Mycoplasma pneumoniae and Its Role as a Human Pathogen

Evidence accumulated since the 1960s through animal models as well as in vitro cell and organ culture systems indicates that attachment of M. pneumoniae to the respiratory epithelium is a prerequisite for later events that culminate in production of disease (403). This close interaction between the mycoplasma and host cells protects it from removal by the host's mucociliary clearance mechanism and allows it to produce a variety of local cytotoxic effects.

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 β, are also involved in binding M. pneumoniae to fibronectin, a very common component of eukaryotic cell surfaces, basement membranes, and the extracellular matrix.

The P1 adhesin is a 170-kDa protein concentrated in the attachment tip that is now known to be the major structure responsible for interaction of M. pneumoniae with host cells (27, 30, 31, 82, 88, 90, 196, 240, 241). In addition to its presence in the attachment organelle, lower concentrations of P1 are also widespread on the cell's surface (15, 375). Loss of P1 activity through spontaneous mutation or by trypsin treatment results in avirluence by reduced adherence of mycoplasmas to eukaryotic cells (26). Spontaneous reversion to the cytadhering phenotype is accompanied by the reappearance of the implicated proteins, restoration of structurally and functionally intact tips, and return of full infectivity. Further proof for the functional role of P1 as an adhesin comes from evidence that monoclonal antibodies against P1 block adherence in a hamster model of mycoplasma respiratory disease, whereas antibodies produced against some other M. pneumoniae proteins have no effect on attachment (242).

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).

Mycoplasmas are known primarily as mucosal pathogens that reside extracellularly on epithelial surfaces. However, during the past few years, the potential for several mycoplasmal species to fuse with and enter host cells that are not normally phagocytic has been demonstrated (355). Such an occurrence should not be unexpected for microorganisms lacking a rigid cell wall that are typically closely associated with host cell surfaces. Rottem (355) has summarized current knowledge concerning the features that enable M. penetrans, a mycoplasma of uncertain pathological significance that has been isolated from urine specimens from human immunodeficiency virus-infected persons, and M. fermentans to invade host cells, some of which may be relevant to enhance understanding of similar events that may occur with M. pneumoniae. Dallo and Baseman (87) recently described the ability of M. pneumoniae to survive, synthesize DNA, and undergo cell replication in artificial cell culture systems over a 6-month period.

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.

Internalization of a cell-associated mycoplasma into a host cell is not a prerequisite for the initiation of local cytotoxic events and clinical manifestation of disease, although cytadherence in the respiratory tract is the initiating event in disease production by M. pneumoniae. It is not known precisely how M. pneumoniae injures the respiratory epithelial cell after attachment, but a number of biochemical and immunological properties of the organism that are likely to be involved have been described. Close approximation of the organism to the host cells, facilitated by the adhesin proteins, appears to be important to facilitate localized tissue disruption and cytotoxicity. Unlike many human pathogens, M. pneumoniae is not known to produce any exotoxins. Hydrogen peroxide and superoxide radicals synthesized by M. pneumoniae act in concert with endogenous toxic oxygen molecules generated by host cells to induce oxidative stress in the respiratory epithelium (420). Consistent with the small genome, M. pneumoniae lacks some enzymes that are associated with virulence of other bacteria, such as superoxide dismutase and catalase. Hydrogen peroxide production in M. pneumoniae occurs as a result of a flavin-terminated electron transport chain (420). Hydrogen peroxide has been known to be important as a virulence factor in M. pneumoniae since Somerson et al. showed it to be the molecule that confers hemolytic activity (385). The ultrastructural effects of peroxide on host cells such as erythrocytes include loss of reduced glutathione, denaturation of hemoglobin, peroxidation of erythrocyte lipids, and eventually lysis of the cells. Almagor et al. (8) suggested that superoxide anion produced by M. pneumoniae acts to inhibit catalase in host cells, thereby reducing the enzymatic breakdown of peroxides produced endogenously and by the mycoplasma, rendering the host cell more susceptible to oxidative damage. M. pneumoniae hemadsorption and lysis of guinea pig erythrocytes, which are low in endogenous catalase, are also mediated by peroxide (420). This property was adapted for use as a diagnostic test to presumptively distinguish M. pneumoniae from other commensal mycoplasmas that are commonly found in the human respiratory tract, which do not produce hydrogen peroxide and therefore do not hemadsorb in this manner.

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-α), gamma interferon (IFN-γ), and various interleukins (including interleukin-1β [IL-1β], 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-α mRNAs and that both proteins were secreted into culture medium. The major proinflammatory cytokine IL-1β 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.

The release of proinflammatory cytokines in association with M. pneumoniae infection has also been implicated as a possible mechanism leading to or exacerbating underlying chronic pulmonary diseases such as bronchial asthma. The concept that chronic infection with M. pneumoniae might play a role in the pathogenesis of asthma was speculated on over 30 years ago (42). The relevant questions are whether M. pneumoniae is a primary cause of asthma or whether mycoplasmal infection is at least a cofactor in its development. Appreciation of the pathogenesis of chronic murine respiratory mycoplasmosis, a naturally occurring mycoplasmal infection of rodents mediated by Mycoplasma pulmonis that is slowly progressive, is greatly influenced by heredity, and has characteristics that are similar in some ways to those of asthma in humans, gives further credence to the potential for mycoplasmas to cause longstanding lung disorders such as asthma (60).

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-α, IFN-γ, 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 β-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-γ 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.

M. pneumoniae possesses both protein and glycolipid antigens that elicit antibody responses in infected individuals. The P1 protein is the target of many of the antibodies that are produced by the host in response to the M. pneumoniae infection, and it has also served as a target for development of serological assays. Following an initial infection, the normal immune system responds by rapidly producing antibodies that peak after 3 to 6 weeks, followed by a gradual decline over months to years. As a result of the long incubation period, an antibody response is often evident by the time symptoms appear. Elevation of M. pneumoniae-specific IgM alone can often be interpreted as evidence of acute infection, since this antibody typically appears within 1 week of the initial infection and approximately 2 weeks before IgG antibody (286, 380). However, the presence of IgM is considered most significant in pediatric populations, where there have been fewer opportunities for repeated exposures. Adults who have been infected repeatedly over a period of years may not respond to mycoplasma antigens with a brisk IgM response (434). In these cases, reinfection leads directly to an IgG response; therefore, the absence of a positive IgM test does rule out an acute infection. When it does occur, the IgM response may persist for months or years following infection (445), and in these cases a positive IgM test result may not reflect a current or recent infection.

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-α 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.

Many mycoplasmal species that infect animals or humans are known for their ability to induce chronic disease states in which clearing of the organism is extremely difficult. Therefore, these organisms must have evolved means by which they can successfully evade or modulate the host immune response. As mentioned above, intracellular localization and immunomodulatory activities are possible means to this end. Another mechanism that has been extensively studied in many other bacteria is variation in surface antigens. High-frequency phase and antigenic variation of surface adhesin proteins made possible by DNA rearrangements in truncated and sequence-related copies of the P1 adhesin genes that are dispersed throughout the genome has been described for M. pneumoniae (396, 397). Recombinational events among the repetitive elements themselves and with regions of the three-gene P1 adhesin operon promote diversity and altered specificities and affinities and maximize the coding potential of the limited mycoplasma genome (31, 451).

Soon after the identification of M. pneumoniae as the etiological agent of primary atypical pneumonia in the early 1960s, considerable interest arose in elucidation and characterization of its incidence, prevalence, mode of spread, and spectrum of disease. M. pneumoniae infections can involve both the upper and the lower respiratory tracts and occur both endemically and epidemically worldwide in children and adults. Climate and geography are not thought to be of major significance. Even though most available data concerning the occurrence of M. pneumoniae infections have come from studies performed in the United States, Europe, and Japan, seroprevalence investigations using CF technology in arctic and tropical zones have also indicated the presence of M. pneumoniae antibody, suggesting that populations in these regions have had infections due to this organism (164, 214, 398). In the United States, there is no national surveillance system tracking M. pneumoniae infections; therefore, much of what we know about rates of endemic disease comes from population-based studies relying primarily on serological measurements (146, 148, 150, 171, 296, 297). Foy (143) used CF antibody determinations and culture to show that M. pneumoniae was responsible for 15 to 20% of all cases of community-acquired pneumonia, or two cases per 1,000 persons on an annual basis between 1962 and 1975, in Seattle, Wash. Additional retrospective serological studies performed in Denmark showed a pattern of M. pneumoniae infections over a 50-year period from 1946 through 1995 with endemic disease transmission punctuated with cyclic epidemics every 3 to 5 years, similar to what was observed in the United States (143, 267). Additional studies in North America and Europe performed over the past 3 decades have also reported similar trends (106, 143, 144, 146, 181, 207, 252, 266, 267, 313, 340). Although the incidence of disease does not vary greatly by season, the proportion of patients with pneumonia due to M. pneumoniae is greatest during the summer in temperate climates due to the lower incidence of other respiratory pathogens at this time (5, 283, 334). Outbreaks of M. pneumoniae infections also tend to occur in the summer or early fall (5, 131, 403). The long incubation period and relatively low transmission rate have been implicated in the prolonged duration of epidemics of M. pneumoniae infections (137).

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.

M. pneumoniae can be transmitted through aerosols from person to person, and disease has been produced experimentally by aerosol inoculation (80). Persons with active mycoplasmal infection will carry the organisms in the nose, throat, trachea, and sputum, indicating diffuse involvement. Spread of organisms is greatly facilitated by the ubiquitous cough. Since the organisms tend to be associated with desquamated cells, relatively large droplets may be required for transmission, as evidenced by the close personal contact typical of outbreak settings, e.g., schools, military barracks, and institutions. M. pneumoniae infections commonly spread gradually among family members within a household (109, 137, 403). In view of the intimate contact needed for droplet transmission and the slow (6-h) generation time of M. pneumoniae, 1 to 3 weeks of incubation for each case is typical, and several cycles may be necessary before intrafamily transmission is complete. Some studies have reported incubation periods from common-source outbreaks of as short as 4 days (363), whereas others (145) have reported longer incubations, with a median of 23 days with intrafamilial spread, where smaller inocula may be involved and transmission may be less effective until the index case has exhibited symptoms for several days. Foy et al. (145) reported that 39% of family contacts may eventually become infected with M. pneumoniae, many asymptomatically. Dorigo-Zetsma et al. (109) found that among 79 asymptomatic household contacts of 30 index cases with acute respiratory tract infection due to M. pneumoniae, 15% harbored the organism, with a significantly greater number being children under 15 years of age. A novel mathematical model for investigating the control and spread of disease transmission has recently been applied to assess the effects of interventions in outbreaks of M. pneumoniae in closed communities (288).

Numerous outbreaks of M. pneumoniae infections have been documented in the community or in closed or semiclosed settings such as military bases (120, 131, 167, 168, 292), hospitals (141, 221, 229), religious communities (253, 299), and facilities for the mentally or developmentally disabled (197, 228, 391). In outbreaks where many more persons, usually living close together in military barracks or similar situations, are exposed to M. pneumoniae aerosols simultaneously, the rate of spread within a facility appears to be higher than in single-family households.

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.

Serological studies performed in the 1960s and 1970s, evaluating the attack rates of M. pneumoniae according to sex and broken down into various age groups, have yielded mixed results, with slight gender differences apparent between some age groups. Overall, there appears to be little reason to suspect that males and females have greatly differing susceptibilities to M. pneumoniae infections (145, 280, 314).

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).

Another study of hospitalized adults with community-acquired pneumonias performed in Israel (334), which used commercial serological kits to detect antibodies, showed M. pneumoniae to be second only to S. pneumoniae, and it was responsible for 29.2% of pneumonias overall.

The clinical entity of pneumonia eventually proven to be caused by M. pneumoniae was recognized many years before the actual identity and nature of the etiological agent were established. The first clues to differentiate pneumonia eventually proven to be due to mycoplasma from classical pneumococcal pneumonia came from the observations that some cases failed to respond to treatment with sulfonamides or penicillin. The lack of response to antimicrobial therapy was deemed “atypical,” and the condition was thought likely to be a primary form of lung disease of uncertain etiology; hence, the term “primary atypical pneumonia” was coined. This term, along with “walking pneumonia,” has been used widely by physicians and the lay public to denote mycoplasmal respiratory disease.

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).

As many as 25% of persons infected with M. pneumoniae may experience extrapulmonary complications at variable time periods after onset of or even in the absence of respiratory illness. Autoimmune reactions have been suggested to be responsible for many of the extrapulmonary complications associated with mycoplasmal infection (403). However, the availability of PCR has greatly enhanced understanding of how M. pneumoniae can disseminate throughout the body. The presence of M. pneumoniae in extrapulmonary sites such as blood, synovial fluid and cerebrospinal fluid, pericardial fluid, and skin lesions has been documented by PCR as well as culture, so direct invasion must always be considered (23, 220, 235, 306, 360). However, the frequency of direct invasion of these sites is unknown because the organism is rarely sought for clinical purposes. It is also important to realize that extrapulmonary complications can be seen before, during, or after pulmonary manifestations or can occur in the complete absence of any respiratory symptoms (62).

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 likely to die or have severe neurological sequelae than other children, second only to cases of herpes simplex virus infection. The presence of peripheral neurological sequelae such as radiculitis and transverse myelitis has been identified as a risk factor for chronic CNS sequelae (59, 331). Central nervous system effects due to mycoplasmas have been reviewed in detail by Talkington (401).

Whereas neurological disorders may be the most severe extrapulmonary manifestations of M. pneumoniae infections, dermatological disorders, including erythematous maculopapular and vesicular rashes, are perhaps the most common clinically significant complications, occurring in up to 25% of patients. Although the disorders are usually self-limited, severe forms of Stevens-Johnson syndrome, conjunctivitis, ulcerative stomatitis, and bullous exanthems have been reported, and the organism has been detected directly in the cutaneous lesions (70, 71, 256, 276, 395). Clinicians should keep in mind that the presence of erythematous maculopapular rashes in M. pneumoniae patients can also be caused by a number of antibiotics commonly used to treat respiratory tract infections.

Nonspecific myalgias, arthralgias, and polyarthropathies occur in approximately 14% of patients with acute M. pneumoniae infection and may sometimes persist for long periods (7). Septic arthritis with detection of the organism directly in synovial fluid has been reported numerous times, occurring most commonly in hypogammaglobulinemic patients but sometimes in immunocompetent persons (92, 183, 209, 249, 304, 329, 351, 374, 408, 410, 441). In view of the well-known associations of animal mycoplasmas and their ability to produce chronic, naturally occurring infection of the joints and the experimental models, joint involvement by M. pneumoniae is not unexpected. Little attention has been paid to invasive infections of bones due to M. pneumoniae, but at least one report of osteomyelitis in a splenectomized patient with hypogammaglobulinemia has been attributed to M. pneumoniae, in which the infection was detected by PCR (250).

Cardiac complications associated with M. pneumoniae are relatively uncommon, but involvement has been reported at rates of from 1 to 8.5% in persons with serological evidence of infection, somewhat more commonly in adults than in children (287, 330). Pericarditis, myocarditis, and pericardial effusion with and without cardiac tamponade have all been described, and the organism has been detected in pericardial fluid (24, 122, 128, 171, 218, 248, 287, 364, 384, 400). According to one study (330), almost half of the patients with M. pneumoniae infection had symptoms or signs of heart abnormalities an average of 16 months later.

Hemolytic anemia is recognized as a rare but severe complication of mycoplasmal pneumonia, occurring more often in children than in adults (70, 122, 381). The mechanism by which M. pneumoniae causes this complication has been attributed to cross-reacting cold agglutinins (80, 122, 381, 403, 424). Two cases of aplastic anemia associated with M. pneumoniae have also been reported (390). A recent report suggests that thrombotic thrombocytopenic purpura associated with M. pneumoniae infection may be the result of cross-reactive antibodies inactivating plasma von Willebrand factor-cleaving protease (23). Fulminant infection leading to fatal disseminated intravascular coagulation has also been reported (73), as has a case of priapism in a 12-year-old boy that was felt to be due to the hypercoagulable state that sometimes occurs in association with M. pneumoniae infection (192). If subclinical forms of hemolytic anemia and intravascular coagulation are considered, over 50% of patients with M. pneumoniae infections may be affected.

Acute glomerulonephritis, renal failure, tubulointerstitial nephritis, and IgA nephropathy, as well as other conditions, have been sporadically reported in association with M. pneumoniae infections (217, 235, 321, 369, 430). Kanayama et al. (217) reported cases of IgA nephropathy in persons in whom the mycoplasma infection was diagnosed serologically. Attempts to demonstrate mycoplasma antigen in damaged renal tissue by immunohistochemical techniques have not been uniformly successful, once again leading to theories that an antibody-mediated pathogenesis is responsible (217, 431). A recent attempt to use PCR to identify mycoplasmas in renal tissue from four children with acute nephritis concomitant with serological evidence of recent M. pneumoniae infection also failed (360). However, the presence of mycoplasma antigen has been demonstrated by immunoperoxidase staining in renal tissue in a patient with acute interstitial nephritis (10).

M. pneumoniae infection may be associated with a variety of nonspecific complaints related to the gastrointestinal system. These include nausea, vomiting, and diarrhea. Rarely, cholestatic hepatitis and pancreatitis have been associated with respiratory infections (11, 174, 381, 386).

Up to one-third of patients with M. pneumoniae infection may have nonspecific ear symptoms, including otitis externa, otitis media, and myringitis (301, 392). Acute rhabdomyolysis was recently reported in association with M. pneumoniae infection in a 15-year-old patient (41). Ocular manifestations have been reported in children occasionally and include conjunctivitis, anterior uveitis, optic neuropathy, retinitis and retinal hemorrhages, iritis, and optic disk swelling, with or without permanent degradation of vision (289, 362). M. pneumoniae has been isolated from the urogenital tracts of males and females and has been cultured from a tubo-ovarian abscess (165). Given the apparent ability of the organism to invade the bloodstream, infections in almost any organ system are possible. Higuchi et al. (184, 185) reported the detection of M. pneumoniae by PCR in ruptured atherosclerotic plaques and stenotic heart valves and speculated about its possible association, along with C. pneumoniae, as a risk factor for embolization and myocardial infarction. Additional data are necessary to determine the significance of these preliminary reports.

Clinical laboratory findings are seldom diagnostic for M. pneumoniae infection. About one-third of persons with lower respiratory tract infections may have leukocytosis (392). An elevated erythrocyte sedimentation rate may also be observed (45). Gram staining of sputum may show mononuclear cells or neutrophils and normal flora. There are no hepatic or renal abnormalities typical of M. pneumoniae infection, although the hemolytic anemia that develops in some patients may be reflected in the hemogram.

Prior to the widespread availability of commercialized antibody assays, and even before the precise bacteriological characterization of the etiological agent was known, clinicians sometimes used the presence of cold agglutinins to confirm their clinical suspicions of primary atypical pneumonia, which was also known as cold-agglutinin disease. Cold agglutinins are actually IgM antibodies that are produced 1 to 2 weeks after initial infection in about 50% of M. pneumoniae infections and may persist for several weeks. One theory is that cold agglutinins are the result of cross-reactive autoantibodies developed against the I antigen of human erythrocytes during acute mycoplasmal infection. Another theory is that they develop directly as a result of antigenic alteration of the erythrocytes caused by M. pneumoniae. The latter theory is supported by the facts that M. pneumoniae is known to interact with and adsorb to human erythrocytes and to produce a hemolysin and because cold agglutinins can be induced in rabbits inoculated with human erythrocytes that were preincubated with M. pneumoniae but not in rabbits inoculated with either M. pneumoniae or erythrocytes alone (16).

Two varieties of cold agglutinin determination have been used. The simplest version of the test was what is referred to as “bedside cold agglutinins.” This consists of collecting blood from a patient suspected of having mycoplasmal pneumonia into a tube containing citrate or another suitable anticoagulant and placing it in ice water in a freezer for approximately 30 s. The tube is then examined for coarse agglutination by being tilted on its side. On warming, the agglutination should resolve, but it can be reproduced by repeating the cooling procedure. A more precise test is to determine the cold agglutinin titer by reacting doubling dilutions of patient sera suspected to contain the cold agglutinins with human type O erythrocytes in the cold and determining the highest dilution at which agglutination occurs. The cold agglutinin response often correlates directly with the severity of pulmonary involvement; patients with extensive lobar involvement will usually have a titer of ≥1:32, whereas those with only minimal illness may not develop cold agglutinins. However, cold agglutinins also occur in persons who have various bacterial and rickettsial infections as well as in those with influenza virus and adenovirus infections (16). Due to the availability of antibody assays that are based on immunological reagents specific for M. pneumoniae, the popularity of testing serum for the presence of cold agglutinins has declined. If used, this test should be limited to persons in whom mycoplasmal pneumonia is a strong possibility. Under these circumstances, a positive test provides reasonable supportive evidence of a mycoplasmal etiology, on the basis of which clinical management decisions can be made.

Primary atypical pneumonia due to M. pneumoniae can be extremely variable and mimic a wide variety of lung diseases. The inflammatory response elicited by M. pneumoniae causes interstitial mononuclear inflammation in the lungs that may be manifested radiographically as diffuse, reticular infiltrates of bronchopneumonia in the perihilar regions or lower lobes, usually with a unilateral distribution, and hilar adenopathy. Bilateral involvement may occur in about 20% of cases (137). However, lobar consolidation with bilateral alveolar involvement has been described, and the degree of consolidation may exceed what would be expected based on the severity of clinical manifestations (97). Among patients with M. pneumoniae pneumonia who require hospitalization, up to 10.9% will require mechanical ventilation (282).

Histopathological examinations from fatal cases of M. pneumoniae pneumonia, biopsy material, examination of tissues from animal models, and tracheal organ cultures have shown lesions of the epithelial lining of the mucosal surfaces with ulceration and destruction of ciliated epithelium of bronchi and bronchioles. Edema of bronchial and bronchiolar walls; bronchiolar and alveolar infiltrates of macrophages, lymphocytes, neutrophils, plasma cells, and fibrin; and bronchiolitis obliterans have been described (65, 77, 93, 259, 277, 352, 372, 432). Type II pneumocyte hyperplasia and diffuse alveolar damage have also been reported. Pleura may contain patches of fibrinous exudates. Pleural effusions and diffuse alveolar damage sometimes occur in association with more severe cases, and long-term sequelae such as pleural scarring, bronchiectasis, and pulmonary fibrosis have been reported (40, 72, 97, 222, 338, 372). Lung abscesses may also occur (258, 379). Recent studies utilizing animal models developed to investigate the potential role of M. pneumoniae in chronic lung conditions such as asthma (discussed above) have also provided insights into the histopathological aspects of M. pneumoniae lung disease (178, 179). Immunosuppressed persons with M. pneumoniae infection may lack pulmonary infiltrates, further attesting to the importance of the host immune response in lesion development (151).

The lack of rapid and accurate diagnostic laboratory tests to detect M. pneumoniae infection has hampered understanding of the epidemiology as well as contributed to unawareness of the potential clinical significance of this common pathogen by many physicians. From the microbiologist's point of view, one might argue in favor of the need to routinely use the best means available to identify persons with acute M. pneumoniae respiratory tract infection under all circumstances. However, from a practical standpoint, one must also consider therapy that will generally be provided empirically. Thus, the performance of diagnostic tests for M. pneumoniae in a typical ambulatory care setting may be unnecessary much of the time. This approach has been taken in the Infectious Disease Society of America diagnosis and treatment guidelines for community-acquired pneumonias (25). However, in the event of an illness in which M. pneumoniae is suspected and that is of sufficient severity as to require hospitalization, especially if the patient has any sort of immune deficiency or underlying condition that may make an unfavorable outcome more likely, attempts to detect M. pneumoniae infection are justified. Unfortunately, most of the diagnostic methods that are currently available for detection of M. pneumoniae infections are somewhat better suited for use in epidemiological studies as opposed to direct management of individual patients due to their prolonged turnaround time, limited availability, and cost. Among currently available test modalities, each has limitations.

Descriptions of the physiological, serological, and morphological procedures for detection and identification of mycoplasmas in clinical specimens have been summarized and updated to include more of the molecular-based methods in Molecular and Diagnostic Procedures in Mycoplasmology, vol. 2, published in 1996 (421). Waites et al. (434, 438, 439) have summarized current methods for clinical diagnosis and susceptibility testing of human mycoplasmas and have discussed commercial products used for culture and serology. Razin (343) has also provided an overview of diagnosis of mycoplasmal infections with commentary on various molecular-based methods. Brief summaries and commentary on culture-based and non-culture-based methods for detection of infections due to M. pneumoniae are presented here.

A summary of the in vitro activities of several antimicrobial agents against M. pneumoniae is shown in Table 3. M. pneumoniae is inhibited by tetracyclines, macrolides, ketolides, and fluoroquinolones, with little variation in MICs among clinical isolates (435, 436). Thus, in vitro susceptibility testing is not indicated for routine patient management purposes for infections of the respiratory tract. Other agents that are active at the bacterial ribosome, such as streptogramins, chloramphenicol, and aminoglycosides, may also show in vitro inhibitory activity against M. pneumoniae but are not widely used for therapeutic purposes against this organism. Clindamycin may be effective in vitro, but limited reports suggest that it may not be active in vivo, and it has not been considered a first-line treatment (80). Due to the lack of a cell wall, all mycoplasmas are innately resistant to all beta-lactams and glycopeptides. Sulfonamides, trimethoprim, polymixins, nalidixic acid, and rifampin are also inactive. Linezolid is the prototype agent of the oxazolidinone class. These agents are much less active against M. pneumoniae than the other agents that inhibit protein synthesis (224). New quinolones such as moxifloxacin, gatifloxacin, garenoxacin, gemifloxacin, and sparfloxacin tend to have somewhat greater in vitro activity than older agents such as ciprofloxacin, ofloxacin, and levofloxacin, although MICs of all fluoroquinolones are severalfold higher than those of macrolides (224, 435-437). Fluoroquinolones have been shown to be bactericidal for M. pneumoniae, whereas macrolides and tetracyclines are primarily bacteriostatic (36, 113, 435, 437).

The extent of naturally occurring, clinically significant acquired antimicrobial resistance of M. pneumoniae to any drug class is not known, but it is generally believed to be uncommon. Treatment failures have been rarely reported for microbiologically proven cases of M. pneumoniae infection, and recent studies evaluating macrolides, tetracyclines, or fluoroquinolones indicate comparable in vitro activities against isolates obtained from North America and Europe, with no change from data reported earlier other than some minor variations that may be due to differences in testing methods, media, and inocula (32, 34, 311, 435). However, organisms from acute M. pneumoniae infections are seldom sought by culture, they are almost never tested for in vitro susceptibilities for clinical purposes, and most in vitro studies reported to date tested relatively small numbers of clinical isolates from limited geographic areas. Organisms can be shed in respiratory secretions for long periods after acute infection, and high-level-macrolide-resistant strains have been isolated following erythromycin treatment, but resistance did not appear to affect clinical outcome (310, 394). Macrolide-resistant M. pneumoniae mutants can easily be selected in vitro (311, 393, 394); such mutants typically exhibit the macrolide-lincosamide-streptogramin B-type resistance, rendering lincosamides and streptogramin B inactive in addition to the macrolides.

Experimental observations of two laboratory-derived erythromycin-resistant M. pneumoniae mutants indicated that macrolide resistance can occur due to point mutations leading to A-to-G transitions in the peptidyl transferase loop of domain V of the 23S rRNA gene at positions 2063 and 2064, which reduces the affinity of these antibiotics for the ribosomes (274, 316). The likelihood of M. pneumoniae developing resistance to macrolides by this mechanism under natural conditions may be enhanced, since there is only a single rRNA operon in the M. pneumoniae genome (163). No plasmids or erm genes to mediate ribosomal modification, or any enzymes that break down macrolides, have ever been described for M. pneumoniae. The well-documented occurrences of the tetM determinant associated with the conjugative transposon Tn916, which confers resistance to the tetracyclines, in M. hominis and Ureaplasma spp. (34, 348, 349) and of mutations in DNA gyrase and topoisomerase that confer fluoroquinolone resistance (34, 37) prove the potential for mycoplasmas to acquire resistance to these drug classes. However, to date no such naturally occurring resistance to tetracyclines or fluoroquinolones in M. pneumoniae has been reported in the published medical literature to our knowledge. Whether the bactericidal effect of various fluoroquinolones against M. pneumoniae shown in vitro is more effective in preventing persistence of the organism in respiratory secretions after clinical resolution has not been documented.

Even though susceptibility testing is not necessary to guide treatment of M. pneumoniae infections, such procedures are needed in order to evaluate new or investigational antimicrobial agents in comparison to existing drugs, occasionally for systemic infections (especially if the host is immunosuppressed), if treatment is not successful clinically, and/or for eradication of the organism from a normally sterile site. There are no official guidelines for performance, interpretation, or quality control of in vitro susceptibility tests for human mycoplasmas published by the National Committee for Clinical Laboratory Standards (NCCLS). However, a subcommittee of the NCCLS was established in 2001 to address this, and work is currently under way to develop in vitro susceptibility protocols and quality control ranges for representative drug classes for M. pneumoniae. M. hominis, and Ureaplasma spp. In the absence of NCCLS-sanctioned methods, the Mycoplasma Chemotherapy Working Team of the International Research Program on Comparative Mycoplasmology has formulated methods for determination of MICs by broth dilution and agar dilution. These methods have been summarized by Waites et al. (434). Procedures for determination of MICs for human mycoplasmas are also available in other publications, which can be used as a basis for development of laboratory protocols (33, 407). Many aspects of susceptibility testing of mycoplasmas are identical to the procedures that have been developed and standardized for conventional bacteria. However, the fastidious nature, complex growth requirements, and incubation length and conditions for mycoplasmal growth have to be considered in optimizing testing for these organisms. Broth- and agar-based methods for determining MICs have been adapted for testing mycoplasmas, and some general comments regarding the procedures to be performed are relevant for both techniques. As with all bacteria, the inoculum of organisms is important. Since mycoplasmas do not produce turbidity in liquid medium, it is somewhat difficult and complex to determine the number of viable cells present in an active culture. Experience has shown that MICs are not greatly affected when M. pneumoniae taken from frozen stocks is used. Thus, some laboratories prefer to test a frozen stock culture that was serially diluted previously to determine the number of viable cells and then dilute it to an inoculum containing 10⁴ to 10⁵ color-changing units/ml for use directly after a 2-h prewarming period (435, 436). SP4 medium and Hayflick's modified medium (pH 7.4 to 7.6) have been used for determining MICs for M. pneumoniae, with similar results (35, 434, 435). MIC test mixtures should be incubated at 37°C. Broth-based test mixtures can be incubated under atmospheric conditions, whereas agar plates should be supplemented with 5% CO₂ for optimum colony development. Mandatory controls that must be used for all types of MIC assays include a sterility control, a growth control, and solvent control. Simultaneous testing of an isolate of M. pneumoniae for which MICs of the drugs of interest are known and consistent is also advisable. Due to the slow growth of M. pneumoniae, MIC test mixtures have to be incubated for 5 days or more and examined daily. No breakpoints to designate susceptibility or resistance have been designated for mycoplasmas. However, some inference about the meaning of the MICs obtained can be made based on breakpoints established for other bacteria and the achievable concentrations within the body for the drugs being tested. Generally speaking, if the MIC is ≤1 μg/ml, the drug may potentially be active against M. pneumoniae.

The agar dilution method of susceptibility testing has the advantages of having a stable endpoint over time, unlike broth dilution, where color change endpoints tend to shift over time. Agar dilution is suitable for testing large numbers of organisms at once. Testing small numbers of isolates by agar dilution is not very practical, since several individual agar plates must be prepared for each drug to encompass all of the dilutions that may be needed and media must be used when fresh. This method of testing is ideally performed by using a Steers replicator to deliver the inoculum to the agar plate, with a goal of generating 30 to 300 colonies in the growth control (434). The MIC is read as the lowest concentration of drug that prevents colony formation when colonies are evident on the growth control plate.

The broth microdilution technique is based on the principle that a constant number of organisms are added to serial doubling concentrations of antimicrobial agents diluted in broth in a 96-well microtiter plate. Broth microdilution is the most practical and widely used method of susceptibility testing for M. pneumoniae. Multiple antimicrobials can be tested in the same microtiter plate at several different dilutions. The inoculated microtiter plates are sealed with an adhesive cover to prevent drying out and are incubated until the growth control shows a color change. The MIC is read as the lowest concentration of drug that inhibits growth at the time the growth control first shows evidence of growth. Minimal bactericidal concentrations can be tested directly by diluting and subculturing wells in the microtiter plate that show no evidence of growth (color change) into fresh medium. When the minimal bactericidal concentration is within two dilutions of the MIC (fourfold), the agent may be considered bactericidal (435).

Waites et al. (435) have described methods for performing time-kill assays in which multiple dilutions of antimicrobials are incubated in broth with inocula of M. pneumoniae. Bactericidal activity over time is assessed by removal of small volumes of broth daily for up to a week and subculturing to agar plates. Colony counts performed on the subcultures will demonstrate the extent of bacterial killing and the time of exposure to the antimicrobial that is necessary to achieve this effect. Traditionally, a reduction of 99.9% or 3 log₁₀ dilutions of the original inoculum has been used to define bactericidal as opposed to bacteriostatic activity (435).

When M. pneumoniae infections were first described soon after the organism was characterized and isolated in culture in the 1960s, there was some sentiment that antibiotic therapy was unnecessary since the disease is self-limiting most of the time. We now know that administration of antimicrobials will generally produce satisfactory results, with a marked reduction in duration of respiratory symptoms compared to nontreatment. Treatment of M. pneumoniae infections on a case-by-case basis has been guided primarily by its well-known and consistent susceptibilities to drugs in the macrolide, tetracycline, and fluoroquinolone classes that are available for oral administration. Appreciation of M. pneumoniae as a significant respiratory tract pathogen and the numerous new drugs in the macrolide and fluoroquinolone classes that have become available since the early 1990s have led to the performance of several trials involving adults and children with community-acquired pneumonia in which the clinical efficacies of various antimicrobials were evaluated. Many published clinical trials have been able to identify relatively small numbers of community-acquired pneumonias proven to be caused by M. pneumoniae and have usually relied upon serological diagnosis, although some recent studies have incorporated culture and/or PCR and thereby improved the numbers of microbiologically proven cases. The use of serology alone as a diagnostic measure precludes determination of whether a treatment regimen actually eradicates the organism, and thus data regarding the microbiological efficacy of any antimicrobial regimen are relatively sparse.

Newer fluoroquinolones are being used extensively for treatment of respiratory tract infections in adults since they can be used empirically to treat infections due to mycoplasmas, chlamydiae, legionellae, M. catarrhalis, and S. pneumoniae (including penicillin- and macrolide-resistant organisms); they have an acceptable safety profile; and they can be given orally once daily with favorable results. However, fluoroquinolones are not recommended for use in children due to possible toxicity to developing cartilage. Likewise, tetracyclines are not approved for use in children younger than 8 years of age. Macrolides are generally considered the treatments of choice for M. pneumoniae in both adults and children.

Table 4 summarizes several comparative and noncomparative studies in which identification of M. pneumoniae infections in adults or children with community-acquired pneumonia was specifically sought and in which the clinical and/or bacteriological response was documented for this organism. The results of these studies have generally shown that newer agents such as clarithromycin or azithromycin are as effective clinically as erythromycin, with cure rates consistently exceeding 90% when such data are available. These recent clinical trials evaluating treatment regimens for M. pneumoniae have not included untreated controls, so the cure rates for various drugs cannot account for the possibility of spontaneous resolution, which may occur in approximately 10 days (137). However, a treatment arm of one study (139) consisted of beta-lactams alone, and all 22 patients with M. pneumoniae infection identified serologically who received these agents achieved clinical cure, suggesting that these infections can indeed resolve spontaneously, since these drugs would have no effect on this organism. However, there could possibly have been another undetected pathogen involved that was inhibited by these agents, thus accounting for a favorable outcome.

Newer macrolides are generally preferred over erythromycin due to their greater tolerability, once- or twice-daily dosing requirements, and shorter treatment duration in the case of azithromycin, even though their costs are considerably greater. The extremely high potency of azithromycin against M. pneumoniae and its long half-life probably account for its ability to cure M. pneumoniae infections with very short treatment courses, and possibly even with a single 1.5-g dose, despite the relatively slow growth of the organism (367, 368). Overall, azithromycin and the investigational ketolide cethromycin (ABT-773) are the most potent drugs against M. pneumoniae in terms of MICs (436). Recently published clinical trials evaluating the investigational ketolide telithromycin alone (57) or in comparison with the fluoroquinolone trovafloxacin (336) for treatment of pneumonia in adults demonstrated that this ketolide produced clinical cure rates of 100 and 93.3%, respectively, for persons whose infection was due to M. pneumoniae.

Some recent studies evaluating fluoroquinolones for treatment of community-acquired pneumonias in adults have included diagnostic tests, primarily serology, for M. pneumoniae (138-140, 322, 326). The general conclusions of these studies are that newer fluoroquinolones such as levofloxacin and moxifloxacin are effective clinically against M. pneumoniae infections, as one might expect from their in vitro potencies. Specific treatment dosage recommendations and therapeutic regiments for M. pneumoniae infections in adults and children are provided in reference texts (433).

Relatively little data are available regarding outcomes of antimicrobial treatment of severely ill children or adults requiring hospitalization for M. pneumoniae pneumonia or treatment of immunosuppressed persons with M. pneumoniae infection. Eradication of M. pneumoniae from persons with immunosuppression can be extremely difficult, requiring prolonged therapy, even when the organisms are susceptible to the expected agents. This difficulty highlights the fact that mycoplasmas are inhibited but not killed by most commonly used antimicrobial agents in concentrations achievable in vivo.

Limited information from case reports suggests that high-dose steroid therapy may be effective in reversing neurological symptoms in children with complicated M. pneumoniae infection (175), and some clinicians recommend use of steroids in combination with an antibiotic that can penetrate the CNS, such as doxycycline or chloramphenicol (105, 208). Azithromycin and clarithromycin are commonly used for M. pneumoniae respiratory infections, and both have been used successfully in M. pneumoniae CNS infections, even though macrolides penetrate poorly into the CNS (175, 384). A recent review of steroid therapy in M. pneumoniae CNS cases suggests that it is often beneficial, particularly in severe cases. Of 14 patients with severe M. pneumoniae CNS disease, 11 (78%) were reported to have a complete or near complete recovery, a better outcome than reported for an earlier series of patients who did not receive steroids (58). The value of using steroids to treat Stevens-Johnson syndrome caused by M. pneumoniae has not been clearly established (257).

Both plasmapheresis and intravenous immunoglobulin therapy might be considered if steroid therapy is ineffective for cases of acute disseminated encephalomyelitis, which is considered to have an important immune component (384). A trial of intravenous immunoglobulin in a critically ill patient with encephalitis and M. pneumoniae pneumonia was associated with neurological improvement within 48 h of treatment (361). A patient with M. pneumoniae infection suffering from bilateral optic neuritis as well as acute Guillain-Barré syndrome recovered after plasmapheresis (325). A survey of commercial intravenous immunoglobulin therapy preparations found that most preparations had significant activity against M. pneumoniae (246).

Antibiotic therapy for persons with asthma and documented infection with M. pneumoniae or other organisms that are amenable to such treatments may prove useful. However, it should be emphasized that antibiotic administration should ideally be reserved for those asthmatic persons who are actually shown to harbor the organism.

A number of trials of prophylactic antibiotics to control outbreaks of M. pneumoniae disease have been performed, with varied success. While some earlier studies used tetracyclines (212), recent investigations used the newer macrolides. Azithromycin has been used effectively for prophylaxis against clinical infection with M. pneumoniae during outbreaks (197, 228) with a 75% protective efficacy against illnesses resulting in physician visits among hospital employees according to one study (197). Weekly oral azithromycin (500 mg) had a 64% protective efficacy based on data from serological tests against M. pneumoniae infections in U.S. marines (169). These findings support the use of prophylactic antibiotics that are highly active against M. pneumoniae in outbreak settings.