Adenovirus ELISA for the evaluation of cerebrospinal fluid in patients with suspected neurosyphilis.

An enzyme-linked immunosorbent assay (ELISA) detecting antibodies against the adenovirus group antigen was used to examine cerebrospinal fluid (CSF) of suspected neurosyphilis patients for serum contamination or blood-brain barrier (BBB) leakage. Adenovirus antibodies are ubiquitous, produce high antibody titers, and rarely cause central nervous system (CNS) infections. Of 52 normal adult sera tested with this ELISA, only one lacked antibodies. CSF from 48 healthy individuals did not present a detectable amount of anti-adenovirus antibodies. CSF from 33 suspected neurosyphilis patients with positive results in the fluorescent treponemal antibody-absorption (FTA-ABS)-CSF test were examined. Eighteen showed anti-adenovirus antibodies indicating contamination of the CSF with peripheral blood or damaged BBB by syphilis or other disease, resulting in questionable CSF treponemal results. The remaining 15 of these patients appeared to be producing their anti-treponemal antibodies in the CNS. This procedure may prove to be of considerable help in excluding false positive FTA-ABS results in CSF samples.

Detection of Chlamydia trachomatis cervical infection by urine tests among adolescents clinics.

PURPOSE: To compare urine ligase and polymerase chain reaction (LCR, PCR) tests for diagnosis of Chlamydia trachomatis cervical infection with PCR and nucleic acid probe (GPA) on cervical specimens in adolescents, as well as risk factors for C. trachomatis infection and prevalence of infection at enrollment. METHODS: Urine and cervical specimens were collected from women aged 13-20 years attending adolescent clinics, and interviews were administered. Urine specimens were tested by PCR and LCR, and cervical specimens by GPA and PCR. Prevalence rates of C. trachomatis infection and gonorrhea were compared by demographic, behavioral, and clinical risk factors. RESULTS: Of 415 women tested, 86 (20.7%) were infected with C. trachomatis as indicated by positive cervical PCR results. A higher prevalence of C. trachomatis infection was seen among adolescents who douched monthly or more frequently, or had gonorrhea; prevalence declined from 25.8% in the first 7 months to 16.3% in the last 14 months of the study (p = .017). A statistically significant protective effect for reported condom use was not observed. Sensitivity of urine PCR was 89.5% and specificity was 100% relative to cervical PCR, compared to 84.9% and 99.4% (urine LCR) and 65.4% and 98.0% (cervical GPA). Sensitivity of urine PCR was higher in women with discharge; urine LCR sensitivity was higher in women < 19 years of age. CONCLUSIONS: Polymerase chain reaction and LCR assays on urine specimens were sensitive, specific, and noninvasive tests in this population of adolescents with high C. trachomatis infection prevalence. Chlamydia trachomatis infection was associated with douching monthly or more frequently. Prevalence of infection declined over the period during which the study was conducted.

Immunofluorescent staining of Treponema in tissues fixed with formalin.

Immunofluorescent examination of formalin-fixed tissue for Treponema pallidum has generally been unsatisfactory because of nonspecific background fluorescence and poor contrast. We examined the process of treating deparaffinized formalin-fixed tissue sections with 1% ammonium hydroxide (NH4OH) to improve fluorescent staining. Treponema pallidum- and Treponema pertenue-infected rabbit testes or human tissue biopsy specimens fixed in 10% buffered formalin and embedded in paraffin were examined. Sections were cut one week to five years after embedment. Tissues were then stained with fluorescein- or rhodamine-labeled human anti- T pallidum globulin for 30 minutes at 37 degrees C. Treponemes were consistently stained and background staining was generally reduced after NH4OH treatment in both fresh and stored tissue. Cutting sections at a thickness of approximately 2 micron was critical to achieve optimal fluorescence.

Detection of Legionella pneumonophila antigen in urine by enzyme-linked immunospecific assay.

An enzyme-linked immunospecific assay "sandwich" technique was developed for detecting soluble antigen from the Legionnaires disease bacterium (Legionella pneumophila). With this technique, antigen was detected in urine specimens from guinea pigs inoculated intraperitoneally with heat-killed Legionnaires disease bacteria and in urine specimens from three of four patients who attended the American Legion Convention in Philadelphia in 1976. Urine from a fifth pneumonia patient who attended the Eucharistic Congress (but who was a dubious seroconverter) was negative. Presumably, the test could also be used for detecting antigen in sputum or respiratory aspirates, but this has not been tried to date.

Detection of antibodies to legionnaires disease organism by microagglutination and micro-enzyme-linked immunosorbent assay tests.

Microagglutination and micro-enzyme-linked immunosorbent assay (ELISA) tests with easily prepared, safe, heat-killed antigens for detecting antibodies to the legionnaires disease organism have been developed. A safranin-stained whole-cell antigen is used in the microagglutination test, and a simply prepared soluble antigen is used in the micro-ELISA tests. The microagglutination test detected elevated titers in 97.2% of the sera from patients with legionnaires disease. Three variations of the micro-ELISA test with anti-human immunoglobulin G, immunoglobulin M, and Fab peroxidase-labeled conjugates revealed elevated titers with 74.3, 82.9, and 88.6% of the sera, respectively. The microagglutination and the micro-ELISA tests used in combination detected 100% of the elevated titers.

Multiple drug-resistant Chlamydia trachomatis associated with clinical treatment failure.

In vitro susceptibility testing and genotyping were done on urogenital isolates of Chlamydia trachomatis from 3 patients, 2 of whom showed evidence of clinical treatment failure with azithromycin and one of whom was the wife of a patient. All 3 isolates demonstrated multidrug resistance to doxycycline, azithromycin, and ofloxacin at concentrations >4.0 microg/mL. Recurrent disease due to relapsing infection with the same resistant isolate was documented on the basis of identical genotypes of both organisms. This first report of clinically significant multidrug-resistant C. trachomatis causing relapsing or persistent infection may portend an emerging problem to clinicians and public health officials.

Four-step enzyme-linked immunosorbent assay for detection of Treponema pallidum antibody.

Further studies of a four-step enzyme-linked immunosorbent assay procedure to detect Treponema pallidum antibody are described. High-titered antibody, produced in rabbits by intravenous injection of T. pallidum, was used to coat polyvinyl chloride microtiter plates. To these plates a known concentration of T. pallidum was added, followed in successive steps by serial dilutions of human sera and appropriately diluted peroxidase-labeled anti-human immunoglobulin G antibody. O-Phenylenediamine was the substrate. A total of 340 sera were obtained from the DeKalb County Sexually Transmitted Diseases Clinic, Atlanta, Ga., and examined within 3 days of receipt. Ninety-six percent test agreement between the enzyme-linked immunosorbent assay and the fluorescent treponemal antibody absorption-double staining test was obtained. A total of 372 additional sera stored at -20 degrees C were examined. The overall sensitivity of the enzyme-linked immunosorbent assay with sera from patients with various stages of syphilis was 96%. With sera from uninfected individuals, the specificity of the enzyme-linked immunosorbent assay was 95%. No antigen instability was noted with the two antigen preparations used during this evaluation.

Double-conjugate enzyme-linked immunosorbent assay for immunoglobulins G and M against Treponema pallidum.

An enzyme-linked immunosorbent assay (ELISA) for the simultaneous measurement of immunoglobulin G (IgG) and IgM was developed to detect antibodies to Treponema pallidum. Wells of polystyrene microtiter plates were coated with T. pallidum antigen, diluted patient serum was added, and IgG and IgM which bound to the T. pallidum antigen were measured by the simultaneous addition of alkaline phosphatase-labeled anti-human IgG and horseradish peroxidase-labeled anti-human IgM. Bound IgG was detected first, followed by bound IgM. After development of the procedure, 145 categorized sera were evaluated: 60 from individuals without syphilis; 62 from patients with syphilis, including 22 with primary, 20 with secondary, and 20 with latent phases of syphilis; and 23 from patients with rheumatoid arthritis. Of the 60 sera from individuals without syphilis, 100% were nonreactive for IgG antibody and 16% were reactive for IgM. Of the 23 sera from patients with rheumatoid arthritis, 3 were reactive for IgG and 3 were nonreactive for IgM. Of the 62 sera from patients with syphilis, 61 (98%) were reactive for IgG antibody with increased titers as the stage of syphilis increased, whereas IgM reactivity decreased. This enzyme-linked immunosorbent assay appears to be a simple method for the simultaneous measurement of antibodies under equal assay conditions.

Rheumatoid factor in syphilis.

Immunoglobulin M (IgM) antibodies directed against IgG antibodies (rheumatoid factor [RF]) are known to occur often in patients with syphilis and to interfere with serological tests measuring specific antibodies of the IgM class. In this study we examined the occurrence and specificity of the RF and demonstrated a simple method to detect and eliminate the RF for a specific Treponema pallidum IgM enzyme-linked immunosorbent assay. We measured the occurrence of the RF with a sensitive enzyme-linked immunosorbent assay and found that it increased with the duration of syphilitic disease: 1 of 13 primary syphilis serum specimens, 3 of 13 secondary syphilis serum specimens, and 10 of 27 latent syphilis serum specimens were reactive in this RF test. Those sera containing IgM RF were immunoprecipitated with anti-human gamma chain antibodies and 2% polyethylene glycol until the RF was removed. One serum specimen from a patient in the secondary stage of syphilis and eight serum specimens from patients with latent disease still presented the RF after immunoprecipitation. Removal of the IgG antibodies also improved the sensitivity of the treponemal IgM test, indicating competition of these antibodies for binding sites of the antigen. The enzyme-linked immunosorbent assays for detection of RF and antitreponemal IgM antibodies are performed on the same plate. Theoretically, only sera positive for both tests have to be immunoprecipitated. But our findings indicated an increase in sensitivity of the IgM enzyme-linked immunosorbent assay after removal of IgG antibodies responsible for competition at the binding sites.

Four serologic tests for syphilis: results with comparison of selected groups of sera.

The specificity of the fluorescent treponemal antibody-absorbed (FTA-Abs) test was assessed for 17 sera from syphilitic patients that were nonreactive in the Treponema pallidum immobilization (TPI) test but reactive in the FTA-Abs test. Thirty-three other sera from syphilitic patients and 19 sera from nonsyphilitic individuals were also examined by fluorescent treponemal and microhemagglutination Treponema pallidum (MHA-TP) tests and by the enzyme-linked immunosorbent assay (ELISA). Specific absorptions of sera with calf thymus DNA or Treponema pallidum biotype Reiter (Reiter treponemes) were performed. In quantitative immunofluorescence assays (IFA) with antihuman IgG and IgM conjugates, results were similar to those for reactive sera from a control group. Results of both the MHA-TP and ELISA tests supported the specificity of the FTA-Abs test; reactivity in the latter was not removed by specific absorption either with calf thymus DNA or with Reiter treponemes. This evaluation suggests a format for serodiagnosis in cases in which test results are discrepant.

Evaluation of sera from patients with Lyme disease in the fluorescent treponemal antibody-absorption test for syphilis.

To determine whether the cross-reactivity between Treponema pallidum and Borrelia burgdorferi affects the specificity of the fluorescent treponemal antibody-absorption (FTA-Abs) test for syphilis, sera from patients with Lyme disease or syphilis were examined in a quantitative FTA-Abs test. Sera were diluted serially in phosphate-buffered saline, then in sorbent, and were tested with T. pallidum and B. burgdorferi antigens. Nine of 40 sera from patients with known Lyme disease were reactive at the 1:5 dilution with antigen from T. pallidum; only one serum was reactive at the 1:10 dilution. When both antigens were tested, the titer against B. burgdorferi was always higher than that against T. pallidum. Similarly, sera from patients with syphilis showed cross-reactivity with B. burgdorferi. Although reactivity could be absorbed with Treponemal phagedenis (Reiter strain), simultaneous titration with both antigens was easily performed and designated the etiologic agent.

The relationship of race, sex, and age to concentrations of serum immunoglobulins expressed in international units in healthy adults in the USA.

Only a few investigations have been made to obtain human serum immunoglobulin values in units compatible with those used by the WHO International Reference Preparation for the Human Immunoglobulins IgG, IgA, and IgM. We report our summary statistics of serum IgG, IgA, and IgM, in international units (IU), for some 800 healthy American adults grouped by age, sex, and race. Our findings are in general agreement with some, but not with all, published data. We found that the mean IgG concentration is markedly higher and the mean IgA concentration is slightly higher in blacks than in whites. Except for white females, there was a significant increase in mean IgA with age for both races. In the younger adults of both races, mean IgM values were markedly higher in females than in males. Statistically significant interactions between race, age, and sex factors were seen for all three immunoglobulin classes. Although we have attempted to estimate the normal population means and variances for the serum concentration of IgG, IgA, and IgM the process we used to select specimens may have resulted in some bias; much larger, truly randomized, and fully documented studies in different geographic areas and in different socioeconomic and racial groups are needed to provide accurate acceptable limits for human immunoglobulins.

Enzyme immunoassay to determine exposure to Chlamydia pneumoniae (strain TWAR).

Recent studies suggest that a group of Chlamydia strains known as TWAR, which are now proposed to be a new species called Chlamydia pneumoniae, may be a frequent cause of respiratory disease in the United States and many other countries. Current serotesting methods do not allow rapid screening of large numbers of samples to distinguish C. trachomatis exposure from C. pneumoniae exposure. We developed an enzyme immunoassay to decrease cross-reactivity between immunoglobulin G antibodies reactive with C. trachomatis and C. pneumoniae. Elementary bodies of C. trachomatis or C. pneumoniae were treated with a detergent-chelating solution to decrease the reactivity of the common lipopolysaccharide antigens. Sera from four groups of patients, totaling 143 persons, were tested by this assay. The prevalences of titers of greater than or equal to 128 to C. trachomatis and C. pneumoniae, respectively, were as follows: (i) for 23 women seropositive for C. trachomatis by the microimmunofluorescence test, 21 (91%) and 18 (78%); (ii) for 50 adult blood donors, 13 (26%) and 39 (78%); (iii) for 40 sexually transmitted disease clinic patients, 20 (50%) and 32 (80%); (iv) for 30 healthy children 5 to 7 years old, 0 (0%) and 8 (27%). Western blots (immunoblots) of each antigen corroborated the differential reactivity of C. trachomatis-positive, C. pneumoniae-negative and C. trachomatis-negative, C. pneumoniae-positive serum samples. Western blots of serum samples from rabbits immunized with either C. trachomatis or C. pneumoniae elementary bodies revealed at least two protein bands (30 and 80 kilodaltons) which appeared to represent unique C. pneumoniae antigens.

Heated versus unheated sera in a microhemagglutination assay for antibodies to Treponema pallidum.

The effect of prior heating of sera on the results of the microhemagglutination assay for antibodies to Treponema pallidum was evaluated. Findings indicate that heating of sera had no significant effect on the results.

Fluorescent treponemal antibody absorption double-staining test evaluation.

The fluorescent treponemal antibody absorption (FTA-ABS) double-staining (DS) test has been developed for microscopes equipped with incident illumination, and the procedure offers many advantages over the FTA-ABS test when tests are performed with this equipment. In this study, 346 fresh sera, including 35 from patients with syphilis, were evaluated by the FTA-ABS DS test. Parameters for investigation included two readers, each using a different microscope; a new FTA-ABS DS test reporting system; sera heated at 56 degrees C for 30 min versus unheated sera; and sera retested after at least 2 weeks of freezer storage. Agreement for FTA-ABS DS test readings between the two microscopes was 99%. Between-test agreement for the FTA-ABS test with the conventional reporting system and the FTA-ABS DS test with the new reporting system was 95%. Sensitivity calculations based on reactivity for the 35 syphilis sera were 94% for the FTA-ABS DS test and 91% for the FTA-ABS test. Specificity calculations based on non-reactivity of nonsyphilis sera were 98% for the FTA-ABS DS test and 93% for the FTA-ABS test. Differences in percentages appeared to be related to borderline readings in the FTA-ABS test. For example, if the same reporting system was used for the reference FTA-ABS test, the specificity was 97%. When sera were examined within 48 h, no difference was observed in results obtained with heated and unheated sera. Sera frozen for 2 weeks showed comparable results in the FTA-ABS DS test and the FTA-ABS test. These findings strongly support the recommendation that the FTA-ABS DS test be accepted as a confirmatory test for syphilis. The new reporting system for the FTA-ABS DS test would be advantageous for the reference FTA-ABS procedure.

Staining intensities in the fluorescent treponemal antibody-absorption (FTA-Abs) test: association with the diagnosis of syphilis.

In 1984 the reporting system for the fluorescent treponemal antibody-absorption (FTA-Abs) test was changed by the Centers for Disease Control (CDC; Atlanta, GA) to eliminate the borderline report. Factors influencing the reliability of the FTA-Abs test results, i.e., sensitivity, specificity, prevalence of syphilis, prescreening of sera with nontreponemal tests, and reproducibility, were considered before the change in the reporting system was recommended and are reported here. The borderline report, when associated with syphilis, was most frequently also associated with the diagnosis of early primary, dark-field-positive, nontreponemal test-nonreactive syphilis. Whereas elimination of the borderline report decreased the sensitivity of the FTA-Abs test as a confirmatory test from 100% to 99.5%, the specificity increased from 82.5% to 88.7%. The 1+ staining intensity had an association of approximately 5% with the diagnosis of syphilis. The changes in the reporting system were designed to assist the clinician in interpreting the results of the FTA-Abs test in those cases that present diagnostic dilemmas.

Antigenic variation among strains of Chlamydia pneumoniae.

The antigenic profiles of six strains of Chlamydia pneumoniae were analyzed by the microimmunofluorescence test (MIF) and immunoblotting with human serum and murine monoclonal antibody. MIF-derived antibody titers in serum samples from culture-positive patients were four- to eightfold higher against autologous isolate antigen than they were against the prototype antigen strain TW-183. Sera of patients with respiratory illness that were culture negative and complement fixation positive for Chlamydia spp. produced higher titers by MIF against a strain of C. pneumoniae isolated in the area than they did against TW-183. For two of five cases, the criteria for establishing the diagnosis of acute infection were met only with use of the antigen from the local strain; TW-183 was inadequate for this purpose. Immunoblot profiles revealed antigenic differences between strains that varied with the human serologic response; i.e., unique antigens were recognized by the sera of some individuals and not by the sera of others. Using the reactivity of a genus-specific monoclonal antibody against a major outer membrane protein, we found that strain CWL-011, isolated in Atlanta, Ga., may possess a major outer membrane protein with a molecular mass between those of C. trachomatis L2 and other C. pneumoniae strains. These data provide evidence of several new and unique serotypes of C. pneumoniae and suggest that the serologic diagnosis of C. pneumoniae infection may require the use of antigens from more than one strain of this species.

Measure of immunoglobulin G-, M-, and A-specific titers against Legionella pneumophila and inhibition of titers against nonspecific, gram-negative bacterial antigens in the indirect immunofluorescence test for legionellosis.

A crude extract of Escherichia coli O13:K92:H4 inhibited 97% of positive indirect immunofluorescence titers against a variety of gram-negative bacterial antigens while lowering Legionella pneumophila titers in only 6% of sera from patients with suspected legionellosis. Legionella-specific titers were the result of immunoglobulins G, M, and A, singly or in combination.

Sodium desoxycholate-extracted treponemal antigen in an enzyme-linked immunosorbent assay for syphilis.

The extraction of Treponema pallidum antigen with sodium desoxycholate, based on a previously described procedure (J. Portnoy and H.J. Magnuson, J. Immuno. 75:348-355, 1955), was used in an enzyme-linked immunosorbent assay (ELISA) test for syphilis. The antigen was prepared from T. pallidum street strain no. 14, and its overall sensitivity and specificity was compared with those of sonicated antigen preparations made with phosphate-buffered saline. The optimum serum dilution for testing and the significant absorbance reading at 490 nm were selected by examination of quantitative dilutions of 91 sera from presumably normal individuals and 92 sera from syphilitics. The time and temperature of serum and conjugate incubations were also examined. With an absorbance reading of greater than or equal to 0.2 at the 1:80 serum dilution, 88 (95.8%) of 92 sera from syphilitics were reactive in the ELISA test with desoxycholate-extracted antigen, and 82 (89.1%) were reactive with the sonicated antigen. Only one nonsyphilitic serum was reactive with each antigen. Greater sensitivity without loss in specificity was obtained with longer serum and conjugate incubations. We concluded that an ELISA test with sodium desoxycholate-extracted antigen is more sensitive than and equally specific to an ELISA with sonicated treponemal antigen.