticle agglutination method, compared to 91.3 % and 97.7 % for the antibody-capture EIA method (p<0.001). The negative predictive value and the sensitivity were 86.3 % and 48.1 % for the microparticle agglutination, not significantly different from the corresponding values of 86.5 % and 61.2 % for the antibody-capture EIA. It is concluded that the overall agreement between the two methods tested is good, but not perfect. The methods complement each other in the identification ofMycoplasma pneumoniae as the causative agent in patients with community-acquired pneumonia.     

Application of a capture enzyme immunoassay in an outbreak of waterborne giardiasis in the United Kingdom

A capture enzyme immunoassay (EIA) for the detection ofGiardia lamblia antigen was used to examine 136 fecal samples collected during an outbreak of waterborne giardiasis in a city in the UK. Six cases ofGiardia lamblia infection were detected that had previously not been diagnosed by microscopy. The capture EIA provides an efficient means of processing large numbers of samples for prompt and accurate assessment of an epidemic. It may also facilitate rapid tracing of epidemic sources.     

Stool diagnosis of giardiasis using a commercially available enzyme immunoassay to detect Giardia-specific antigen 65 (GSA 65).

A commercially available enzyme immunoassay for the diagnosis of giardiasis was evaluated in a clinical trial. The ProSpecT/Giardia diagnostic test (Alexon, Inc., Mountain View, Calif.) was compared with the standard ova and parasite (O&P) microscopic examination. Additionally, several widely used stool fixatives and a commonly used transport medium were assessed for compatibility with the immunoassay. A total of 325 stool specimens were collected and used to evaluate assay performance. Of those, 93 specimens were collected from symptomatic Giardia O&P-positive patients and 232 specimens were randomly collected from patients as part of a routine health screening procedure. All 93 Giardia O&P-positive stool specimens were strongly positive by visual and spectrophotometric examination using the immunoassay. Of the 232 randomly collected specimens, 16 were positive by O&P examination and immunoassay, 6 were negative by O&P examination but positive by immunoassay, and 1 was positive by O&P examination and negative by immunoassay. There was substantial supportive evidence that indicated that the six immunoassay-positive, O&P-negative specimens were true-positives. When these six specimens were accepted as true-positives, the immunoassay detected almost 30% more cases of Giardia infection than did O&P examination. Its sensitivity and specificity were 96 and 100%, respectively, while the sensitivity and specificity of O&P examination were 74 and 100%, respectively. The immunoassay also performed well on specimens treated with 10% neutral Formalin, sodium acetate-Formalin fixative, and Cary-Blair transport medium. However, the test was not compatible with polyvinyl alcohol-treated specimens. Overall, the ProSpecT/Giardia test was a sensitive, specific immunoassay which was easy to run and interpret. It offers a simple solution to traditional difficulties encountered in diagnosing Giardia infection.     

Human rotavirus detection by counterimmunoelectrophoresis versus enzyme immunoassay and electron microscopy after direct ultracentrifugation.

The sensitivity of the counterimmunoelectrophoresis test with NCDV, Wa, and SA-11 rotavirus antisera was 60, 60, and 67%, respectively. The counterimmunoelectrophoresis specificity was greater than 99%, but the low sensitivity is a limiting feature of this test as a first-line immunodiagnostic test for rotavirus detection.     

Choice of reference assay for the detection of rotavirus in fecal specimens: electron microscopy versus enzyme immunoassay.

Two previously demonstrated sensitive and specific enzyme immunoassays (EIAs) for rotavirus, one using polyclonal and monoclonal antisera (TestPack Rotavirus [TPK]; Abbott Laboratories) and the other using only monoclonal anti-rotavirus antibodies (Rotaclone [RTC]; Cambridge BioScience Corporation), were evaluated as potential reference assays for rotavirus testing in comparison with direct negative-staining electron microscopy (EM), the current laboratory standard. Two hundred and seven stool samples collected consecutively during the winter of 1989 from children with acute diarrhea admitted to a ward for infants from 0 to 2 years of age were tested by the EIAs and by EM. TPK specimens were read visually; RTC results were read spectrophotometrically. Specimens with discordant EIA and EM results were further evaluated by a fluorescent focus assay. Specimens positive by EM and those negative by EM but positive by fluorescent focus assay were considered to be positive for rotavirus. Of the 207 stools tested, 35 (17%) were positive for rotavirus by these criteria. EM had a sensitivity of only 80%. Specificities were 100% for RTC and EM and 89% for TPK. These findings indicate that EM, although very specific, is relatively insensitive compared with a highly sensitive monoclonal antibody-based EIA. An EIA with high sensitivity and specificity, such as RTC, is a more appropriate reference standard for rotavirus testing.     

Toxocariasis: serological diagnosis by enzyme immunoassay.

An enzyme-immunoassay was developed to measure the concentration of serum antibody specific for the secretory antigens released by migrating toxocaral larvae. This technique was evaluated by testing sera from healthy UK adults, and from patients with and without toxocariasis. In 922 healthy adults, 2.6% were found to have elevated specific antibody levels. Elevated values were observed twice as frequently in males as in females but showed no significant regression with age between 20 and 65 years. Of 62 patients with non-toxocaral helminthic infections, all had antitoxocaral antibody levels within the range of values observed in healthy controls and had a mean level which was not significantly elevated. All of 13 patients with clinical toxocariasis had enzyme-linked immunosorbent assay (ELISA) antibody levels above the 100th percentiles of both the healthy population and the helminth-infected group and had a significantly high mean value (p less than 0.001) more than 12 times that of the healthy or infected controls. The high degree of sensitivity and specificity of the toxocariasis enzyme-immunoassay indicates that this new test should be useful in reference immunodiagnostic applications and in large-scale seroepidemiological surveys.     

Sensitive enzyme immunoassay for early diagnosis of tuberculous meningitis

Cerebrospinal fluid from patients with tuberculous, pyogenic, and viral meningitis, as well as from appropriate control individuals, were assayed for immunoglobulin G and immunoglobulin M antibody activity to Mycobacterium bovis BCG by an enzyme-linked immunosorbent assay. BCG linked covalently to plastic disks served as the antigen in a classical indirect enzyme-linked immunosorbent assay. A significant difference was found between the tuberculous meningitis group and the nontuberculous meningitis and control groups. All samples from the tuberculous meningitis group gave a positive reaction, and none of the known negative samples gave false-positive reactions. Because of its sensitivity, specificity, and predictive value, this test may be useful in the early diagnosis of tuberculous meningitis.     

Competitive enzyme immunoassay for diagnosis of human brucellosis.

The methods commonly used for human brucellosis serological testing are agglutination tests and the complement fixation test (CFT). Among the newer serological tests, primary binding assays were developed to improve sensitivity and specificity. The competitive enzyme immunoassay (CELISA) for the detection of serum antibody to Brucella is a multispecies assay which appears to be capable of differentiating vaccinal and cross-reacting antibodies from antibodies elicited by field infection in cattle. The competing monoclonal antibody used in this assay is specific for a common epitope of smooth lipopolysaccharide (S-LPS). In this study, we compared the CELISA to the classical tests for the diagnosis of human brucellosis. The CELISA cutoff value was determined to calculate its diagnostic specificity and sensitivity. A survey was performed with 911 sera. Of the sera, 341 were from an asymptomatic population that tested negative with conventional serological tests (screening and confirmatory). Based on these samples, the CELISA specificities were determined to be 99.7 and 100% with cutoff values of 28 and 30% inhibition (%I), respectively. In a further study with 393 additional sera from an asymptomatic population found negative by the conventional screening tests, the CELISA specificities were calculated to be 96.5 and 98.8% with cutoff values of 28 and 30%I. The CELISA sensitivities were determined to be 98.3 and 94.8% with cutoff values of 28 and 30%I, respectively, for sera from 116 individuals found positive by the classical tests. For the 51 culture-positive patients, CELISA was positive for 100%, the CFT was positive for 92%, and the standard tube agglutination test (TAT) was positive for 100%. The CELISA specificity was 100% for 31 sera from patients found negative by conventional serological tests but with brucellosis-like symptoms. The CELISA is fairly rapid to perform, somewhat faster than TAT, and cross-reacts less with other antigens (or antibodies) than the conventional tests. Further, the CELISA is simpler to perform that the CFT and may readily be standardized by the use of purified S-LPS antigen and monoclonal antibody for competition.     

Lipase versus teichoic acid and alpha-toxin as antigen in an enzyme immunoassay for serological diagnosis ofStaphylococcus aureus infections

Titres of IgG antibodies toStaphylococcus aureus lipase were analysed in 448 sera from patients suspected of havingStaphylococcus aureus infections and the results compared to those for the routinely used staphylococcal antigens teichoic acid and alpha-toxin. The results indicated that determination of serum antibodies to lipase is a sensitive assay for serological diagnosis of staphylococcal infections and increased sensitivity may be achieved by selection of optimal antigen combinations.     

Serological diagnosis of Mycoplasma pneumoniae infection by enzyme immunoassay.

Antibodies against Mycoplasma pneumoniae antigen obtained by Tween-ether treatment from purified M. pneumoniae were measured by means of enzyme-linked immunosorbent assay (ELISA). Paired sera from 19 patients with pneumonia and from 13 patients with acute pancreatitis with a significant rise in complement fixing antibodies against M. pneumoniae were studied. Single sera from healthy 1-year-old children were used as controls. High levels of IgG and IgM class antibodies were seen in sera from patients with pneumonia while most patients with acute pancreatitis and all the children showed low levels of antibodies. The results indicate that ELISA using Tween-ether treated M. pneumoniae antigen could be used successfully in the specific laboratory diagnosis of M. pneumoniae infection.     

Evaluation of a commercial enzyme immunoassay versus culture for the detection ofChlamydia trachomatis

A commercial EIA (Chlamydiazyme) for detection ofChlamydia trachomatis was evaluated in comparison to culture using genital specimens from 472 men and 279 women. The sensitivity of the EIA compared with culture was 66.0% in men and 71.4% in women, while the specificity was 99.7% and 95.9% respectively. The EIA failed more often to detect chlamydial antigen when the number of inclusion bodies found in the corresponding cultures was 100/well. A direct test (MicroTrak) was performed on the EIA samples which showed discordant results compared to corresponding cultures. One of 17 EIA positive samples, and 12 of 36 EIA negative samples were positive in the direct test (p<0.05). A cut-off absorbance value of 0.1 is recommended by the manufacturer in the EIA. However, 84.2% of the EIA negative samples in the negative absorbance interval 0.05–0.099 corresponded with a positive culture. In view of variations in the sensitivity of the culture technique between laboratories and the low sensitivity of the EIA found in this study, it is recommended that each laboratory using the EIA compare it to culture. It is also recommended that an equivocal zone around the cut-off value be used in the EIA, the zone to be established by each laboratory using the test.     

Determination of lymphocyte subpopulations by enzyme immunoassay. Comparison with conventional fluorescence microscopy

An enzyme immunoassay for measuring lymphocyte subpopulations was evaluated and the results compared with those obtained with conventional fluorescence microscopy, using two different panels of antibodies. The enzyme immunoassay is a photometric method which expresses the results using a standard curve with known amounts of cells. The method was reproducible and accurate. The intra-assay variation for the standard curve ranged from 3.2 to 5.7% and the interassay variation from 9.5 to 13.8%. The intra-assay variation for clinical samples ranged from 3.3 to 8.2%. Results obtained with the enzyme immunoassay and with conventional fluorescence microscopy showed a significant correlation (P less than 0.05) for all the subclasses of lymphocytes tested using two different panels of monoclonal antibodies. We conclude that the choice of method should be related to the particular needs and manpower in the laboratory.     

Evaluation of a recombinant antigen-based enzyme immunoassay for the diagnosis of noninvasive aspergillosis

Antibody detection is a key diagnostic tool for noninvasive aspergillosis (NIA) such as allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis. Specific immunoprecipitin detection (IPD) is considered as the reference but lacks standardization and is time-consuming. To evaluate the performance of a new anti-Aspergillus fumigatus IgG enzyme immunoassay (EIA) kit using a recombinant A. fumigatus antigen (Bio-Rad), a retrospective study was performed on 551 sera collected from patients with a definite diagnosis of NIA (group 1; n = 64), bronchial Aspergillus colonization (group 2; n = 26), and probable aerial Aspergillus contamination (group 3; n = 44); from patients suspected of NIA with negative serological and mycological investigations (group 4; n = 49); and from a group of 222 patients not suspected of NIA (group 5). The EIA exhibited excellent reproducibility with coefficients of variation below 10%. Agreement with IPD was calculated between 62.5 and 84.4% according to the group of patients with Cohen's kappa coefficient at 0.6196 ± 0.077. Taking as reference a composite status including clinical, radiological, mycological, and serological data, sensitivity (group 1) and specificity (other groups) were calculated between 90.2 and 93.8% and 54.3 and 100%, respectively. Lower specificity was observed for patients with Aspergillus colonization. However, Yule Q coefficients estimating the correlation between EIA result and the definite diagnosis of NIA were calculated between 0.97 and 0.98. The method is a highly useful screening tool for the diagnosis of NIA, reducing the need for confirmatory IPD tests.     

Examination of the Rotazyme II enzyme immunoassay for the diagnosis of rotavirus gastroenteritis.

Rotazyme II, which is a shorter version of Rotazyme (less than 3 h), was compared with electron microscopy and Rotazyme for sensitivity and specificity on 229 human stool specimens. Compared with electron microscopy, the newer assay was found to be 99.4% sensitive and 97.3% specific for an overall agreement of 98.7%. After resolution of discordant results by blocking tests, the Rotazyme II enzyme immunoassay was shown to be more sensitive than electron microscopy and, therefore, 100% specific.     

Semiquantitative polymerase chain reaction enzyme immunoassay for diagnosis of disseminated Candidiasis

A polymerase chain reaction enzyme immunoassay (PCR-EIA) was developed for the semiquantification of circulating candidal DNA in disseminated candidiasis due toCandida albicans. Polymerase chain reaction was based on primers from the internal transcribed ribosomal region. Binding of the product to a streptavidin-coated microtitration plate was mediated by a biotinylated capture probe. The product was digoxigenylated during PCR; this was the tag to which antibody was bound in the subsequent EIA. The optical density (OD) endpoint was <0.1 in 15 sera from patients with no evidence of candidal infection (group 1) and in 13 of the 16 sera from colonised patients (group 2); it was >0.1 in the other three sera from group 2 blood culture-negative patients who required intravenous amphotericin B for cure. The OD was positive in 28 patients with disseminated candidiasis (group 3), defined as positive blood cultures and successful treatment with amphotericin B (n=11), positive blood culture confirmed at autopsy (n=11), or negative blood culture first proven at necropsy (n=6). In patients from whom multiple samples were available, recovery correlated with an optical density of <0.1 by day 4 in four patients and by day 13 in the rest. In the five patients with fatal outcome from whom multiple samples were available, the mean OD rose from 0.174 to 0.668. Samples seeded withCandida albicans blastoconidia demonstrated that an OD of 0.220 was equivalent to 10 cfu. Assay of the group 3 sera by a commercial antigen detection test gave a corresponding sensitivity of 60%, which rose to 67.9% when an in-house reverse passive latex agglutination test was used.     

An improved membrane-filtration enzyme immunoassay for the rapid serological diagnosis of viral infections

A one-step modification of the membrane-filtration enzyme immunoassay (MF EIA) (Barnett et al. J. Clin. Microbiol., 23:385–399, 1987), for estimation of virus-specific antibody is described. The modified MF EIA allowed serum, antigen and enzyme-conjugated anti-globulin to be incubated together in membrane-based 96-well plates to enable the formation of immune complexes in solution at 37°C. The assay required only 45 min for completion and polyethylene glycol was shown to be an essential component in reaction mixtures for IgG assays to enhance immune complex formation. The modified MF EIA was as sensitive as the previous two-step method for monitoring responses to influenza vaccin, and control antigen backgrounds were significantly reduced. The one-step procedure was also shown to be suitable for the rapid serodiagnosis of naturally acquired influenza A and B infections. However, MF EIA detected cross-reactive H1N1 responses in 57.7% of naturally-acquired H3N2 infections, suggesting that responses to common internal antigens were being measured. Cross-reactive responses to influenza A viruses could not be detected in volunteers receiving subunit vaccines.