Chlamydia DNA extraction for use in PCR: stability and sensitivity in detection.

We evaluated multiple procedures for extracting chlamydial DNA from specimens for detection in PCR tests. Commercial kits and an in-house method were tested for their sensitivity and utility. Quantifiable chlamydial elementary bodies (EB) were used for spiking buffy coats from EDTA-collected blood. EBs of Chlamydia pneumoniae at 2,500 and 25 EB/ml were used as specimens for DNA extraction using seven different procedures. These included either columns (3 procedures), centrifugation (1), glass (1), or patented extraction matrices (2), coupled with either alcohol precipitation (6) or heat-detergent treatment (1). Five procedures required 10-40 minutes manipulation; two required 2-5 hours. PCR results for DNA extracts using chlamydial 16S genus primers were generally more intensely positive with denser bands on electrophoresis gels for the higher concentrations of EB (up to 4+ for stained product on gels) than was PCR with lower EB concentrations (up to 2+). Further, the incidence of procedures with positive results was: 5 of 7 for chlamydial genus primers with 5 EB vs. 6 of 7 with 500 EB. Maximal sensitivity for one of the extractions was in the range of 2.5-5.0 EB/ml of test specimen with 4 of 5 replicates being positive with EB controls or extracts. Extracts were stable up to 2+ weeks at 4 degrees C and were effective in multiplexing with fluorescent-tagged primers. Taking into consideration the time factor and sensitivities, the two procedures with extraction matrices are favored for routine laboratory use.

Application of a nested, multiplex PCR to psittacosis outbreaks.

We developed a nested, multiplex PCR for simultaneous detection of three species of chlamydiae in human and avian specimens. The PCR was designed to increase sensitivity and to circumvent inhibitors of PCR present in clinical specimens. The target sequence was the 16S rRNA gene. The first-step PCR was genus specific, and the second-step PCR was multiplexed (i.e., had multiple primer sets in the same tube) and could discriminate among Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia trachomatis on the basis of the molecular weight of the amplicon. The limit of detection of each of the two PCR steps was 5 inclusion-forming units. We used PCR and serologic evidence during outbreaks of psittacosis to infer that C. psittaci had been transmitted from birds purchased in pet stores to humans. We also used this method to test both live and dead birds from pet stores for infection with C. psittaci. Compared with culture, the application of PCR to avian specimens increased the rate of C. psittaci detection.

ELISPOT assay for Chlamydia-specific, antibody-producing cells correlated with conventional complement fixation and microimmunofluorescence.

Chlamydia antigens cross-reactive with pneumoniae (TWAR), psittaci, and trachomatis strains were used to evaluate the ELISPOT assay for detecting antigen-specific, antibody-secreting cells (ASC). Human blood specimens from healthy and hospitalized persons were randomly collected and tested by coating the nitrocellulose membrane at the base of microtiter wells. Ficoll-separated mononuclear cells from blood specimens collected in EDTA were incubated in the wells with Iscove's growth medium in CO2 atmosphere at 37 degrees C. An IgG-specific conjugate labeled with biotin was used in an avidin-peroxidase chromogen system for indicating the areas (spots) of immunologically committed lymphocytes. Positive specimens had median levels of ASC above 8 per 10(6) cells (range 15-23 ASC/10(6) cells). Evidence that the ELISPOT is reliable, sensitive, and specific includes the following:(1) immunized animal and clinical human specimens in control experiments were selectively reactive in the presence of antigen, but negative without antigen, (2) serologically characterized reference sera demonstrated homologous rather than heterologous reactions with the antigens, (3) conventional complement fixation and microimmunofluorescence on serum fractions of clinical specimens correlated well (P < 0.02) with ELISPOT results that were both TWAR- and psittaci-positive, and (4) the array of specimens (from healthy donors, community hospitalized, and pulmonary service patients) selected for their increasing likelihood in that order for being positive due to illness was then confirmed and supported by their respectively increasing positivity rates (6, 15, and 25%) for TWAR/psittaci combined. The incidence of positive specimens for either TWAR or psittaci was greatest (23/54, 43%) in specimens from the hospitalized patients and least (8/33, 24%) in specimens from healthy individuals. These findings suggest that ELISPOT detects chalmydial antibody production at the cellular level. ELISPOT positivity thus indicates previous exposure and would favor earlier detection.

Utility of complement fixation and microimmunofluorescence assays for detecting serologic responses in patients with clinically diagnosed psittacosis.

The serodiagnosis of human psittacosis was considerably improved by a microimmunofluorescence (MIF) assay that uses selected strains of Chlamydia psittaci, C. pneumoniae, and C. trachomatis as antigens. The 78 patients examined in the study were clinically diagnosed as having psittacosis on the basis of compatible clinical symptoms following exposure to sick birds. The conventional complement fixation (CF) test identified 36 patients, or 46% (36 of 78) of the total, as positive. Antibody responses to C. psittaci were demonstrated by the MIF test in all 36 CF-positive patients. The MIF test also detected antibody responses to C. psittaci in 12 patients (15% of the total) whose sera were negative or anticomplementary in the CF test. Seven patients, or 9% (7 of 78) of the total, were identified by the MIF test as having C. pneumoniae infections. About 30% of the study patients (23 of 78) showed no serologic evidence of either C. psittaci or C. pneumoniae infection by both the CF and the MIF tests. Four distinctive serologic reaction patterns were observed in the study patients. Recognition of these reaction patterns and judicious corroboration of serologic responses to the chlamydial species by the MIF test with epidemiologic and clinical information will increase the efficiency and accuracy of serodiagnosis for human psittacosis.

Morphologic and staining characteristics of a cyanobacterium-like organism associated with diarrhea.

A spherical organism 9-10 microns in diameter, seen in three outbreaks of diarrhea in Southeast Asia and the United States during the past 2 years, bore characteristics of a cyanobacterium when observed in formalin-preserved stool specimens and by electron microscopy. Organisms in freshly passed stool specimens showed an internal morula of lipid-containing globules. In fresh water, the morula divided into two sausage-shaped structures resembling the sporocysts of an isosporid coccidian. After 7 months, the organisms had not developed the crescentic sporozoites seen in the Coccidia but had begun to multiply slowly in culture. It was impossible to stain the internal structures of the organisms because the outer cyst wall ruptured during desiccation, releasing the contents of the cysts. The organisms were readily identified by their intense blue autofluorescence under UV light, but they were also recognizable by bright-field microscopy and by a modified acid-fast stain. Almost all infected persons suffered intermittent diarrhea for 2-3 weeks and many emphasized a feeling of intense fatigue during the course of their illness.

Comparative study with in situ hybridization and immunocytochemistry in detection of HIV-1 in formalin-fixed paraffin-embedded cell cultures.

The sensitivities of three immunohistological techniques were compared in this study for detecting human immunodeficiency virus (HIV-1) in infected cultured human lymphocytes that had been formalin-fixed and paraffin-embedded. The techniques included in situ hybridization (ISH) with HIV-1 cDNA; immunocytochemistry with HIV-1 p24 monoclonal antibody (ICC-m); and immunocytochemistry with HIV-1 polyclonal antibody from a patient with acquired immunodeficiency syndrome (AIDS) (ICC-p). Procedures were optimized for enzyme digestion and for antibody reaction conditions. HIV-1--infected cells and noninfected control cells were tested. Noninfected controls were uniformally negative by all three methods. Infected cells had the highest positivity rate by the ISH method (p less than or equal to 0.0001), and the ICC-p method was more positive than the ICC-m (p less than or equal to 0.0001). Both the ICC-p and the ICC-m techniques were more positive with the cocultivated cell cultures than the ISH, which was more sensitive with the infected continuous cell line (P less than or equal to 0.0001). The ICC-p method had a lower standard deviation on positive results than either the ICC-m or ISH method. The variability observed with these test procedures, reagents, and specimens suggests that these are important technological parameters in detecting p24, with implications for detecting other HIV-1 markers in infected tissues.

Immunoglobulin bound to platelets as immune complexes or specific antibody in specimens from acquired immunodeficiency syndrome and immune thrombocytopenic purpura.

Acquired immunodeficiency syndrome (AIDS), lymphadenopathy syndrome (LAS), and immune thrombocytopenic purpura (ITP) specimens were tested by an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (Ig) bound to platelets. All specimen evaluations were performed with General Diagnostic's newly developed kit procedure. The test measured but did not distinguish immune complex (IC) binding with platelet Fc receptor sites from platelet-specific antibody (PAb) binding with platelet antigen Fab-binding sites. Alkaline phosphatase-labeled antihuman IgG as conjugate detected IgG as low as 2.0 ng/ml or platelet-adsorbed, heat-aggregated IgG, simulating IC, at 2-10 ng/ml. There was a high prevalence of platelet-bound Ig in AIDS specimens (25/25) compared with normals (2/15), detected primarily by the indirect ELISA (p less than 0.001), and a preponderance of PAb in ITP specimens (5/5) compared with normals (6/22), by the direct ELISA (p less than 0.01). AIDS specimens had a geometric mean titer (GMT) of 173 ng of IgG bound/10(7) platelets, compared with the Ig from ITP, which had a GMT of 20 (p less than .0002). Monoclonal antibody to human receptors for IgG Fc fragment (anti Fc gamma R) inhibited 69% of specimens tested as having platelet-bindable antibody. Thus, the ELISA procedure would be useful in assessing but not in differentiating platelet-bound Ig in patients with AIDS and ITP and certain other clinical groups tested.

Autoimmune implications of immune complexes in clinical variants of hepatitis B.

Immune complexes (IC) were investigated in sera from 208 individuals with various clinical types of viral hepatitis diagnosed by clinical and laboratory criteria, including liver biopsy. Immune complexes were assessed by platelet aggregation (PI A) and by radioimmunoassay (RIA). The data were related to autoimmune phenomena (especially rheumatoid factors) and to the role that the IgM class of hepatitis B (HB) antibody might have in IC formation. Although the highest frequency of P1 A was in the few sera from patients with cirrhosis or hepatoma, the next highest was in sera from acute hepatitis patients (71%), and the lowest in sera from chronic active (57%) and chronic persistent (46%) hepatitis patients. A proportional number of patients with IC's were positive for hepatitis B surface antigen (HBs). A parallel prevalence was noted between P1 A and autoantibodies, with anti-Ig's being found more frequently in sera from acute hepatitis and chronic active hepatitis patients. The relationship between RIA results for complexes and RIA results for anti-IgG was inverse, as though anti-IgG interfered with IC reactivity by RIA. Anti-IgM pre-incubated with sera increased the amount of P1 A in sera from patients with acute hepatitis as well as in those from patients with chronic persistent hepatitis, suggesting a more frequent IgM involvement in IC's in these diseases than in chronic active hepatitis. Whereas liver cell damage in acute and active hepatitis may reflect elevated autoantibodies, the IgM class of HBs antibody may be involved in acute as well as chronic persistent hepatitis.

A survey for circulating immune complexes in patients with acute myocardial infarction. Use of a C1q-binding assay with soluble protein A as indicator.

A new assay for the detection of circulating C1q-binding immune complexes (IC) is described. The assay makes use of solid-phase C1q and iodinated soluble protein A, extracted from the cell wall of Staphylococcus aureus. In a model system the assay could detect heat-aggregated IgG down to a concentration of about 50 ng/ml. This method and three other assays, previously described, were used to survey the appearance of IC activity in sera from hospitalized patients with acute myocardial infarction. Depending on the assay system used, from 56% to 66% of the patients investigated were found to develop circulating IC. The earliest appearance of circulating IC was noted 5 days after infarction. The highest incidence of positive reactions and the strongest reactions occurred 2 to 3 weeks after hospitalization; thereafter the IC positiveness tapered off, and all patients were negative 6 weeks after infarction.

Platelet aggregation by hepatitis B surface antigen-antibody complexes.

Platelet aggregation indicating antigen-antibody complex formation was observed when hepatitis B surface (HBs) antigen and antibody were mixed. Platelet aggregation titers were determined for serum specimens found positive by radioimmunoassay for either HBs antigen or HBs antibody. From these determinations, incidence of HBs antigen-antibody complexes was found to be higher in HBs antigen seraas than in HBs antibody sera. There was an inverse correlation between platelet aggregation titers and radioimmunoassay values that was statistically significant for HBs antigen sera but not for HBs antibody sera. The incidence of anti-complementary activity was twice as high for platelet aggregation-positive HBs antigen and antibody sera as for platelet aggregation-negative sera. HBs antigen sera that were positive by platelet aggregation exhibited nearly three times the incidence of anti-complementary activity as did HBs antibody sera. However, the low incidence of anti-complementary activity was distributed about equally between HBs antigen and antibody sera that were negative by platelet aggregation. Additional HBs antigen preincubated with HBs antigen-positive sera effectively inhibited platelet aggregation, whereas additional HBs antibody was somewhat less effective. On the other hand, preincubation of HBs antigen sera with anti-IgG serum effectively enhanced platelet aggregation, whereas preincubation of HBs antigen sera with HBs antibody did not.

Interactions of standard antibody with Australia antigens in Au Ag-Ab radioimmunoassay.

Human antisera against Australia (Au) antigen have been characterized by liquid-phase radioimmunoassay (RIA) for their precipitation of (125)I-labeled Au antigen. The end-point dilutions of sera (anti-Au) which precipitated 50% of (125)I-Au antigen by RIA correlated well with complement fixation titers but had a much wider range, indicating a greater precision and perhaps a better sensitivity of assay. Anti-Au serum diluted to precipitate 50% of (125)I-labeled Au antigen was used as standard antibody in RIA tests to detect either inhibition or enhancement of the reaction by preincubated mixtures of Au antigen and antibody specimens. Without free Au antigen or antibody in the resultant mixtures there was no inhibition or enhancement; the mixtures presumably contained immunoreactively equivalent proportions of Au antigen and antibody. RIA data for diagnostic specimens indicated an end-point sensitivity which was proportional to the dilution of the standard anti-Au sera used in the test. High concentrations of the standard antibody permitted detectable inhibition of (125)I-Au antigen precipitation at lower antigen specimen concentrations. Similarly, low concentrations of the standard antibody permitted detectable enhancement of (125)I-Au antigen precipitation at lower antibody specimen concentrations. Omitting the standard antibody altogether resulted in a more sensitive RIA for Au antibody in test sera.

Mumps class-specific immunoglobulins in radioimmunoassay and conventional serology.

Antibodies against mumps virus have been studied by using immunoglobulin class-specific indicators labeled with (125)I in the radioimmunoassay (RIA) procedure. The immunoglobulins in paired acute and convalescent sera were allowed to react with mumps virus in a solid-phase RIA system. Class-specific immunoglobulin indicators (anti-immunoglobulin M [IgM] and anti-immunoglobulin G [IgG]) labeled with (125)I revealed that immunoglobulins of early antisera were preponderantly IgM, whereas immunoglobulins of late antisera were predominantly IgG. These indicators detected antibodies of the early (IgM) and late (IgG) phases of the immune response. These findings are consistent with the classical temporal order of appearance of 19s (IgM) and 7s (IgG) globulins. Specificity of these indicators for reacting with fractionated 7s and 19s globulins is also presented. Mumps virus RIA obtained with anti-IgG correlated well with conventional serological data obtained by neutralization and hemagglutination inhibition, but most strongly with complement-fixation data. In addition, antibody bound by solid phase was capable of distinguishing between related antigens of the myxovirus group.

Mumps class-specific immunoglobulins in radioimmunoassay and conventional serology.

In assessing the host cell range of bovine parvoviruses, these viruses were found to replicate optimally in actively dividing bovine fetal lung and spleen cells. Other primary bovine fetal cells supported growth to a lesser extent, but bovine line cells and line cells of other animal species tested did not. Minimal infectivity remained after passage of bovine parvovirus in cells from chicken embryos and guinea pig fetuses. During bovine parvovirus replication in bovine fetal lung and spleen cells, production kinetics of infectious virus and hemagglutinins were determined. An eclipse period of 16 h occurred, and viral release from cells was not detected until 30 h after inoculation of bovine fetal lung cells and 36 h after inoculation of bovine fetal spleen cells. Cell-associated virus titers were always higher than extracellular virus titers. Hemagglutinins were detected in parallel to infectious virus.

Solid-phase radioimmunoassay of total and influenza-specific immunoglobulin G.

An antigen-antibody system of polystyrene tubes coated with immunoglobulin antibody was used for quantitating immunoglobulins. A similar radioimmunoassay method was adapted for a viral antigen-antibody system. The viral system can be used for quantitating viruses and for measuring virus-specific antibodies by reacting with (125)iodine-labeled anti-immunoglobulin G (IgG). Optimal conditions for coating the solid phase, specificity of the immune reaction, and other kinetics and sensitivities of the assay method were investigated. Comparison of direct and indirect methods of assaying for immunoglobulins or viral antibody indicates that the indirect method is more sensitive and can quantitate a minimum of 0.037 mug of IgG per ml. Results of solid-phase radioimmunoassay for influenza antibody correlate well with hemagglutinin antibody titers but not with complement-fixing antibody titers. Radioimmunoassay results for influenza antibody by solid phase are likewise in agreement with results by the carrier precipitate radioimmunoassay method. The simplicity, reproducibility, and versatility of the solid-phase procedure make it diagnostically useful.