Trudy O. Messmer

Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen.

The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Centers for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum. The qualified ELISA had a minimum detection limit of 0.06 µg/mL, a reliable lower limit of detection of 0.09 µg/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 µg/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 94.2%. A competitive inhibition anti-PA IgG ELISA was also developed to enhance diagnostic specificity to 100%. The anti-PA ELISAs proved valuable for the confirmation of cases of cutaneous and inhalational anthrax and evaluation of patients in whom the diagnosis of anthrax was being considered.

Outbreak of Pneumonia in a Long‐term Care Facility: Antecedent Human Parainfluenza Virus 1 Infection May Predispose to Bacterial Pneumonia

OBJECTIVES: To determine the causes of an outbreak of lobar pneumonia.

Comparison of two commercial microimmunofluorescence kits and an enzyme immunoassay kit for detection of serum immunoglobulin G antibodies to Chlamydia pneumoniae.

ABSTRACT We compared the MRL and the Labsystems Chlamydia pneumoniae microimmunofluorescence (MIF) immunoglobulin G (IgG) kits and the Labsystems enzyme immunoassay (EIA) kit in a blinded study of 83 serum samples in which we evaluated titers, cross-reactivity to other species, and reproducibility. There was no statistically significant difference between the MRL and the Labsystems MIF kits in the endpoint titers of IgG antibody to C. pneumoniae. The correlation between the results obtained with these two MIF kits was excellent (r = 0.95; P = 0.001). The cross-reactivity of the C. pneumoniae-positive sera with C. trachomatis- and C. psittaci-positive sera was assessed for each MIF kit. ForC. pneumoniae-positive sera with titers of ≥32, the Labsystems MIF kit exhibited more cross-reactivity to C. psittaci than the MRL kit did. The values obtained with the Labsystems EIA kit represented single dilutions of serum specimens expressed as enzymeimmuno units on a continuous scale. The results obtained with the Labsystems EIA kit correlated moderately well with those obtained with each MIF kit when they were compared for their abilities to detect IgG antibodies to C. pneumoniae(for the MRL MIF kit, r = 0.79 [P = 0.001]; for the Labsystems MIF kit, r = 0.78 [P = 0.001]). The results obtained with the commercial MRL and Labsystems MIF kits and the Labsystems EIA kit tested were reproducible; and the kits were standardized, had quality control reagents, and are suitable for detection of C. pneumoniae antibodies in serum and for use in interlaboratory studies. Validation of the use of these kits for clinical diagnosis still needs further evaluation.

Newly Characterized Species-Specific Immunogenic Chlamydophila pneumoniae Peptide Reactive with Murine Monoclonal and Human Serum Antibodies

ABSTRACT A monoclonal antibody (MAb) directed against an unknown Chlamydophila pneumoniae epitope has been characterized, and the respective peptide mimotope has been identified. A murine MAb specific for C. pneumoniae was used to select peptides from phage display libraries. The peptides identified from the phage display library clones reacted specifically with the respective target murine MAb and with human sera previously identified as having antibody titers to C. pneumoniae. The selected peptide mimotope sequences tended to be composed of charged residues surrounding a core of hydrophobic residues. The peptide with the best binding could inhibit >95% of binding to the MAb, suggesting that the selected peptide binds the paratope of the respective MAb. The peptide reacted with human sera previously determined by microimmunofluorescence to have anti-C. pneumoniae antibodies. The peptide was competitively competed with the MAb against Renografin-purified, sonicated C. pneumoniae in an enzyme-linked immunosorbent assay and with whole-cell C. pneumoniae in an indirect fluorescence assay format, demonstrating its potential utility in the development of diagnostics. The use of this novel peptide may allow investigators to establish standardized assays free from cross-reactive Chlamydia trachomatis and Chlamydophila psittaci epitopes and immunoreactivity.

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°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. J. Clin. Lab. Anal. 12:47–53, 1998.

A tale of discrimination: Differentiation of chlamydiaceae by polymerase chain reaction

We developed a nested, multiplex polymerase chain reaction (PCR) for simultaneous detection and discrimination of three species of chlamydia in human and avian specimens to determine transmission of chlamydophila psittaci from infected birds to humans. The nested PCR strategy was used to increase sensitivity and to circumvent inhibitors of PCR present in clinical specimens. The target sequence of our PCR is the 16S ribosomal DNA gene. The first-step PCR is genus-specific, and the second-step is multiplexed (ie, has multiple primer sets in the same tube) and can simultaneously discriminate and detect Chlamydophila pneumoniae, C psittaci , and Chlamydia trachomatis on the basis of the molecular weight of the amplicon. With available tests, pet stores can fail to identify birds infected with C psittaci that are asymptomatic before sale. We used PCR and serological evidence during outbreaks of psittacosis to infer that C psittaci had been transmitted from birds purchased in pet stores to humans. We also used PCR to detemine whether both live and dead birds from pet stores in West Virginia were infected with C psittaci . Finally, PCR showed that a human lung sample from a man with atypical pneumonia in a rural town in Victoria, Australia, was infected with C psittaci and not with C pneumoniae , sometimes a difficult diagnosis for clinicians to distinguish.

Discrimination of Streptococcus pneumoniae from other upper respiratory tract streptococci by arbitrarily primed PCR

OBJECTIVES In the clinical laboratory, identification of Streptococcus pneumoniae can be confused with other streptococci. Conventional biochemical tests such as optochin sensitivity and bile solubility can give inconsistent results. This report presents a method to distinguish true S. pneumoniae from other upper respiratory tract streptococci when conventional tests fail. DESIGN AND METHODS We used arbitrarily primed polymerase chain reaction with the single primer M13 universal as a method to distinguish S. pneumoniae from other upper respiratory tract streptococci. RESULTS The fingerprint pattern of S. pneumoniae was established by amplifying DNA of S. pneumoniae type strains 1-48 and of other common upper respiratory tract streptococci at three different DNA concentrations with the single primer M13 universal. From these type strains, a common arbitrarily primed-polymerase chain reaction pattern was identified characterized by two predominant bands of equal intensity at 800 base pairs and at 1100 base pairs. Fingerprint patterns of viridans streptococci were easily distinguishable from those of S. pneumoniae. Many of the clinical isolates used in this study were equivocal by conventional tests but were distinguishable by their fingerprint patterns. CONCLUSIONS Our results indicated that the fingerprint pattern of S. pneumoniae is species specific and distinguishes true S. pneumoniae of clinical isolates from other streptococci when conventional biochemical tests are unclear.

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 Iscoves growth medium in CO2 atmosphere at 37°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 106 cells (range 15–23ASC/106 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 chlamydial antibody production at the cellular level. ELISPOT positivity thus indicates previous exposure and would favor earlier detection. J. Clin. Lab. Anal. 11:45–52.