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.

Chlamydia pneumoniae strain TWAR, the new third species of Chlamydia, is a common cause of pneumonia and other acute respiratory tract infections. About 10% of hospitalized and outpatient pneumonia cases have been associated with TWAR infection. TWAR is among the four or five most commonly identified causes of all pneumonia. Most TWAR infections are mild or asymptomatic, but occasionally severe pneumonia with death has been observed. Laboratory diagnosis is not generally available. Vigorous treatment with tetracycline or erythromycin is recommended. Both epidemic and endemic infections have been described in North America and the Nordic Countries. Population prevalence antibody studies suggest that TWAR infection is wide-spread throughout the world, that nearly everyone is infected and reinfected during their life-time, and that infection is common in all ages except those less than 5 years in temperate zone countries. The infection is transmitted from person to person, apparently with a long incubation period.

Chlamydia pneumoniae is a human respiratory pathogen that causes acute respiratory disease and approximately 10% of community-acquired pneumonia. The infections are geographically widespread. Antibody prevalence studies have shown that virtually everyone is infected with the C. pneumoniae organisms at some time and that reinfection is common. In addition to respiratory disease, seroepidemiologic studies have shown an association of this organism with coronary artery disease. C. pneumoniae was detected in coronary artery atheromas by immunocytochemistry (15/36) and by polymerase chain reaction (PCR) (13/30) in 20 of 36 autopsy cases from Johannesburg, South Africa. Sequence analysis of the C. pneumoniae rRNA genes amplified by PCR confirmed that the amplified gene products were C. pneumoniae. Electron microscopy revealed typical pear-shaped C. pneumoniae elementary bodies in 6 of 21 atheromatous plaques. These findings support the seroepidemiologic studies and offer further evidence that C. pneumoniae may be involved in the atherosclerotic process.

During a 2 1/2-year period, we studied 386 University of Washington students with acute respiratory disease, to determine whether a Chlamydia psittaci strain, here designated TWAR, is an important respiratory pathogen. Serologic evidence of recent TWAR infection was found in 13 students, and the organism was isolated from 8 of these. TWAR infection occurred in 12 percent of the students who had pneumonia (9 of 76), 5 percent of those with bronchitis (3 of 63), and 1 percent of those with pharyngitis (1 of 150). The TWAR infections occurred throughout the study period. Pharyngitis, often accompanied by laryngitis, was a common first symptom. Clinically, the infections resembled those with Myco-plasma pneumoniae; therefore, the patients were given courses of erythromycin used for the treatment of M. pneumoniae infections. This therapy proved to be inadequate. The limited data available suggest that the TWAR strain is a "human" C. psittaci that is spread from human to human, without a bird or animal host.

Chlamydia pneumoniae (TWAR) is a recently recognized third species of the genus Chlamydia that causes acute respiratory disease. It is distinct from the other two chlamydial species that infect humans, C. trachomatis and C. psittaci, in elementary body morphology and shares less than 10% of the DNA homology with those species. The organism has a global distribution, with infection most common among children between the ages of 5 and 14 years. In children, TWAR infection is usually mild or asymptomatic, but it may be more severe in adults. Pneumonia and bronchitis are the most common clinical manifestations of infection, and TWAR is responsible for approximately 10% of cases of pneumonia and 5% of cases of bronchitis in the United States. The microimmunofluorescence serologic assay is specific for TWAR and can distinguish between recent and past infections. The organism can be isolated in cell culture; however, PCR techniques have recently facilitated its detection in tissues and clinical specimens.

Cardiovascular disease, of which atherosclerosis is an important component, is the leading cause of death in the western world. Although there are well-defined risk factors for atherosclerosis, these factors do not account for all incidences of the disease. Because atherosclerotic processes are typified by chronic inflammatory responses, which are similar to those that are elicited by chronic infection, the role of infection in promoting or accelerating atherosclerosis has received renewed attention. This review focuses on the accumulating evidence that chronic infection with Chlamydia pneumoniae, a ubiquitous human respiratory pathogen, might contribute to atherosclerotic lesion progression.

Recent evidence has shown the presence of Chlamydia pneumoniae antigens and nucleic acid in coronary artery atheromas from autopsy patients in South Africa. In this study, the immunocytochemical technique was used to demonstrate C pneumoniae antigens in atheromas of the aorta in autopsy patients from retrospective aortic atherosclerosis studies at the University of Washington. Hie patients were 34 to 58 years old. Immunoperoxidase staining using Chlamydia-spetiRc monoclonal antibodies showed one of four fatty streaks and six of 17 fibrous plaques were positive for C pneumoniae antigens; four control aortic tissues were negative. Two of the positive plaques were from the same patient Double-label immunocy* tochemical staining using Chlamydia-and tissue type-specific monoclonal antibodies demonstrated the antigens in the cytoplasm of macrophages and smooth muscle cells in the atheromatous lesion. This study suggested a wider involvement of Cpneumoniae organisms in atherosclerotic lesions of the arterial system than has previously been documented. (Aitenosdtr Thromb. 1993;13:1501-1504 KEY WORDS • atherosclerosis of aorta • Chlamydia pneumoniae • immunocytochemistry

Chlamydia pneumoniae has been postulated to cause systemic disease by infection of monocytes/macrophages and spread via the blood or lymphatics. To investigate how C. pneumoniae disseminates, the ability of the organism to infect murine macrophages in vivo and whether infection can be transferred via macrophages were determined. C. pneumoniae was detected by direct plating, isolation, and polymerase chain reaction in alveolar macrophages from intranasally inoculated mice and peritoneal macrophages from intraperitoneally inoculated mice. C. pneumoniae were also detected in peripheral blood mononuclear cells, but not plasma, of intranasally and intraperitoneally inoculated mice. When alveolar or peritoneal macrophages were adoptively transferred by intraperitoneal injection from infected to uninfected mice, C. pneumoniae DNA was detected by polymerase chain reaction in lung, thymus, spleen, and/or abdominal lymph nodes. These results demonstrate the ability of C. pneumoniae to infect macrophages in vivo and to disseminate systemically via infected macrophages by hematogenous and lymphatic routes.