Chlamydia pneumoniae has been associated with atherosclerosis and several other chronic diseases, but reports from different laboratories are highly variable and "gold standards" are lacking, which has led to calls for more standardized approaches to diagnostic testing. Using leading researchers in the field, we reviewed the available approaches to serological testing, culture, DNA amplification, and tissue diagnostics to make specific recommendations. With regard to serological testing, only use of microimmunofluorescence is recommended, standardized definitions for "acute infection" and "past exposure" are proposed, and the use of single immunoglobulin (Ig) G titers for determining acute infection and IgA for determining chronic infection are discouraged. Confirmation of a positive culture result requires propagation of the isolate or confirmation by use of polymerase chain reaction (PCR). Four of 18 PCR assays described in published reports met the proposed validation criteria. More consistent use of control antibodies and tissues and improvement in skill at identifying staining artifacts are necessary to avoid false-positive results of immunohistochemical staining. These standards should be applied in future investigations and periodically modified as indicated.

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.

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Avian chlamydiosis was detected in a shipment of ú700 pet birds from a Florida bird distributor that were sold to nine Atlanta-area pet stores in August 1995. Respiratory illness among persons who had recently acquired birds from this shipment was reported to local public health officials. The attack rate of acute respiratory illness was 10.7% among persons in households exposed to birds from the implicated flock vs. 1.8% among control households (odds ratio, 6.60; 95% confidence interval, 1.39 -31.2). Illness and serological evidence of infection in the absence of symptoms were more common among persons in households with recently purchased birds that were sick or that had died and among persons who had had direct contact with the birds. Clinical psittacosis or serological evidence of Chlamydia psittaci infection was found in 30.7% of households with birds from the infected flock. Mild illnesses and asymptomatic infections in exposed persons were unusual features of this outbreak.

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.

Background: Laboratory diagnosis of Chlamydophila psittaci, an important turkey respiratory pathogen, is difficult. To facilitate the diagnosis, a nested PCR-enzyme immunoassay (PCR-EIA) was developed to detect the Cp. psittaci outer membrane protein A (ompA) gene in pharyngeal swabs.

Noninfluenza viral infections may play a role in the pathogenesis of some bacterial pneumonias. S. pneumoniae was the cause of at least two pneumonias; lack of preantibiotic cultures may have interfered with isolation of S. pneumoniae in others. Recent HPIV1 infection was epidemiologically linked to subsequently developing pneumonia. Spread of HPIV1 in the facility may have contributed to increased susceptibility to S. pneumoniae and, potentially, to other bacterial pathogens.

A nested primers strategy was used to develop a two-step PCR test for the direct species-specific detection of the 16s rRNA gene of Chlamydia pneumoniae. This test was applied to 58 nasopharyngeal or oropharyngeal swab specimens collected from patients in studies of community-acquired pneumonia and in a local outbreak of respiratory disease. Twelve patients (21%) showed evidence of Chlamydia pneumoniae infection in serological tests (7/56; 13%), culture (8/58; 14%) or PCR (10/58; 17%). Nested PCR but not single-step PCR was found to be as sensitive as culture or serology for detection of infection with this organism. In summary, nested PCR can be useful in direct testing of clinical specimens for Chlamydia pneumoniae, making additional DNA purification steps unnecessary.