A seroepidemiological study of Mycoplasma pneumoniae infections in Denmark over the 50-year period 1946-1995.

The epidemiological pattern of Mycoplasma pneumoniae infections in Denmark over the 50-year period 1946-1995 is described. The study is based on blood specimens received at the central laboratory at Statens Serum Institut for titration of cold agglutinins (CA), initially for the diagnosis of CA positive primary atypical pneumonia, and during the 1960s of M. pneumoniae infection; in addition, specimens from the last 38 years were tested for antibodies specific to M. pneumoniae. By retrospective analysis of the test results compiled over the years it was found that intervals of regular periodicity have been interrupted by an era of changes in the pattern. Attention is paid to the significance of CA for this study, and the possible background of the epidemiological pattern is described.

Clinical overview of typical Mycoplasma pneumoniae infections.

A number of clinical and epidemiological factors permit the tentative identification of Mycoplasma pneumoniae infections. These selective factors can be considered the mainstay of diagnosis because they facilitate intelligent and efficient use of laboratory tests. The laboratory, in turn, through identification of the causative agent, augments both clinical and epidemiological data. The laboratory can better serve its purpose as more rapid, sensitive, and specific diagnostic methods come into use.

Mycoplasmal pericarditis: evidence of invasive disease.

Although the pathogenic mycoplasmas usually infect the respiratory and urogenital tracts, these organisms also can cause disease in remote sites. Such infections are difficult to diagnose because of both the fastidious nature of the mycoplasmas and the failure to consider their presence. Pericarditis is an uncommonly diagnosed and rarely confirmed example of invasive mycoplasmal infection. As part of a prospective study of large pericardial effusions, we discovered two cases with Mycoplasma pneumoniae infection. Subsequently, two cases of pericarditis due to Mycoplasma hominis and one due to Ureaplasma urealyticum were diagnosed. For all five patients, cultures of pericardial tissue and/or fluid were positive. In addition, four of the five patients either were immunocompromised or had undergone cardiac surgery previously. Appropriate antibiotic therapy was uniformly effective. We report here our experience with mycoplasmal pericarditis, provide evidence of an invasive pathogenesis for this syndrome, and suggest that pericardial disease caused by these organisms may not be an uncommon finding when sought in an aggressive manner.

Epidemic pneumonia in university students.

Longitudinal surveillance of pneumonia in a university student health service was conducted from 1965-1971 and 1984-1987. Of 104 pneumonia cases documented by chest x-ray, only six were presumed to have bacterial etiology; the remaining 98 were characteristic of atypical pneumonia syndrome. Mycoplasma pneumoniae was the etiology in 51% of the pneumonias in the 1960s and 13% in 1984-1987. Pneumonia incidence was highest in the fall semester in seven of 11 years studied. Annual incidence followed a three- to four-year periodicity. Both of these observations mirror the epidemiology of M. pneumoniae in the world population. Symptoms of cough, headache, malaise, and absence of the physical finding of wheezing were seen more consistently in M. pneumoniae pneumonia than in other atypical pneumonias; other clinical features varied among epidemics. Rapid cold agglutinin tests were positive in 27% of our clinically diagnosed pneumonias and in 36% of those with documented mycoplasmal infections. This study appears to provide a basis for predicting future epidemics of atypical pneumonia in student populations.

Chlamydial and mycoplasmal pneumonias.

Chlamydia species and Mycoplasma pneumoniae are among the most common agents of community-acquired pneumonia, as well as causes of various nonpneumonic syndromes. Both can be considered "exotic" bacteria: Chlamydiae because they depend on host cell energy, hence their obligate intracellular replication; and M pneumoniae because it is an extracellular parasite that lacks the standard protective bacterial cell wall. The unusual biology of these organisms complicates laboratory diagnosis, but because both are susceptible to selective antimicrobials, therapy often proceeds empirically on clinical suspicion. Generally the respiratory diseases produced are self-limited without significant complications or known sequelae.

A Mycoplasma genitalium protein resembling the Mycoplasma pneumoniae attachment protein.

In previous studies with hyperimmune rabbit sera and monoclonal antibodies against the P1 protein of Mycoplasma pneumoniae, we obtained evidence of a shared antigenic determinant with a single protein of Mycoplasma genitalium. Because of biologic and morphologic similarities between these two human Mycoplasma species, attempts were made to characterize this cross-reacting protein of M. genitalium (designated MgPa). The protein was surface exposed and had an estimated molecular size of 140 kilodaltons. Electron microscopy with monoclonal antibodies produced against either MgPa or P1 demonstrated that MgPa is located over the surface of the terminal structure of M. genitalium which is covered by a nap layer. These immunologic and morphologic findings suggest that the MgPa protein of M. genitalium could be the counterpart of the P1 protein of M. pneumoniae.

Ureaplasma colonization: definitions and epidemiologic implications.

Genital mucosal colonization with Ureaplasma species complicates interpretation of their role as pathogens. From an epidemiologic point of view, colonization should be considered a dynamic process in which knowledge about the habitat colonized is an integral part. The classical criteria of pathogenicity need to be broadened to include qualitative, quantitative, and temporal aspects as disease associations of ureaplasmas are explored.

Antigenic determinants of the attachment protein of Mycoplasma pneumoniae shared by other pathogenic Mycoplasma species.

In previous studies with hyperimmune rabbit antisera, we found evidence of serologic cross-reactivity among Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma gallisepticum. Because of certain biologic and morphologic similarities of these species, attempts were made to determine if this cross-reactivity related to the attachment protein (P1) of M. pneumoniae. Monoclonal and monospecific antibodies against P1 were used to probe proteins of the other species by immunoblotting. One of the P1 monoclonal antibodies was reactive with a smaller protein of M. genitalium; rabbit antiserum raised by immunization with P1 excised from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel was found to react with a similar-sized protein of M. gallisepticum. These preliminary findings suggest antigenic sharing among the species examined; however, limitations of the methods used are discussed.

Demonstration of multiple antigenic determinants on Mycoplasma pneumoniae attachment protein by monoclonal antibodies.

Distinct multiple antigenic determinants of the attachment protein of Mycoplasma pneumoniae have been identified by limited proteolytic cleavage, using specific monoclonal antibodies. Western blots prepared from the gels containing the cleaved fragments were probed with antiserum against M. pneumoniae or monoclonal antibodies. Five distinct bands with intact antigenic determinants were detected by the antiserum, of which two bands were each reactable with two monoclonal antibodies. A sequential binding assay suggested that these monoclonal antibodies recognized different antigenic sites of each band. These results demonstrate the existence of multiple antigenic sites on the attachment protein and describe procedures that should prove useful for identifying those antigenic sites critical to the specific attachment of M. pneumoniae.

The changing pathogenicity of Mycoplasma pneumoniae with passage in vitro. Correlates of virulence.

An avirulent strain of Mycoplasma pneumoniae isolated by broth passage failed to produce pneumonia in hamsters. The major biological property lost in this avirulent strain is its ability to attach to the respiratory epithelium. Although the surface protein responsible for the specific attachment of virulent M. pneumoniae has been identified, protein analysis by gel electrophoresis has failed to produce evidence that could account for the loss of virulence in the avirulent strain. It is possible that the binding sites of the avirulent strain have been altered by mutational event(s) without affecting the molecular weight or electrophoretic mobility of this protein. Antigenic determinant analysis of the membrane proteins by the use of monoclonal antibodies is suggested as a relevant approach, which may lead to a better understanding of the molecular basis of attachment.

Conservation of pathogenic mycoplasma antigens.

The protein profiles of different mycoplasmas are generally species-specific; however, several pathogenic species share certain biologic properties and/or virulence factors. We compared species of human, avian and animal origin to determine if there was evidence for conservation of key antigens. Log-phase cultures of Mycoplasma pneumoniae, M. genitalium, M. gallisepticum and M. pulmonis were subjected to SDS-gel electrophoretic analysis. Protein profiles were compared with silver-stained gels. To examine antigenic cross-reactivity, Western blots were prepared by transferring separated proteins to nitrocellulose filters and incubating these with homotypic and heterotypic antisera. Localization of cross-reacting antigens was done by immunoradioautography using 125I-labeled antiglobulins. Several two-way cross-reactions were observed among M. pneumoniae, M. genitalium, and M. gallisepticum; M. pulmonis shared no significant cross-reactions with the other three species. Preliminary evidence suggests that the cross-reactivity was due to conservation of certain antigenic determinants, although the overall protein composition of the four species was different.

Suppression of respiratory syncytial virus infection in cotton rats by bis(5-amidino-2-benzimidazolyl)methane.

Intraperitoneal administration of bis(5-amidino-2-benzimidazolyl)methane at well-tolerated daily doses of 25 mg/kg subsequent to challenge and for 3 days thereafter effected over a 1-log reduction in the amount of virus recovered from lungs of cotton rats inoculated intratracheally with respiratory syncytial virus. When animals were immunosuppressed to prolong virus shedding, the reduction in recovered virus achieved with a 7-day dosing schedule of bis(5-amidino-2-benzimidazolyl)methane exceeded 2 logs.

Serological comparison of virulent and avirulent Mycoplasma pneumoniae by monoclonal antibodies.

An avirulent strain (M129-B175) of Mycoplasma pneumoniae is morphologically indistinguishable from its virulent parent strain (M129-B7). Functionally, the avirulent strain has lost its ability to attach to respiratory epithelium and does not produce pneumonia in hamsters. Biochemical analyses by one- or two-dimensional SDS-gel electrophoresis have so far failed to produce evidence that could account for the changes in the avirulent strain. It is possible that proteins of the avirulent strain have been altered by spontaneous mutations to nonfunctional states, events which would not necessarily alter their physical properties, i.e., molecular weight. To examine this possibility, Western blots prepared from proteins of the avirulent strain, separated on SDS-gels, were exposed to a collection of monoclonal antibodies to the virulent strain. Immunoradioautographs showed that two protein bands of the avirulent strain lost their reactivity to the monoclonal antibodies, although overall protein profiles were identical. This preliminary observation suggests that spontaneous mutations that lead to structural changes in protein molecules occurred during the derivation of the avirulent strain.

A new animal model for human respiratory tract disease due to adenovirus.

Cotton rats (Sigmodon hispidus) were tested as a model for human respiratory tract infection due to adenovirus. After intranasal instillation of 10(6.1) 50% tissue culture infectious doses (TCID50) of adenovirus type 5 into one-month-old cotton rats, groups were killed at intervals for nasal and lung titration of virus and lung histopathology. In lung, eclipse occurred at 8 hr followed by peak viral titer (10(7.5) TCID50/g of lung) on day 5. Titers fell to 10(3.2) TCID50/g by day 10 and persisted at that level through the remainder of the study (day 28) despite appearance of serum neutralizing antibody after day 6. Interstitial pneumonia paralleled viral growth, and peribronchial mononuclear infiltration followed one to two days later. Titers in nasal mucosa peaked on day 3 but were undetectable beyond day 21. Pulmonary histopathology and viral replicative patterns paralleled findings in natural human disease.

Mycoplasma pneumoniae infections of man: integration of attachment mechanism, cellular responses and clinical manifestations.

Research advances of recent years are permitting new understanding of M. pneumoniae disease pathogenesis, although our knowledge remains incomplete. Colonization of the respiratory tract mucosa, mediated by an attachment protein, leads to specialized cell injury and altered muco-ciliary clearance. Pulmonary cellular infiltrates and airway exudates are a mixture of both non-specific and specific host immune responsiveness to the mycoplasma. It is now possible to begin integration of the organism's molecular biology and the clinical manifestations of infection.

Mycoplasma pneumoniae respiratory disease symposium: summation and significance.

The symposium on M. pneumoniae respiratory disease has examined the clinical expression of infection in adults and children, the pathophysiologic disturbances which occur, and the laboratory diagnosis by isolation and serology. That these infections are very common has been well documented; however, a variable incidence over periods of several years tends to minimize importance of the disease for many clinicians. While good laboratory diagnostic methods exist, they provide retrospective insight predominantly and are not useful for early diagnosis or therapeutic decision making. Development of rapid diagnostic methods which are sensitive and specific is an important goal for future research. Success would facilitate our understanding and control of M. pneumoniae disease.

Location of attachment moiety on Mycoplasma pneumoniae.

Mycoplasma pneumoniae initiates infection in the human host by attachment to respiratory epithelium. The organism attaches by a specialized terminal structure. Monoclonal antibodies to an organism surface protein (P1) inhibited attachment to respiratory epithelium and were localized to the tip structure by a ferritin antibody label. The P1 protein was degraded by trypsin treatment to smaller polypeptides that possessed the same antigenic determinants as the larger P1 protein when reacted with the specific monoclonal antibody, and evidence has been provided for the existence of multiple antigenic determinants on the attachment protein.