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Mycoplasma pneumoniae and Its Role as a Human Pathogen

Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35249,¹ Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333²

SUMMARY

INTRODUCTION

MOLLICUTE TAXONOMY AND CLASSIFICATION

CELL BIOLOGY

PATHOGENESIS OF DISEASE

Cytadherence

Intracellular Localization

Cytotoxicity and Inflammation

Asthma and Other Chronic Lung Conditions

Immune Response and Immunomodulatory Effects

Antigenic Variation

EPIDEMIOLOGY

Geographic Prevalence and Seasonality of Disease

Disease Transmission

Disease Outbreaks

Demographics and Spectrum of Disease

CLINICAL SYNDROMES

Respiratory Tract Infections

Extrapulmonary Manifestations

DIAGNOSIS

General Laboratory Features

Radiographic Findings

Pathological Findings

Microbiological Tests

Culture.

Antigen detection techniques.

DNA probes.

PCR.

Serology.

ANTIMICROBIAL SUSCEPTIBILITIES AND CHEMOTHERAPY

Antimicrobial Susceptibility Profiles

In Vitro Susceptibility Testing

Treatment of Infections Due to M. pneumoniae

VACCINES

FUTURE NEEDS

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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The first mycoplasma to be isolated in culture was the bovine pleuropneumonia agent now known as Mycoplasma mycoides subsp. mycoides, which was described initially by Nocard and Roux in 1898 (315). Over the next 50 years, evidence accumulated relating to the importance of the parasitic bacteria known at that time as pleuropneumonia-like organisms (PPLO) in various diseases of animals and their possible involvement in human disease. In the 1930s Klieneberger introduced the concept that mycoplasmas were "L-forms" of bacteria lacking cell walls and living symbiotically with other, walled bacteria (232). This theory started a spirited debate pitting those who believed that mycoplasmas were unique species against those who believed that mycoplasmas were wall-less variants of other known bacterial species and were undeserving of a unique taxonomic representation. This controversy was not completely settled until the 1960s, when guanine-plus-cytosine (G+C) content assays and DNA-DNA hybridization assays showed that mycoplasmas were indeed unique forms of life and lacked the ability to produce cell walls under any circumstances. A summary of the mycoplasma and ureaplasma species known to occur in humans, excluding occasional isolates or mycoplasmas of animal origin that sometimes infect humans, is shown in Table 1.

The numerous commensal mycoplasmal species that commonly inhabit the human oropharynx, especially the most common species, Mycoplasma orale and M. salivarium, may occasionally cause diagnostic confusion with M. pneumoniae if they happen to find their way to the lower respiratory tract or if diagnostic specimens from the lower respiratory tract are contaminated with oropharyngeal secretions. However, with rare exceptions, usually involving immunosuppressed persons, the oropharyngeal commensal mycoplasmal species are not pathogenic.

Among human mycoplasmas, M. pneumoniae is by far the best known and most carefully studied. Much has been learned during the past several years about its cell biology, the host immune response that it elicits, laboratory techniques for detection, disease epidemiology, and its role as a respiratory tract pathogen. These developments are summarized in this review.

MOLLICUTE TAXONOMY AND CLASSIFICATION

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The term "mycoplasma" (Greek; "mykes" = fungus and "plasma" = formed) emerged in the 1950s (117) and replaced the older PPLO terminology. The allusion to a fungus-like growth pattern in the name "mycoplasma" happens to describe only the growth of M. mycoides, but the term was nevertheless adopted and has persisted to this day. In the 1960s, mycoplasmas were designated members of a class named Mollicutes, which derives from Latin words meaning soft ("mollis") and skin ("cutis"). The current taxonomic designations included in class Mollicutes comprise 4 orders, 5 families, 8 genera, and about 200 known species that have been detected in humans, vertebrate animals, arthropods, and plants. M. pneumoniae is a member of the family Mycoplasmataceae and order Mycoplasmatales (435).

Members of the class Mollicutes are characterized by their small genomes consisting of a single circular chromosome containing 0.58 to 2.2 Mbp, a low G+C content (23 to 40 mol%), and the permanent lack of a cell wall (213). The taxonomy of this class has been extensively revised based on 16S rRNA analysis and is discussed in greater detail elsewhere (213). Studies of 16S rRNA sequences suggest that mycoplasmas are most closely related to the gram-positive eubacterial subgroup that includes the bacilli, streptococci, and lactobacilli. According to Maniloff (278), the Mollicutes diverged from the Streptococcus branch of gram-positive bacteria with low G+C contents and relatively small bacterial genomes about 605 million years ago. Their small genomes are now believed to be the result of a gradual reduction in genome size from a common ancestor in a process known as degenerative evolution (278). The nature of the selective pressure that led to the evolution of Mollicutes is not precisely known. Figure 1 shows the mycoplasma phylogeny reconstructed from 16S rRNA sequence comparisons.

View larger version (24K): [in this window] [in a new window]   FIG. 1. Phylogeny of mycoplasmas reconstructed from 16S rRNA sequence comparisons. Branch lengths are proportional to evolutionary distance (the number of base changes per 1,000 nucleotides). The scale at the bottom denotes the branch distance corresponding to five base changes per 100 nucleotides. Reprinted from reference 278 with permission.

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View larger version (116K): [in this window] [in a new window]   FIG. 2. Spherical colonies of M. pneumoniae growing on SP4 agar. Magnification, x95.

The small genome of M. pneumoniae and its limited biosynthetic capabilities are responsible for many of the biological characteristics and requirements for complex medium supplementation in order for the organism to be cultivated in vitro. Mollicutes have no ability to synthesize peptidoglycan cell walls, since the genes responsible for these processes are not present in the genome. The lack of a rigid cell wall confers pleomorphism on the cells and makes them unable to be classified as cocci or bacilli in the manner of conventional eubacteria. Mollicutes have never been found as freely living organisms in nature, since they depend on a host cell to supply them with the things they need for their parasitic existence. Another characteristic of most mollicutes and all members of the genus Mycoplasma is the requirement for sterols in artificial growth media, supplied by the addition of serum. Sterols are necessary components of the triple-layered mycoplasmal cell membrane that provide some structural support to the osmotically fragile mycoplasma.

Maintenance of osmotic stability is especially important in mollicutes due to the lack of a rigid cell wall. Although these organisms can flourish within an osmotically stable environment in their chosen eukaryotic host, they are extremely susceptible to desiccation, a fact that has a great impact on the need for proper handling of clinical specimens from which cultural isolation is to be attempted and the need for close contact for transmission of infection from person to person by airborne droplets. Another structural component of the M. pneumoniae cell that is important for extracellular survival is a protein network that provides a cytoskeleton to support the cell membrane.

One aspect of M. pneumoniae cell biology that is not widely appreciated is the fact that this organism, along with several other mollicute species, may elaborate capsular material external to the cell membrane. The first report indicating the presence of capsular material in M. pneumoniae appeared in 1976 in a review of its ultrastructure as determined by electron microscopy (444). It was suggested that this capsular material may have a role in adherence, but this has not been conclusively proven (444).

M. pneumoniae possesses very limited metabolic and biosynthetic activities for proteins, carbohydrates, and lipids in comparison to conventional bacteria. Like other mollicutes, it scavenges for nucleic acid precursors and apparently does not synthesize purines or pyrimidines de novo (91). For many years it has been known that fermentation of glucose to lactic acid by means of substrate phosphorylation effected by phosphoglyceric acid kinase and pyruvate kinase is a means by which M. pneumoniae generates ATP. Beyond that, there has been considerable speculation about which enzyme systems were actually present. Much of what is now known about the metabolic properties of M. pneumoniae has been made possible through annotation of its genome and direct identification of the genes encoding enzyme systems responsible for various metabolic pathways (91, 187). However, some interesting and perhaps unexpected findings have occurred since the sequence and annotation of the genome have been published. For example, there is genomic evidence for enzymes such as arginine deiminase, even though biochemical activity evidenced by ammonia production has not been directly observed in M. pneumoniae (328). Pollack et al. (327) have recently reviewed the central carbohydrate pathways of mollicutes and reported that M. pneumoniae possesses all 10 reactions of glycolysis but that the tricarboxylic acid cycle and a complete electron transport chain containing cytochromes are absent. Thus, the lactic acid end product of fermentation is still relatively reduced with electrons whose energy could be trapped if the pyruvate precursor could be diverted to the tricarboxylic acid cycle.

M. pneumoniae reduces tetrazolium either aerobically or anaerobically, and this has been one of several characteristics that have been used historically to identify the species and distinguish it from commensal mycoplasmas of the oropharynx. The availability of nucleic acid amplification techniques such as the PCR assay has made older methods of identification such as tetrazolium reduction and hemadsorption with guinea pig erythrocytes less important than they were previously. A pathway listing all of the relevant enzymes encoded in the M. pneumoniae genome is available at www.bork.embl-heidelberg.de/Annot/MP/ (91). Another unique property of Mycoplasma spp. is the use of the universal stop codon UGA as a codon for tryptophan (200).

M. pneumoniae, like other mollicutes, has developed specialized reproductive cycles as a result of its adaptation to existence with a limited genome and a parasitic life style that requires attachment to host cells (91). It reproduces by binary fission, temporally linked with duplication of its attachment organelle, which migrates to the opposite pole of the cell during replication and before nucleoid separation (15). A graphic model proposed for M. pneumoniae cell division, illustrating the formation and migration of the attachment organelles, is shown in Fig. 3. M. pneumoniae has been shown to bind and glide on glass and other solid surfaces, with the organism moving with the attachment organelle at the leading end. Neither genomic analysis nor electron microscopy of M. pneumoniae has demonstrated the presence of structures such as flagella or pili, suggesting that gliding motility occurs by an unknown mechanism involving the attachment organelle (290).

View larger version (27K): [in this window] [in a new window]   FIG. 3. Proposed scheme for cell division and duplication of the terminal attachment structure in M. pneumoniae. Reprinted from reference 375 with permission.

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Because M. pneumoniae is primarily an extracellular pathogen that depends on close association with host cells to survive, it has evolved a complex and specialized attachment organelle to facilitate its parasitic existence, as shown by electron microscopy (Fig. 4 and 5). This attachment organelle consists of a specialized tip structure with a central core of a dense rod-like central filament surrounded by a lucent space that is enveloped by an extension of the organism's cell membrane. The tip structure is actually a network of adhesins, interactive proteins, and adherence accessory proteins that cooperate structurally and functionally to mobilize and concentrate adhesins at the tip of the organism. The host cell ligand for mycoplasmal adhesins has not been characterized conclusively, although sialoglycoconjugates and sulfated glycolipids have been implicated (247, 347). Recent data (89) have shown that two proteins expressed on the M. pneumoniae cell surface, elongation factor TU and pyruvate dehydrogenase E1 ß, are also involved in binding M. pneumoniae to fibronectin, a very common component of eukaryotic cell surfaces, basement membranes, and the extracellular matrix.

View larger version (118K): [in this window] [in a new window]   FIG. 4. Scanning electron micrograph of M. pneumoniae cells. Whole mycoplasmas are shown, with terminal attachment structures indicated by arrowheads. Reprinted from reference 245 with permission.

View larger version (154K): [in this window] [in a new window]   FIG. 5. Transmission electron micrograph of M. pneumoniae-infected hamster tracheal ring, demonstrating the close association of the attachment structure to the epithelium (arrow). (Copyright J. L. Jordan and D. C. Krause.)

Jacobs (202) postulated that the immunodominant epitopes of the M. pneumoniae adhesins differ from the highly conserved adherence-mediating domains, thereby suggesting that one reason for the lack of protective immunity against reinfection even in the midst of a serological response could lie in the fact that antibodies directed against the variable domains are unlikely to generate effective cytadherence-blocking antibodies. Studies by Baseman and coworkers suggest that the P1 protein expression alone is not sufficient to mediate adherence of M. pneumoniae to certain host cells and cause disease and that the cooperative activity of other proteins is needed (29, 243, 244). P30 is one of several additional proteins that have been implicated in the adherence process, based on the knowledge that antibodies developed against P30 can block M. pneumoniae hemadsorption (29, 88, 298). Balish and Krause (15) recently suggested that P30 may be involved in gliding motility as well as coordination of cell division with biogenesis of the attachment organelle.

Other structures produced by M. pneumoniae that have been studied as mediators in cytadherence in M. pneumoniae include proteins HMW1, HMW2, HMW3, HMW4, HMW5, P90, and P65, which, in addition to P30, are believed to participate in the establishment of the polar structure. Once this polar structure is established, an independently assembled complex of proteins B, C, and P1 is drawn to the structure to complete formation of the functional terminal attachment organelle (15). A more in-depth discussion of mycoplasma interactions with host cells and the process of cytadherence at the subcellular level was published recently by Rottem (354).

An intracellular existence that sequesters M. pneumoniae could facilitate the establishment of latent or chronic states, circumvent mycoplasmacidal immune mechanisms, facilitate its ability to cross mucosal barriers and gain access to internal tissues, and impair the efficacy of some drug therapies, accounting for difficulty in eradicating the mycoplasmas under clinical conditions (28, 355, 403, 420). Fusion of the mycoplasmal cell membrane with that of the host may also result in release of various hydrolytic enzymes produced by the mycoplasma as well as insertion of mycoplasmal membrane components into the host cell membrane, a process that could potentially alter receptor recognition sites and affect cytokine induction and expression (355). At present, the extent to which M. pneumoniae invades and replicates intracellularly in vivo is not known. Therefore, the clinical significance of these theoretical events associated with cell fusion remains to be proven.

Host cell lactoferrin acquisition by M. pneumoniae is yet another possible means by which local injury may occur, through generation of highly reactive hydroxy radicals resulting from the introduction of iron complexes in a microenvironment rendered locally acidic by cellular metabolism that also includes hydrogen peroxide and superoxide anion (419).

Mammalian cells parasitized by M. pneumoniae exhibit a number of cytopathic effects that may occur as a result of the local damage mediated biochemically following cytadherence. M. pneumoniae infection leads to deterioration of cilia in the respiratory epithelium, both structurally and functionally. Cells may lose their cilia entirely, appear vacuolated, and show a reduction in oxygen consumption, glucose utilization, amino acid uptake, and macromolecular synthesis, ultimately resulting in exfoliation of all or parts of the infected cells (80, 83) These subcellular events can be translated into some of the clinical manifestations of respiratory tract infection that are associated with this organism, such as the persistent, hacking cough that is so commonly associated with M. pneumoniae.

Once M. pneumoniae reaches the lower respiratory tract, the organism may be opsonized by complement or antibodies. Macrophages become activated, begin phagocytosis, and undergo chemotactic migration to the site of infection. High percentages of neutrophils and lymphocytes are present in alveolar fluid. CD4⁺ T lymphocytes, B lymphocytes, and plasma cells infiltrate the lung (65, 318), manifested radiologically as pulmonary infiltrates. Further amplification of the immune response in association with lymphocyte proliferation, production of immunoglobulins, and release of tumor necrosis factor alpha (TNF-), gamma interferon (IFN-), and various interleukins (including interleukin-1ß [IL-1ß], IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and IL-18) occurs, based on evidence from clinical and in vitro studies and from animal models (193, 195, 263, 308, 309, 406, 420, 447, 448). Many of these reactive substances will have elevated levels in both alveolar fluid and serum (233, 263, 406, 448). Yang et al. (448) recently reported that M. pneumoniae infection of human epithelial carcinoma cells in vitro resulted in increased levels of IL-8 and TNF- mRNAs and that both proteins were secreted into culture medium. The major proinflammatory cytokine IL-1ß mRNA also increased and the corresponding protein was synthesized, but its secretion was cell type specific due to an endogenous caspase-1 inhibitory component in the lung epithelial cells studied (447).

The role of cytokines and other reactive substances in the pathogenesis of M. pneumoniae lung disease has been a topic of considerable interest during the past several years, and a number of clinical studies involving humans as well as investigations based on animal models have been reported. Current evidence from human and animal studies suggests that cytokine production and lymphocyte activation may either minimize disease through the enhancement of host defense mechanisms or exacerbate disease through immunological lesion development. Thus, the more vigorous the cell-mediated immune response and cytokine stimulation, the more severe the clinical illness and pulmonary injury (78, 201, 305, 338, 342, 404-406). This concept of immune-mediated lung disease provides a basis for consideration of immunomodulatory therapeutics in addition to conventional antimicrobial therapies in management of disease due to M. pneumoniae.

Multiple lines of evidence suggest why M. pneumoniae may play a role in the pathogenesis of asthma beyond simple, acute exacerbation. M. pneumoniae can be detected by PCR and/or culture more often from the airways of patients with chronic, stable asthma than from matched control patients. Kraft et al. (238) detected M. pneumoniae by PCR in respiratory secretions of 10 of 18 stable adult asthmatics (56%) and in only 1 of 11 healthy controls. In another study, throat cultures for M. pneumoniae were positive in 24.7% of children and adults with asthma exacerbation, compared with 5.7% of healthy controls (158). However, other studies of children with acute asthma exacerbation showed that while rhinoviruses and respiratory syncytial virus may be detected frequently, M. pneumoniae played a minor role and was detected in just a few patients (48, 155, 414). Limitations of some of these studies were the use of complement fixation tests alone to identify patients with M. pneumoniae (414) and inclusion of very young children in whom viral bronchiolitis instead of asthma may have been present (155).

Treatment of asthma patients in whom M. pneumoniae has been detected with macrolide antimicrobials resulted in improvement in pulmonary function tests in comparison with asthma patients who did not have evidence of M. pneumoniae in airways (239), owing perhaps to both the antibacterial and the anti-inflammatory effects of macrolides. Macrolides are known to reduce airway hyperresponsiveness in asthmatic patients, attenuate pulmonary inflammation by protecting ciliated epithelium against oxidative damage, stabilize cell membranes, and decrease sputum purulence (133). Mycoplasmas have been detected by PCR in airways even when cultures and serological results are negative, suggesting that low numbers of organisms may evade detection by the immune system (239). The lack of a measurable serological response may also facilitate the organism's persistence in the lower respiratory tract.

Lung abnormalities, including reduced pulmonary clearance and airway hyperresponsiveness, may persist for weeks to months after an infection with M. pneumoniae (227, 279, 295, 359, 377). Marc et al. (279) reported abnormalities in pulmonary function tests in up to 50% of children, and Kim et al. (227) described abnormal computerized axial tomography studies for 37% of children months to years after an episode of M. pneumoniae respiratory tract infection, thus establishing the ability of mycoplasmas to induce chronic and possibly permanent lung damage long after resolution of respiratory tract symptoms.

In animal models of M. pneumoniae infection, Hardy et al. (178) demonstrated that an initial pneumonia lasted 3 to 4 weeks, similar to the case for human disease, characterized by histological lung inflammation and elevated cytokine and chemokine levels. At 530 days following inoculation of M. pneumoniae into the respiratory tract, 78% of mice demonstrated peribronchial and perivascular mononuclear infiltrates that were significantly more pronounced than those in controls by a histopathological severity score, concomitantly with increased airway reactivity and obstruction. Such information provides further evidence for the potential for this organism to produce chronic lung disease of clinical significance.

M. pneumoniae is known to induce a number of the inflammatory mediators implicated in the pathogenesis of asthma that may play a role in exacerbations, which often include wheezing (74, 126, 373, 377). Esposito et al. (126) studied 225 children with an acute episode of wheezing, 16 of whom had mycoplasmal infection as determined by serology and/or PCR on nasopharyngeal secretions, and compared them to 8 asymptomatic children with M. pneumoniae and 8 uninfected controls. Children with wheezing and acute M. pneumoniae infection had a statistically significant increase in IL-5 compared to children with M. pneumoniae who were asymptomatic and to the controls without wheezing. Those authors therefore proposed that M. pneumoniae might trigger the wheezing process by means of IL-5 secretion in persons who are genetically predisposed or are otherwise susceptible. This seems plausible since IL-5 is the cytokine that has been shown to be essential for development of airway hyperresponsiveness in association with infection caused by respiratory syncytial virus (370, 371).

Hardy et al. (179) provided further evidence that the presence of M. pneumoniae in the lower respiratory tract stimulates production of a wide array of inflammatory mediators, including TNF-, IFN-, IL-6, and IL-8, using a murine model of infection. By plethysmography, intranasal inoculation of live M. pneumoniae into mice triggered greater pulmonary airflow resistance or obstruction for a longer duration than what was observed in animals that were inoculated with dead organisms or SP4 broth alone. Significant airflow resistance persisted for the entire 28-day observation period, even after histological evidence of pulmonary inflammation subsided.

M. pneumoniae can be associated with significantly greater numbers of mast cells in patients with chronic asthma, according to one study (284), and experimental evidence from a rodent mast cell line suggests that the organism can induce activation of mast cells with release of serotonin and ß-hexosaminidase (193). Elevated serum immunoglobulin E (IgE) levels as well as production of IgE specific to M. pneumoniae or common allergens may also occur during mycoplasmal infection in children with onset of asthma. Koh et al. (233) showed that levels of the cytokine IL-4 and the ratio of IL-4 to IFN- were significantly higher in children with M. pneumoniae than in those with pneumococcal pneumonia or uninfected controls, suggesting that a TH2-like cytokine response represents a favorable condition for IgE production.

There are a relatively small number of very limited studies that have implicated M. pneumoniae in other chronic lung conditions. Respiratory tract infections with bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis have been associated with acute exacerbations of chronic obstructive pulmonary disease (COPD) for many years. Over 20 years ago, Gump et al. (176) and later Buscho et al. (54) and Smith et al. (383) made some of the earliest observations that mycoplasmal infections could be associated with some cases of COPD exacerbation. However, little attention was given to this possibility for several years. Since the late 1990s, some investigations have been undertaken to assess the possible contribution of M. pneumoniae and Chlamydophila pneumoniae in COPD (260, 261, 293). Mogulkoc et al. (293) used serology to assess the presence of M. pneumoniae and C. pneumoniae in 49 ambulatory patients with acute purulent exacerbations of COPD. They found evidence of acute C. pneumoniae in 11 patients (22%), sometimes in association with other bacteria. M. pneumoniae infection was detected in only three patients (6%). Lieberman et al. (260) also used serology to assess the presence of M. pneumoniae, evaluating a group of 219 patients hospitalized with acute exacerbations of COPD. A total of 34 patients (14.2%) had serological evidence of acute M. pneumoniae infection, making it the third most common bacterial pathogen detected. More than one agent was detected in one-third of the cases. The findings of this study were particularly significant in that they showed that respiratory viruses and atypical bacteria, mainly Legionella species and M. pneumoniae, were involved in most cases and that the classical bacteria were responsible for a minority of cases. These same investigators (261) further described 34 hospitalizations of COPD patients with evidence of M. pneumoniae and showed that 3 had pneumonia, 3 required intensive care management, and 1 died. Since the majority of these patients had serological evidence of an additional pathogen and since the study relied entirely upon serological measurements, it is impossible to determine the precise contribution of M. pneumoniae to these clinical conditions. Specific short-term treatment with drugs known to be active against mycoplasmas did not appear to be beneficial in reducing the duration of hospitalization, but this is not too surprising in view of the chronicity of many mycoplasmal infections and the difficulty in their eradication in many instances.

The occurrence of bacterial infections of the respiratory tract is considered the main cause of progressive pulmonary failure in patients with cystic fibrosis (124). Although the role of M. pneumoniae in community acquired infections of the lower respiratory tract is well known, very little information is available about its occurrence and pathogenic significance in patients with cystic fibrosis. Petersen et al. (324) detected M. pneumoniae antibody by complement fixation (CF) in only 2 of 332 episodes of acute exacerbations in patients with cystic fibrosis. Subsequently, Efthimiou et al. (121) noted a fourfold rise in antibody titers against M. pneumoniae. Coxiella burnetii, and various viruses in a small number of young adults with cystic fibrosis who experienced deterioration in lung function and increase in lower respiratory tract symptoms. Ong et al. (317) and Pribble et al. (335) detected antibodies against M. pneumoniae in 1 of 19 and in 4 of 80 acute pulmonary exacerbations, respectively. Although these studies were limited in the respect that serology was the sole means of assessing the presence of mycoplasmas and the test methods employed present some difficulty in proper interpretation to define a recent infection, taken together they suggest that mycoplasmas may occur but are fairly uncommon causes of these complications in persons with cystic fibrosis. Emre et al. (124) confirmed this assumption in that they were unable to demonstrate the presence of M. pneumoniae by PCR of oropharyngeal secretions in 16 patients, and only 1 of 16 showed serological evidence of recent infection. C. pneumoniae was detected by nasopharyngeal culture in 4 of 32 cases (12.5%), and three of these four patients had elevated IgM or IgG antibodies that were suggestive of acute infection. Clearly, more work must be done to clarify the importance of M. pneumoniae and other atypical bacteria in the epidemiology and pathogenesis of exacerbations of chronic lung diseases, using a more comprehensive diagnostic strategy that would include direct tests for the presence of the organism by PCR so that very low numbers of organisms might be detected in the airways, culture, and serology.

IgA, while often overlooked as a diagnostic antibody class, may actually be a better indicator of recent infections in all age groups (380). IgA antibodies are produced early in the course of disease, rise quickly to peak levels, and decrease earlier than IgM or IgG (166, 440). Research into IgA responses in adult and pediatric populations and assessment of antibody levels in other body fluids such as urine are warranted. The importance of an intact immune response in protection against mycoplasmal disease is apparent in view of prolonged disease and dissemination in persons with hypogammaglobulinemia (410).

In addition to M. pneumoniae-specific antibodies, a variety of cross-reactive antibodies may develop in association with M. pneumoniae infection. The extensive sequence homology of the M. pneumoniae adhesin proteins and glycolipids of the cell membrane with mammalian tissues is a well-known example of molecular mimicry that may trigger autoimmune disorders that involve multiple organ systems through formation of antibodies against substances such as myosin, keratin, fibrinogen, brain, liver, kidney, smooth muscle, and lung tissues (16). Mycoplasmal adhesins also exhibit amino acid sequence homologies with human CD4 and class II major histocompatibility complex lymphocyte proteins, which could generate autoreactive antibodies and trigger cell killing and immunosuppression (353). Cold agglutinins and their historical use as a crude diagnostic test for M. pneumoniae infection are discussed in a subsequent section. Circulating immune complexes also occur during acute phases of M. pneumoniae diseases (80, 291).

Specific T-cell-mediated immunity is also involved in the host reaction to infection by M. pneumoniae. Lymphocytes from persons known to have had a prior mycoplasmal infection will undergo blast transformation when cultured in the presence of M. pneumoniae (134). Leukocytes from individuals with M. pneumoniae infections will show evidence of chemotaxis in the presence of the organism, and these individuals will respond with IFN- in their blood (285, 302).

A property of many species of mycoplasmas that affects the immune responsiveness of the host is their propensity for mitogenic stimulation of B and T lymphocytes, thereby inducing autoimmune disease through the activation of anti-self T cells or polyclonal B lymphocytes (403). This property is associated with the ability of M. pneumoniae to stimulate production of cytokines in the initiation of the acute inflammatory response as described above.

EPIDEMIOLOGY

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Layani-Milon et al. (252) used data obtained by PCR assays on nasal swabs to define the incidence of M. pneumoniae and several respiratory viruses among persons presenting with evidence of acute respiratory tract infection in a region of France over a 5-year period. The distributions of organisms varied from year to year, with M. pneumoniae ranking second to influenza A virus as the most frequent pathogen encountered during the surveillance period. The rates of M. pneumoniae infections varied from year to year, unrelated to rates of other pathogens, suggesting cycles of epidemics as have been described in earlier studies. In some instances, both viral and mycoplasmal organisms were detected simultaneously in the same specimens.

Some recent changes in the incidence of M. pneumoniae infections described by Lind et al. (267), in which high numbers of cases occurred between epidemics without a return to lower endemic levels, have led to speculation as to the reasons for this occurrence. Jacobs (202) suggested that the availability of new information on virulence factors and the P1 adhesin along with the deciphering of the complete genome might enhance understanding of why these changes have occurred. Dorigo-Zetsma et al. (107) genotyped M. pneumoniae clinical isolates and grouped them into eight subtypes within two genomic groups based on P1 adhesin subtypes. Studies from Germany (207) and Japan (365) have shown that different P1 adhesins subtypes may be operating in the development and cycling times of M. pneumoniae epidemics. Such gene divergences within the P1 adhesin and development of subtype-specific antibodies following initial infection might account for the frequency of reinfections, which may be due to another subtype (202). Further work by Cousin-Allery et al. (86) and Dumke et al. (114), using techniques including restriction fragment length polymorphism, rapid amplified polymorphic DNA analysis, multilocus sequence typing, Western blotting, and two-dimensional gel electrophoresis to characterize over 200 M. pneumoniae isolates collected over several years from several European countries and the United States, showed that M. pneumoniae is a rather uniform microorganism, such that most of the isolates could be classified into two groups or subtypes based on the sequences of the P1 adhesin gene, the ORF6 gene, and the P65 gene and by a typical DNA restriction fragment pattern. Both studies found that one or the other of the two subgroups tended to predominate in specific geographical regions to some extent and that there were changes over time with respect to which subgroups most of the isolates belonged to. Dumke et al. (114) found four variants, which were identical to one another but did not belong to either subtype. These studies support the earlier findings (207, 365) that suggested that different M. pneumoniae subtypes might be operative in the cycling times of epidemics. Ovyn et al. (320) described the use of nucleic acid sequence-based amplification to classify 24 M. pneumoniae isolates into two types, yielding results in general agreement with those obtained by the other techniques.

Historically, M. pneumoniae has not been considered part of the normal flora of humans, and its detection by culture could usually be considered abnormal and of etiological significance if in a person with a clinical condition known to be caused by the organism. However, it can persist for variable periods in the respiratory tract following infections that have resolved clinically with appropriate antimicrobial therapy (143). The usual explanation for such persistence has been that the organism attaches strongly to and invades epithelial cells and that macrolide or tetracycline antibiotics commonly used for treating mycoplasmal infections are bacteriostatic and unable to kill all of the organisms. Surveillance studies using culture and/or PCR indicate that a prolonged asymptomatic carrier state may occur in some persons, providing a reservoir for spread of the organism to others (109, 143, 162, 254). Gnarpe et al. (162) demonstrated that 13.5% of 758 healthy volunteers harbored the organism. During a subsequent period of 11 months, the incidence of M. pneumoniae decreased to 4.6% of 499 volunteers, indicating the fluctuating occurrence of this organism over time.

Powerful molecular techniques such as PCR have extremely high sensitivity, theoretically being able to detect a single organism or a single copy of the targeted gene when purified DNA is used; this greatly exceeds the detection threshold of culture, which is approximately 100 to 1,000 cells under optimum conditions (53, 211, 343, 434). Since the mid-1990s, the widespread use of PCR for studies of M. pneumoniae has greatly enhanced, but also complicated, our understanding of its epidemiology, necessitating a reconsideration of the meaning of a "gold standard" for M. pneumoniae diagnosis.

Attack rates of M. pneumoniae among military recruits and other closed or semiclosed populations can be quite high, with reports ranging from 25 to 71% in some settings (5, 120, 131, 228). Some studies have shown M. pneumoniae to be the leading cause of bacterial pneumonia among hospitalized and nonhospitalized military personnel (168, 170). Although long-term morbidity is uncommon, these outbreaks can be very disruptive and can consume significant resources. Strategies to control these outbreaks have included cohorting and use of antibiotics for symptomatic persons and for prophylaxis.

M. pneumoniae causes up to 40% or more of cases of community-acquired pneumonias and as many as 18% of cases requiring hospitalization in children (5, 47, 137, 156, 180, 182, 199, 273, 357, 403, 446). Older studies relying upon serology and culture reported M. pneumoniae pneumonia to be somewhat uncommon in children aged less than 5 years and greatest among school-aged children 5 to 15 years of age, with a decline after adolescence and on into adulthood (5, 143-145). However, M. pneumoniae may occur endemically and occasionally epidemically in older persons, as well as in children under 5 years of age (47, 143, 180, 199, 446). Detection of the organism in 23% of community-acquired pneumonias in children 3 to 4 years of age in a study performed in the United States during the 1990s (47) and documentation of its occurrence in children less than 4 years of age in France (252), without significant differences in infection rates for other children or adults, may reflect the greater number of young children who attend day care centers on a regular basis than in previous years and the ease with which young children share respiratory secretions with older household members or contacts. These recent findings may also reflect improved detection abilities that were unavailable in the 1960s and 1970s, when the first descriptions of M. pneumoniae epidemiology and age distribution were published. Although M. pneumoniae is generally not considered to be a neonatal pathogen, Ursi et al. (426) described probable transplacental transmission of M. pneumoniae, documented by PCR, in the nasopharyngeal aspirate of a neonate with congenital pneumonia.

Whereas pneumonia may be the most severe type of M. pneumoniae infection, the most typical syndrome, especially in children, is tracheobronchitis, often accompanied by a variety of upper respiratory tract manifestations. Esposito et al. (125) demonstrated acute M. pneumoniae infection in 44 of 184 children with nonstreptococcal pharyngitis (23%), using the criteria of an elevated IgM antibody titer or a fourfold increase in IgG antibody titer and/or a positive PCR assay on the nasopharyngeal aspirate. Since the testing for M. pneumoniae was performed only on specimens that were negative for Streptococcus pyogenes, it is possible that an even greater proportion of infections due to M. pneumoniae might have been detected, since some cases may be mixed. No other clinical manifestations or laboratory analyses other than a history of recurrent episodes of pharyngitis were useful in predicting the presence of M. pneumoniae.

M. pneumoniae infection is ordinarily mild, and many adult cases may be asymptomatic, whereas this is much less common in children, perhaps reflecting some degree of protective immunity for reinfections. Moreover, subsequent infections may be more common following initial mild infections as opposed to infection in which pneumonia develops, perhaps due to lesser stimulation of the immune response (147).

Although most mycoplasma infections occur among outpatients (hence the colloquial term "walking pneumonia"), M. pneumoniae is a significant cause of bacterial pneumonia in adults requiring hospitalization in the United States. Marston et al. (283) reported that M. pneumoniae was definitely responsible for 5.4% and possibly responsible for 32.5% of 2,776 cases of community-acquired pneumonia in hospitalized adults in a two-county region of Ohio, using CF antibody determinations for detection. Extrapolation of these data nationally provides an estimated 18,700 to 108,000 cases of pneumonia in hospitalized adults due to M. pneumoniae annually in the United States. Since the majority of patients with pneumonia in the United States are treated as outpatients, the total number of pneumonias due to M. pneumoniae is almost certainly many times greater, and as many as half of all infections in adults may even be asymptomatic. An additional striking finding of the study by Marston et al. (283) was their observation that the incidence of pneumonias due to M. pneumoniae in hospitalized adults increased with age, and it was second only to S. pneumoniae in elderly persons (Fig. 6).

View larger version (14K): [in this window] [in a new window]   FIG. 6. Data from an active surveillance study performed in Ohio in 1991, showing age-specific rates of community-acquired pneumonia due to the major bacterial pathogens. M. pneumoniae infections were diagnosed by seroconversion, using CF tests. These data demonstrate that M. pneumoniae causes a relatively large proportion of pneumonias of sufficient severity to warrant hospitalization among persons younger than 50 years but that it is also an important cause of pneumonia in older age groups. Sp, S. pneumoniae; Mp, M. pneumoniae; Lp, L. pneumophila; Cp, C. pneumoniae. Reprinted from reference 283 with permission of the publisher.

CLINICAL SYNDROMES

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M. pneumoniae infections may be manifested in the upper respiratory tract, the lower respiratory tract, or both. The frequency of nonspecific upper respiratory tract infection manifestations has varied among numerous studies published since the mid-1960s, with some reports indicating that as many as 50% of patients with M. pneumoniae infection present with upper respiratory tract illness (132). Symptomatic disease typically develops gradually over a period of several days, often persisting for weeks to months. The most common manifestations include sore throat, hoarseness, fever, cough which is initially nonproductive but later may yield small to moderate amounts of nonbloody sputum, headache, chills, coryza, myalgias, earache, and general malaise (80, 137, 273, 392, 403). Dyspnea may be evident in more severe cases, and the cough may take on a pertussis-like character, causing patients to complain of chest soreness from protracted coughing (80). Inflammation of the throat may be present, especially in children, with or without cervical adenopathy, and conjunctivitis and myringitis sometimes occur (7, 125, 152). Children under 5 years of age are most likely to manifest coryza and wheezing, and progression to pneumonia is relatively uncommon, whereas older children aged 5 to 15 years are more likely to develop bronchopneumonia, involving one or more lobes, sometimes requiring hospitalization (137, 273, 392). Mild infections and asymptomatic conditions are particularly common in adults, and bronchopneumonia involving one or more lobes develops in 3 to 10% of infected persons (61). As mentioned above, M. pneumoniae is an important cause of pneumonia sufficiently severe to require hospitalization, especially in elderly persons (282, 283, 334). Several studies from the 1960s and 1970s indicate that M. pneumoniae may cause up to 5% of cases of bronchiolitis in young children (102, 112, 161, 271).

Chest auscultation may show scattered or localized rhonchi and expiratory wheezes. Since the alveoli are usually spared, rales and frank consolidation are fairly uncommon unless atelectasis is widespread. In uncomplicated cases, the acute febrile period lasts about a week, while the cough and lassitude may persist for 2 weeks or even longer. The duration of symptoms and signs will generally be shorter if antimicrobial treatment is initiated early in the course of illness (80).

It is important for clinicians to understand that the clinical presentation of M. pneumoniae respiratory disease is often similar to what is also seen with other atypical pathogens, particularly C. pneumoniae, various respiratory viruses, and bacteria such as S. pneumoniae. M. pneumoniae may also be present in the respiratory tract concomitantly with other pathogens (47, 109, 137, 180, 182, 252, 446), and there is some evidence from humans and animal models indicating that infection with M. pneumoniae may precede and somehow intensify subsequent infections with various respiratory viruses and bacteria, including S. pyogenes and Neisseria meningitidis (78). Potential explanations for such a synergistic effect include immunosuppression or alteration in respiratory tract flora due to the presence of M. pneumoniae (78, 255, 269, 358). Children with functional asplenia and immune system impairment due to sickle cell disease, other conditions such as Down syndrome, and various immunosuppressive states are at risk of developing more fulminant pneumonia due to M. pneumoniae (38, 137, 151, 192, 273, 378, 403).

Children with hypogammaglobulinemia are also known to be at greater risk for development of respiratory and joint infections due to M. pneumoniae, demonstrating the importance of functional humoral immunity in protection against infections due to this organism (137, 351, 403, 410). Roifman et al. (351) reported that 18 of 23 patients with hypogammaglobulinemia had one or more episodes of acute respiratory illness during which Ureaplasma urealyticum. M. orale, or M. pneumoniae was isolated from sputum. Resolution occurred followed institution of specific antibiotic therapy and elimination of the mycoplasmas. M. pneumoniae was isolated from the joint of a patient with arthritis and from six patients with chronic lung disease. Clinical improvement, albeit transient, coincided with negative mycoplasma culture results. There are a few case reports of M. pneumoniae infections in pediatric AIDS patients (49, 210), but is not known whether the incidence or severity of pulmonary or extrapulmonary M. pneumoniae infections in AIDS patients is increased significantly or how any immunosuppressed state specifically affects host resistance to M. pneumoniae infection. Fulminant infections with multiple organ involvement and deaths due to M. pneumoniae, usually in otherwise healthy adults and children, have been reported but are uncommon (73, 93, 103, 149, 198, 364, 372, 402, 403, 416, 442).

Central nervous system (CNS) complications are recognized as among the most common of extrapulmonary manifestations of M. pneumoniae infection (384) and have been known to occur since the first report appeared in 1943, even before the true identity of the causative organism was known (56). Approximately 6 to 7% of hospitalized patients with serologically confirmed cases of M. pneumoniae pneumonia may experience neurological complications of varying severity (237, 294, 332, 387). Such complications have included encephalitis, cerebellar syndrome and polyradiculitis, cranial nerve palsies, aseptic meningitis or meningoencephalitis, acute disseminated encephalomyelitis, coma, optic neuritis, diplopia, mental confusion, and acute psychosis secondary to encephalitis (39, 98, 159, 226, 264, 331, 386, 392). A number of motor deficiencies have also been described, including cranial nerve palsy, brachial plexus neuropathy, ataxia, choreoathetosis, and ascending paralysis (Guillain-Barré Syndrome) (2, 4, 9, 12, 39, 44, 223, 226, 319). Encephalitis has been the most common neurological manifestation in children (237). Most patients with neurological complications experience them 1 to 2 weeks after the onset of respiratory signs, but 20% of patients or more have no preceding or concomitant diagnosis of respiratory infection (333). This figure may be higher yet in children (413).

Most of the first descriptions of CNS complications were based on serology and later on occasional isolation of M. pneumoniae from the respiratory tract rather than the CNS. The lack of clear evidence that mycoplasmas were actually present in neurological tissues led to theories that damage to brain tissue occurred as a result of cross-reacting or autoimmune antibodies (129, 142) and even to concern that neurological infections by other bacterial pathogens were causing false-positive mycoplasmal serology (230). The potential role of immunological sequelae of M. pneumoniae infection that can lead to neurological complications cannot be discounted, and some CNS complications are very likely due to this mechanism as opposed to direct invasion (312, 323). Antibodies against galactocerebroside, a component of CNS myelin, has been detected in 100% of patients with M. pneumoniae and CNS involvement and in only 25% of those without CNS involvement (312). Postinfectious leukoencephalopathy due to M. pneumoniae also suggests a role for autoimmunity in some cases (325).

Proof that viable organisms or M. pneumoniae DNA can be detected directly in neural tissues and CSF provides convincing evidence that this organism does indeed disseminate from the respiratory tract in some instances (3, 105, 198, 251, 307, 401, 403, 415). Neurological manifestations associated with M. pneumoniae infections usually resolve completely, but they can result in chronic debilitating deficits in motor or mental function (384). These conditions can be severe and life threatening. Rautonen et al. (341) reported that children with M. pneumoniae were seven times more