PAEDIATRIC RESPIRATORY TRACT INFECTION BY MYCOPLASME PNEUMONIA

Abstract and Introduction
Abstract

Mycoplasma pneumoniae is one of the most common agents of community-acquired pneumonia in children and young adults. Although M. pneumoniae is a small bacterium that can reproduce in an artificial culture medium and is known to be sensitive to certain antibiotics in vitro as well as in vivo, the immunopathogenesis of M. pneumoniae in the human host is not fully understood. The epidemiologic characteristics, including periodic epidemics, and some clinical characteristics of M. pneumoniae are similar to those observed in systemic viral infections. Many experimental and clinical studies have suggested that the pathogenesis of lung injuries in M. pneumoniae infection is associated with a cell-mediated immune reaction, including high responsiveness to corticosteroid therapy. This paper presents an overview of M. pneumoniae infections, with emphasis on epidemiology, pathogenesis and treatment.
Introduction

Mycoplasma pneumoniae is an important causative organism of respiratory infections in children and young adults. M. pneumoniae infections display a spectrum of symptoms and signs, ranging from asymptomatic infection to severe and potentially fatal pneumonia or extrapulmonary manifestations.[1-3] M. pneumoniae pneumonia has been reported in 10-40% of cases of community-acquired pneumonia and shows an even higher incidence during epidemics.[4-8] M. pneumoniae is a small bacterium that can be artificially cultured and is very sensitive to certain antibiotics in in vitro assays. However, the immunopathogenesis of M. pneumoniae in humans is still poorly understood. Children have a relatively well-equipped adaptive immune system at birth, but some immune functions mature during childhood. The interaction between an infectious agent and the maturing immune system may result in the appearance of different age-related phenotypes.[9] The epidemiologic characteristics, including periodic epidemics, and some clinical characteristics of M. pneumoniae infection, including the appearance of lymphopenia with anergy to tuberculin skin testing and extrapulmonary manifestations, are similar to those observed in systemic viral infections, such as measles.[10-12] Experimental and clinical evidence supports the idea that the pathogenesis of lung injuries in M. pneumoniae infection is associated with cell-mediated immunity, including high responsiveness to corticosteroid therapy.[10,13] In this paper, a brief review of infections due to M. pneumoniae is presented from the standpoints of bacteriology, epidemiology, pathogenesis, diagnosis, clinical features and therapy, with the emphasis on the epidemiology and pathogenesis of cell mediated-immunity, and corticosteroid therapy.



Abstract and Introduction
Bacteriology
Epidemiology
Pathogenesis
Clinical Manifestations
Diagnosis
Treatment
Expert Commentary
Five-year View

References
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Bacteriology

Mycoplasmas may have evolved as normal flora inside the human host, as have certain species of bacteria and viruses, living symbiotically with host cells and/or other neighboring normal flora.[3,14,15] Over 150 mycoplasma species have been discovered in animals and humans to date, but only a few of these, including M. pneumoniae, are human pathogens. After the discovery of M. pneumoniae as an agent of atypical pneumonia, M. pneumoniae is now recognized as a small bacterium that can be grown and isolated in artificial culture medium.[16,17] It is exceptionally small in size, at 1-2 µm long and 0.1-0.2 µm wide, compared with the 1-4 µm length and 0.5-1 µm width of a typical bacillus. Accordingly, the volume of M. pneumoniae is approximately 5% of that of a typical bacillus and is not much larger than the large Poxviridae viruses (diameter: 0.25-0.4 µm). Since M. pneumoniae has no cell wall, it is insensitive to β-lactam antibiotics and is not stained by Gram staining. M. pneumoniae has one of the smallest-known bacterial genomes, at approximately 0.8 Mbps and with only 688 genes. This genomic capacity represents less than 20% of other bacterial genomes. As a result, M. pneumoniae is restricted in its biosynthetic capability, required not only for the essential replication of compounds but also for the production of pathogenic substances, such as exotoxins.[18,19] These genomic limitations make M. pneumoniae a fastidiously growing bacterium, requiring the presence of a variety of substances, both in the host and in in vitro culture systems. In contrast to viruses, M. pneumoniae is primarily a mucosal pathogen, residing extracellularly on epithelial surfaces, although the bacterium can survive in the cytoplasm of certain cell lines.[20] The colonies of M. pneumoniae in in vitro culture systems appear approximately 2-3 weeks after inoculation, whereas adding feeding cells into the culture system can accelerate the colony formation of M. pneumoniae within a week.[14,20] It is unknown whether M. pneumoniae is supported by the host cells and/or the neighboring normal flora for its colonization and replication. The host and normal flora in a variety of regions, including the airway and gut of the host, are closely linked and the normal microflora of the enteric mucosal immune system is now recognized as an important part of the host immune system.[21-23] As well as the enteric mucosal immune system, microflora distribution in the respiratory tract may also affect the local mucosal immunity of the respiratory system against invading pathogens, including M. pneumoniae.[24]

The existence of asymptomatic patients, a variety of clinical phenotypes and the varied incubation period of M. pneumoniae infection (1-3 weeks) indicate that the initial inoculated dose of the organism, the immune status of the host and, possibly, the distribution of normal flora in the respiratory tract may be important for the establishment of M. pneumoniae infections.

Abstract and Introduction
Bacteriology
Epidemiology
Pathogenesis
Clinical Manifestations
Diagnosis
Treatment
Expert Commentary
Five-year View

References
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Epidemiology

The incidence of overt M. pneumoniae infection may be dependent upon differences in the background rate of infection in a community (herd immunity), in host immunity and, possibly, in the organism's virulence. M. pneumoniae infection is endemic in the larger communities of the world, with cyclic epidemics lasting several months to years.[1-3] Although there is some geographical and temporal variation, long-term epidemiologic studies of M. pneumoniae in a variety of populations have indicated that M. pneumoniae epidemics periodically occur every 3-7 years.[25-31] In the USA, Foy et al. reported that M. pneumoniae infection was endemic without significant seasonal fluctuations and between 1963 and 1974, two epidemics occurred in 1967 and 1974.[26] In Europe, a retrospective study in Denmark examining a 50-year period from 1945 to 1995 revealed a 4.5-year epidemic periodicity from 1958 to 1972.[28] Similar findings have been reported from other regions of Europe.[29,30] In Japan, epidemic peaks recurred regularly at 3-4-year intervals from 1979 to 1999.[31] In Korea, approximately 3-year cycles have been observed from the mid-1980s to the early 2000s.[12,32,33] Interestingly, several long-term studies indicate that since the 1990s, this strict epidemic pattern has been interrupted, with a trend toward a more endemic occurrence being reported.[28,30,31]

Epidemiologic characteristics of an infection, including age distribution and variation of cyclic intervals, may result from environmental factors such as socioeconomic status and population density. The cyclic occurrence and changes in age distribution of M. pneumoniae epidemics suggest that the epidemiologic characteristics of M. pneumoniae infections are similar to those of viral diseases, such as measles, in the prevaccination era. The susceptible groups of the population, especially the young age group, would accrue during the 3-4 years of the interepidemic period and the infection of this age group may be responsible for the reservoir of epidemics.[34,35] As time elapsed, the susceptible groups would become younger and most of the adult generation would acquire immunity to M. pneumoniae. Populations that have experienced longer cyclic epidemics may have a broader age distribution of susceptible groups, including more adult patients.

Studies examining the seroprevalence of M. pneumoniae IgG antibodies have shown progressively greater rates with increasing age.[36-38] The study reported by Brunner et al. documents that the detection rates of an IgG antibody to M. pneumoniae are 28% for infants aged 7-12 months, 55% for infants from 13 to 24 months of age, 67% for children from 25 to 60 months of age and 97% for those over 17 years of age, although this antibody is suggested not to be protective against the disease caused by M. pneumoniae.[36] However, the pattern of M. pneumoniae antibodies in groups of children indicate that mycoplasma infections begin in early childhood, similar to common viral infections. Recent studies for selected patients with M. pneumoniae infection indicate that young children (<5 years of age) also have a predisposition to M. pneumoniae, as well as school-aged children.[7,39,40] The interaction between an infectious agent and the maturing immune system may account for the age-related differences in the clinical and laboratory features of the infection. For example, hepatitis A, severe acute respiratory syndrome (SARS) due to corona virus and scrub typhus are largely less symptomatic in younger children and produce more severe clinical manifestations in older children and adults.[41-43] In addition, there are several early-childhood immune-mediated disorders that have strict age restrictions, mainly between 6 months and 4 years of age, with a self-limited clinical nature; these include transient erythroblastopenia of childhood,[44] autoimmune neutropenia of infancy,[45] childhood immune thrombocytopenic purpura,[46] transient hypo-γ-globulinemia of infancy[47] and Kawasaki disease.[48] These disorders may be related to infections and provide evidence to suggest that the maturing immune system in early childhood may be involved in the phenotype of infectious and immune-mediated disorders.[48] In M. pneumoniae infections, younger children appear to have milder or subclinical infections and older children (>5 years of age) are more likely to develop pneumonia than younger children when they are infected with M. pneumoniae.[1-3] Since the majority of clinical studies for M. pneumoniae epidemics are based on pneumonia patients, the real prevalence of M. pneumoniae infection in younger children may be an underestimate.[39,40] Although recurrence of M. pneumoniae infection has been reported in adult groups, with increasing incidence in the elderly,[49,50] the immunopathogenesis of mycoplasma pneumonia in the elderly requires further study. The pattern of age distribution might also be changing, for example, through socio-economic development; although, there have been few studies of this subject. Indeed, in Korea, the peak age of incidence in the most recent mycoplasma pneumonia epidemic appears to be falling, with low incidence in the adult group.[12,32,33,51] The changes and differences in the epidemiologic pattern among the population seem to be a natural phenomenon.

Since M. pneumoniae has a small genome, emergence of new subtypes of this organism that induce subtype-specific immunity in the host may be restricted. Two subtypes of M. pneumoniae, based on the P1 protein, have been documented.[52] It has been reported that one subtype of M. pneumoniae was predominant in an outbreak and the subtype was changed in other outbreaks but, in endemic periods, both of the subtypes were detected in the human population at a low level.[53,54] Recently, macrolide-resistant M. pneumoniae strains have been detected; the strains are mutated in nucleic acid sequences of the 23S rRNA or ribosomal proteins.[55] These strains have been discovered mainly in Japan and, possibly, in Korea, but not yet in Western countries. Currently, it is unknown whether the strains can spread worldwide and affect macrolide therapy. The infected children with macrolide-resistant strains showed significantly prolonged duration of fever compared with children with macrolide-sensitive M. pneumoniae; but they showed defervescence with initial macrolide therapy.[56]
Pathogenesis

M. pneumoniae can be cultured in tissue cultures, organ cultures, chick embryo lungs and small animals, with no overt injury to host cells in these instances.[57] From culture-based studies, it is clear that the first step in the pathogenic process of M. pneumoniae involves cytoadherence between M. pneumoniae and the ciliated respiratory epithelium. However, the mechanism for lung injury and progressive pneumonia is still unclear.[3,14,15] M. pneumoniae has an elongated, snake-like structure with an attachment tip, in which a complex of adhesion and interactive adhesion-accessory proteins is localized. These proteins decide the structural and functional motif at the tip and permit attachment to the target ciliated respiratory epithelial cells. It is believed that the virulence of Mycoplasma spp. depends on the genetic differences of adhesion- and specific cytoadherence-related proteins.[58,59] After adherence, M. pneumoniae numbers need to increase in order to establish an infection, involving colonization and further inflammation in human tissues, if the components of M. pneumoniae are responsible for the cause of lung inflammation. Animal model and in vitro cell line studies have revealed that Mycoplasma spp. preferentially attach to ciliated respiratory epithelial cells, inducing ciliostasis and eventual sloughing of the epithelial cells, caused by oxidative substances or other factors, including toxins and gliding motility.[60-62] Recently, an ADP-ribosylating and vacuolating cytotoxin (community-acquired respiratory distress syndrome toxin) was identified from M. pneumoniae and it shares homology with other bacterial toxins, including pertussis toxin. This toxin is cytopathic and highly immunogenic, and may be responsible for symptoms of chronic cough, such as those of whooping cough in children.[61]

The host immune responses to etiologic agents and the pathogenesis of pneumonia may be different according to etiologic agents. Patients with typical bacterial pneumonia manifest more toxic clinical symptoms, such as leukocytosis, neutrophilia and bacteremia.[63] Bacterial exotoxins, components of the bacterial wall (endotoxins), oxygen radicals, proteolytic enzymes or cytokines from activated neutrophils and phagocytes may induce lung injury and these cells are also observed predominantly in pathologic tissues.[64,65] In systemic viral infections, such as measles and SARS, which mainly manifest respiratory symptoms, patients display leukopenia with lymphopenia and pathologic findings show a predominance of mononuclear cells, especially T-cell infiltrations, in severely affected cases.[66,67] It is well known that cell-mediated immunity has a crucial role in full recovery from such viral infections.[68,69]

In contrast to the direct cytopathic effect on lung injury due to M. pneumoniae, the involvement of the cell-mediated immune reaction has been proposed. In a cell-mediated immune reaction, various pro-inflammatory cytokines, including IL-2, other reactive substances and immune cells, such as antigen presenting cells and T cells, may be involved in the immune reaction to pathogens.[70-72] In one murine model, M. pneumoniae-infected mice produced cytokines and chemokines in bronchoalveolar lavages (BALs) within a week following intranasal inoculation. Some cytokines are correlated with the histopathologic score of lung lesions, anti-M. pneumoniae IgG levels and lung function (assessed by respiratory plethymography).[72] Elevated levels of various cytokines, including IL-1, IL-5, IL-6 and IL-18, in sera or pleural effusions of patients with M. pneumoniae pneumonia have also been reported.[70,71,73,74] It has been recognized that Toll-like receptors (TLRs) have crucial roles in the early innate immune reaction and inflammatory responses by the host against invading pathogens.[75,76] The lipoproteins derived from mycoplasmas, such as macrophage-activating lipopeptide 2, induce cytokines and chemokines from macrophages via TLRs. These cytokines recruit immune cells, including T cells, leading to further production of cytokines, and may be involved in the inflammatory responses toward M. pneumoniae infection.[77,78]

Phenomena suggesting depressed cell-mediated immunity, including transient anergy to purified protein derivative (PPD), have been observed in adults as well as children following M. pneumoniae infection.[79,80] Adult patients with severe pulmonary lesions (airspace consolidation) display higher PPD negativity over patients with milder pulmonary lesions (nodular opacities), when assessed by computed tomography.[81] Analysis of a 2003-2004 M. pneumoniae pneumonia outbreak reported that children with severe pulmonary lesions (segmental or lobar patterns) showed significant lower white blood cell and lymphocyte counts than children with milder pulmonary lesions (brochopneumonic patterns). In addition, the severity of pulmonary lesions in M. pneumoniae pneumonia correlates inversely with the lymphocyte count.[10,82] These findings were also observed in a 2006-2007 outbreak and an age-matched study of both pneumonia groups did obtain similar results (Lee K-Y, Hwang J-Y, Yim J-W, Yoon Y-S, Unpublished Data). Lymphopenia with possibly depressed cell-mediated immune testing has been described in systemic viral infections, including measles, chicken pox and SARS.[83-85] In addition, the reported adult patients of M. pneumoniae pneumonia consistently exhibit lymphopenia.[86-89] These findings may be a secondary immune phenomenon of infections that are associated with a cell-mediated immune reaction.

In animal model studies, the establishment of pneumonia appears at the end of the second week following intranasal inoculation.[57] The animals deprived of the cell-mediated immunity induced by thymectomy or irradiation show less-severe pulmonary lesions than the controls,[90] although passive transfer of sensitized lymphocytes from vaccinated animals failed to prevent respiratory diseases of the recipient animals.[91] Administration of IL-2 or cyclosporine A affects periarteriolar lymphocyte infiltration and pulmonary lesions in mycoplasma-infected mice.[92] Animals previously sensitized with experimental vaccines demonstrate strong immune reactions with live mycoplasmas as well as with a component of mycoplasma, signifying that immunological memory of the host from experimental vaccines is intact, with inclusion of the cell-mediated immune response.[93,94]

Pathologically, infected animals initially show a large number of lymphocytes, mainly CD4+ T-cell infiltration around the peribronchiolar and periarteriolar regions and, later, phagocytes appear in the bronchiolar lumens.[95,96] Similar results have been reported in human pathologic findings from patients with severe mycoplasma pneumonia, that is, the infiltration of peribronchiolar lymphoplasmocytic cells and the exudation of prominent neutrophils in the bronchiolar lumens (cellular bronchiolitis).[97,98] The pathologic finding of lymphocyte dominance may be similar to findings for other autoimmune diseases, which may also demonstrate the pathogenesis of cell-mediated immunity.

Direct evidence of M. pneumoniae in pathologic tissues is limited; although, positive PCR has been acquired from some extrapulmonary lesions or transthoracic lung aspirates.[3] M. pneumoniae are neither found beneath nor inside respiratory epithelial cells in animal models of infection[95] and it may be difficult to find material associated with M. pneumoniae from electronic microscopic examination[98] and from lung aspirate specimens of children with community-acquired pneumonia.[99]

Clinically, corticosteroid treatment of severe mycoplasma pneumonia induces rapid improvement of the clinical symptoms and pulmonary lesions in adults[13,87,88] and children.[10,100] One study reported that 15 children with severe progressive mycoplasma pneumonia who were nonresponsive to antibiotics responded to additional corticosteroid (prednisolone) therapy. In this series, more than half of the children showed negative antimycoplasma antibody or cold agglutinin titers at presentation, suggesting that the humoral immune response may not be associated with the pathogenesis of M. pneumoniae pneumonia.[10] Recently, the use of corticosteroids has demonstrated clinical benefits for the treatment of severe pneumonia caused by corona virus (SARS) and avian influenza infection. The pathogenesis of pulmonary lesions in these viral infections, as well as in M. pneumoniae infection, may be associated with a hyperimmune reaction of the host, such as cytokine dysregulation, toward the agents.[85,101,102]

Some clinical characteristics of M. pneumoniae pneumonia also help to postulate the pathogenesis of lung injury due to M. pneumoniae. Children under the age of 5 years show milder nonpneumonic respiratory tract symptoms, whereas older children and young adults develop pneumonia when they are affected by M. pneumonia. The pneumonic lesions and extrapulmonary lesions appear, in some cases, within several days to 1 week after initiation of constitutional symptoms, including fever, sore throat, malaise and cough.[1-3] A variety of clinical phenotypes containing pleomorphic pneumonic lesions is a characteristic of M. pneumoniae infection, akin to certain immune-mediated disorders.

M. pneumoniae may remain in the respiratory tract for long periods of time after primary infection[34,35,103] and a similar phenomenon has also been found in other bacterial and viral infections, such as salmonellosis and hepatitis B.[104,105] In addition, the nasopharyngeal carriage rate of Streptococcus pneumoniae, the most common pathogen of bacterial pneumonia, has been reported to range from 15 to 50% in healthy children.[106,107] Chronic carriers of these infections, without clinical signs and symptoms, may act as a reservoir for the infection, facilitating transfer to other susceptible hosts. From the point of view of pathogens, a successful outcome is the establishment of a symbiotic life with the host, although the mechanism of inducing immune tolerance is unknown. The pleomorphic pulmonary lesions, including the involvement of other lung areas, lobar consolidation and pleural effusion seen in mycoplasma pneumonia suggest that other mechanisms for the spread of M. pneumoniae may exist; although, it is believed that M. pneumoniae, as well as some virus particles, spread along bronchial trees and invade alveoli.[108]

To summarize, the pathogenesis of M. pneumoniae infection is postulated to occur as follows: the inducers (components of M. pneumoniae and/or other inflammatory mediators) of the cell-mediated immune reaction are initially produced at the primary inflammatory sites of the upper respiratory tract during the incubation period. This process may be conducted by the innate immune system as a first-line defense mechanism of the host. The substances may then spread directly or, more commonly, via the systemic circulation and reach the lower respiratory tract cells, which is the main target organ, and, possibly, other organs that are responsible for extrapulmonary manifestations, such as skin rash. As for removal of these substances, the adaptive immune reaction may be operated by immune cells, mainly T cells, various inflammatory cytokines and counterinflammatory cytokines, together with the initiation of systemic symptoms. Since this immune response may be dependent on antigenic doses, distribution of target organs and the effectiveness of the immune system (immune maturation), this process may be expressed as clinical phenotypes from asymptomatic to severe pneumonia with difference of severity due to age. During this process, the host acquires immunologic memory and certain pathogens may acquire immune tolerance. The disturbances of adaptive immune reactions, expressed as 'over-regulation' or 'misregulation', may be associated with disease progression or extrapulmonary (autoimmune) manifestations.

Abstract and Introduction
Bacteriology
Epidemiology
Pathogenesis
Clinical Manifestations
Diagnosis
Treatment
Expert Commentary
Five-year View

References
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Clinical Manifestations

Pneumonia is the most significant clinical presentation of M. pneumoniae infection, although pharyngitis, otitis media and bronchitis have been observed clinically in children and adults. Since clinical manifestations reflect the host immune response to invading pathogens, younger children (<5 years of age) show milder or nonpneumonic respiratory tract symptoms, whereas older children and young adults often develop pneumonia when they are infected with M. pneumoniae.[1-3] The severity of pneumonia may also represent the intensity of the host's immune reaction; older groups who have a more mature immune system may be prone to the development of more severe pulmonary lesions when pneumonia develops. We found that older children (>6 years of age) had a more severe pneumonic pattern (segmental or lobar patterns) than children under the age of 5 years during two recent outbreaks.[10,82]

In the case of pneumonia patients, illness presents with relatively acute onset of fever, sore throat, malaise and headache, but coryza is not usual.[109] Cough and radiographic evidence of pneumonia appears 3-7 days after onset of constitutional symptoms. Clinical symptoms and physical findings of suspected pneumonia, including wheezing, rales and rhonchi, are often poorly correlated with the radiographic finding of pneumonia (‘walking pneumonia'). The clinical course in untreated pneumonia patients is quite variable, but most symptoms will resolve within 2-4 weeks, even in progressive pneumonia.[1-3,10] The rare fatality of M. pneumoniae pneumonia in adults has been debated on the selection or addition of macrolide antibiotics for community-acquired pneumonia treatment.[110,111]

Some younger children are likely to manifest cough and wheezing as well as show abnormal lung function after M. pneumoniae infection.[112] In addition, M. pneumoniae, as well as viral agents, is associated with the exacerbation of symptoms in asthmatics.[113] Children with immunodeficiencies or sickle cell disease have a more severe clinical course when infected with M. pneumoniae.[114]

Extrapulmonary involvement of M. pneumoniae infection has been defined in neurologic, dermatologic, hematologic and other immunologic disorders. Skin rash is most frequently seen (in ~10% of infected children) and CNS complications, such as meningoencephalitis and Guillain-Barré syndrome, are rarely seen.[115,116] M. pneumoniae is the most common agent of pediatric encephalitis.[117] The onset of extrapulmonary manifestations occurs from 2 days to several weeks following the initiation of illness; however, no respiratory symptoms have been noted in some patients. Although M. pneumoniae has, in limited cases, been discovered at various extrapulmonary sites via PCR or culture, the majority of extrapulmonary manifestations may result from the disturbance of the adaptive immune reaction. Cell-mediated and humoral immunity, including tissue-specific T cells and autoantibodies, may be involved in the pathogenesis of extrapulmonary manifestations of M. pneumoniae.[115,116]

The radiologic findings of mycoplasma pneumonia are varied, usually presenting with interstitial and bronchopneumonic patterns similar to viral pneumonia; although, in some instances, segmental and/or lobar pneumonia patterns with pleural effusion similar to bacterial pneumonia are evident.[10,118] Viral pneumonia, including respiratory syncytial virus, is predominantly seen in younger children and its radiographic findings are mainly interstitial and/or bronchial (bronchial wall thickening, peribronchial shadowing and patchy pulmonary shadowing, with lobar or sublobar consolidation and, rarely, pleural effusion).[119,120] Some bacterial pneumonias display characteristic findings according to the etiologic agent. For example, the radiographic findings of typical pneumococcal pneumonia in children and adults show clear lobar consolidation with frequent pleural effusion. Staphylococcal pneumonia in infants is highly toxic and pulmonary lesions rapidly progress to empyema and pneumatoceles. Considering similar radiolographic findings among atypical pneumonias,[119-122] radiological findings alone cannot discriminate between M. pneumoniae and other etiologic agents.[118,122,123] With decreasing incidence of a typical pneumococcal pattern in developed countries, recent comparative studies have reported that the clinical, laboratory and radiological findings between atypical pneumonias, including mycoplasma pneumonia and bacterial pneumonias, are similar in children and adults.[124,125]

As for laboratory results, there are no specific findings for M. pneumoniae infection; the white blood cell counts are within normal levels but lymphopenia appears in severe cases. C-reactive protein may be related to the severity of lung injury at the time of examination.Diagnosis

Early diagnosis of an infectious disease is important for treatment modality, including selection of proper antibiotics. A variety of serologic assays and PCR techniques have been developed for the diagnosis of M. pneumoniae infections.[2,3] However, each test has some limitations because of the absence of IgM antibodies in the early stage of infection and the existence of normal carriers of M. pneumoniae in clinical settings.
Serology

During the past few decades, a number of commercial serologic assays for M. pneumoniae have been developed. The serologic assays for the detection of IgM and IgG include the complement fixation assay (CFA), microparticle agglutination assay (MAA, including Serodia Myco II), ELISA, enzyme-linked immunoassay (EIA) and indirect immunofluorescence assay. The antigenic compositions used in the serologic assays are slightly different according to the manufacturers. The antigenic compositions used in kits can constitute crude whole-cell antigens, including glycolipids, or more purified proteins, including P1 membrane proteins.[2,3] Most immunoassays detect IgG and IgM but some assays, such as CFA and MAA, are known to detect mainly IgM. CFA has been used as a routine and standard serologic test for comparison with other serologic assays. It has been documented that the sensitivity and specificity of some serologic tests are compatible to CFA, if the study is properly designed.[2,3] However, it is still doubtful as to whether the sensitivity of the detection of M. pneumoniae antibodies is similar among various serologic assays.[38,127,128] Beersma et al. recently evaluated the 12 commercially available EIAs (IgM and IgG) and CFA, using M. pneumoniae DNA detection by real-time PCR as the 'gold standard' for adult patients with lower respiratory tract infections due to M. pneumoniae.[128] The IgM detection rates of the kits in the acute stage ranged from 16 to 42%, but IgG detection rates ranged from 37 to 89% in the patient group. In the control group with other serologically proven infections, the rates of IgM and IgG detection ranged from 0 to 51% and from 36 to 93%, respectively. The IgG seroprevalence in both groups was similar. Since the appearance of IgM to pathogens may precede IgG in M. pneumoniae infections as well as other primary infections, paradoxically, the immunoassays that show lower detection rates of IgG in the acute stage of infection may be more sensitive diagnostic tools. This finding may be important for the diagnosis of adult patients who are IgM negative, should reinfection occur. Sensitivity of the IgM EIAs is related to the duration of illness; positive rates of IgM ranged from 7 to 25% within 1-6 days, 31 to 69% within 7-15 days and 33 to 87% within 16 or more days after initiation of illness.[128] These observations indicate that the seroconversion rate of IgM during the early phase is greater than 50% within 1-2 weeks following initiation of disease. We found similar findings in children with mycoplasma pneumonia diagnosed by a MAA assay (Serodia Myco II) and the cold agglutinin titers of paired samples obtained at admission and discharge (mean 6 days apart). Approximately 30% of children with mycoplasma pneumonia presented as seroconverters, that is, IgM negative to IgM positive, for both assays during admission and they tended to show more severe pulmonary lesions than those who had increased antibody titers during admission. The period of seroconversion in some patients was more than 1 week (Lee K-Y, Hwang J-Y, Yim J-W, Yoon Y-S, Unpublished Data). If investigators select the M. pneumoniae patients based on IgM-positive titers at presentation, they may omit patients who would progress to more severe clinical symptoms. Negative antibody testing in the early stages of infectious disease has been well documented.[83,85,129] In the case of measles, approximately 20% of children with measles were antimeasles IgM-negative at presentation, with no clinical and laboratory differences compared with IgM-positive patients.[129]

The detection of cold agglutinins is an old serologic test and is nonspecific for M. pneumoniae. Cold agglutinins usually appear by the end of the first week of initiation of illness and disappear by 2-3 months. The pathogen-specific IgM antibodies of various infectious diseases may be similar to this pattern of cold agglutinins. Although cold agglutinins are observed in a number of infections, such as Epstein-Barr virus infection,[130] this assay may be used as an adjuvant serologic test not only for diagnosis of M. pneumoniae infection but also as a base of sensitivity for diagnostic kits, even at the present time. Both earlier and more recent studies have indicated that the detection of cold agglutinins (IgM antibodies) in M. pneumoniae infection has a relatively higher sensitivity (~80%) when seroposivities of the serologic assay and the cold agglutinins in patients with M. pneumoniae pneumonia are compared, in particular, in mycoplasma pneumonia outbreaks.[131-133] We also found similar patterns of seroconversion rates and increased antibody titers between cold agglutinins and mycoplasma antibodies.[10] In addition, in children with lower respiratory tract infections during mycoplasma pneumonia outbreaks, there were few patients who showed only cold agglutinin changes (Lee K-Y, Hwang J-Y, Yim J-W, Yoon Y-S, Unpublished Data). Interestingly, in our preliminary study of 50 pneumonia patients during a recent mycoplasma pneumonia outbreak, the positive rate of an EIA kit (IgG and IgM) that was being considered as a replacement for the Serodia Myco II in our institution was significantly lower than those of even cold agglutinins.[134]

Confirmation of a greater than fourfold increase in IgG antibody titers during a 4-6-week interval has been recommended for serologic diagnosis of infectious diseases, including M. pneumoniae infection. In children, a positive IgM test in the acute stage can be regarded as indicative of recent infection. However, because of a higher rate of seroconverters and the existence of false-positives,[40] the paired examination of all children with pneumonia may be mandatory. With confirmation of seroconversion, the appearance of increased IgM titers, over two- to fourfold, with corresponding cold agglutinin titers signifies an acute M. pneumoniae infection within 1 week, in the majority of patients.[10] For severely ill, hospitalized patients, repeated examination within a short interval may help with relatively early diagnosis.[10] Although serologic assays have some handicaps for the early diagnosis of M. pneumoniae infection, the assays truly represent the host immune responses to M. pneumoniae and cannot be replaced with other assays.[135]
Polymerase Chain Reaction

M. pneumoniae may spread through the respiratory tract after colonization. The existence of M. pneumoniae in various lesions of the respiratory tract has been reported through culture methods and nucleic acid-amplification techniques.

Development of molecular-based PCR assays has replaced the culture method for detection of M. pneumoniae, which is rarely performed in clinical settings.[136,137] Owing to limitations of early diagnosis by serologic assays, especially in adult groups that have no IgM response, PCR assays are currently widely used for the rapid diagnosis of respiratory infections. From the first detection of M. pneumoniae DNA in animals and simulated clinical samples in 1989,[138] numerous in-house PCR assays to detect M. pneumoniae have been developed. The target genes for detection include the ATPase operon, P1 and 16S rRNA genes. PCR assays have high specificity and relatively high sensitivity in children with M. pneumoniae infection, when compared with serologic assays.[139,140]

The materials for diagnosis of respiratory infections - sputum, a throat swab, nasopharygeal aspirates and BALs - consist of various agents, including the actual pneumonia-inducing pathogen. If the pathogenesis of pneumonia is associated with the host immune reaction, colonization itself may not signify a pathogen-mediated systemic infection. Some Mycoplasma species, possibly M. pneumoniae, may be part of the normal flora in the respiratory tracts of healthy individuals.[3,14,103] Also, M. pneumoniae can persist for a long time after the initial infection, especially in children involved in an outbreak.[34,35] In PCR assays, including multiplex PCR, other viral or bacterial pathogens are also detected together with M. pneumoniae.[6,141] Owing to the higher carriage state of bacterial pathogens, including S. pneumoniae and Haemophilus influenzae, in healthy children,[106,107] it is unclear which agent is predominantly responsible for disease expression.

PCR results are affected by the samples obtained from different respiratory tract sites[142] and by diverse technical errors.[136] In addition, curiously, given the length of time the technique has been in use, there is no standard method for performing PCR. Despite these limitations, the PCR assay for M. pneumoniae is a useful tool for early diagnosis, especially in outpatient clinics.



Abstract and Introduction
Bacteriology
Epidemiology
Pathogenesis
Clinical Manifestations
Diagnosis
Treatment
Expert Commentary
Five-year View

References
Sidebar: Key Issues

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Treatment

Most M. pneumoniae infections are mild and self-limiting, without the need for specific treatment. However, earlier double-blinded controlled studies based on young adult groups have provided evidence that antibiotic therapy with tetracyclines or macrolides is associated with more rapid resolution of fever, reduced hospitalization and less-severe cough.[143-145] M. pneumoniae is highly sensitive to antibiotics, such as macrolides, tetracyclines and quinolones, but is not affected by β-lactams (penicillins and cephalosporins) in in vitro assays. Since tetracyclines have associated risks, such as teeth staining, and quinolones may damage growing cartilage, only macrolides are recommended for use in children with M. pneumoniae infection (recommended macrolides include erythromycin, clarithromycin, roxithromycin and azithromycin). Comparative studies between macrolides have demonstrated no difference in therapeutic effect among macrolides in children.[146,147] Recently, one study in Japan reported that children infected with macrolide-resistant M. pneumoniae had significantly longer fever duration than those with macrolide-sensitive M. pneumoniae (9.3 vs 5.5 days; p = 0.02), when treated with macrolides.[56] However, this report did not present other comparisons of clinical symptoms, such as duration of cough or progression of pneumonia, and the age distribution of the groups may differ. Despite the increase of resistant strains, there are few reports on apparent treatment failure or progression of illness in patients with M. pneumoniae infections. One might anticipate that the immunomodulatory effect of macrolides or the self-limiting nature of the illness is responsible for this phenomenon.[56,148]

On the other hand, together with the unknown pathogenesis of pneumonia, there is still controversy concerning the effects of antibiotics on M. pneumoniae-mediated pneumonia.[149,150] Several studies, including a Cochrane database study, have reported no differences in the therapeutic outcome of children with lower respiratory tract infections due to M. pneumoniae, randomized to macrolides or nonmacrolide antibiotics.[150-152] The response to antibiotics, including macrolides, in some patients with M. pneumoniae pneumonia tends to be more delayed than expected, despite M. pneumoniae being extremely sensitive to macrolides in vitro.[6,10,56,153] Moreover, occasionally, M. pneumoniae pneumonia in previously healthy children and young adults can progress to severe and fatal pneumonia, despite appropriate antibiotic therapy, including macrolides.[10,86,88,100,154] For this small group of patients, additional corticosteroid treatment can induce rapid improvement of clinical symptoms and pulmonary lesions in adults and children.[10,86,88,100] Corticosteroids have many modes of action as immunomodulatory and anti-inflammatory drugs for use in almost all immune-mediated diseases. Corticosteroid treatment of extrapulmonary manifestations of M. pneumoniae infection, including CNS involvement, is also beneficial for early recovery from complications.[115,116] Although corticosteroid therapy is generally avoided for infectious diseases, it has been used as an adjunctive therapy in severe bacterial infections, such as typhoid fever and tuberculous meningitis,[155] and viral diseases, including SARS.[101] Given that the pathogenesis of M. pneumoniae infection is immune mediated, both in terms of pneumonia and extrapulmonary manifestations, an immunosuppressive therapy would be valid for selected patients with M. pneumoniae infection.

As an alternative treatment modality for immune modulation, the use of intravenous immunoglobulin (IVIG) as an indication of immune-mediated diseases has been extended.[156,157] Recently, IVIG has been used in extrapulmonary manifestations of M. pneumoniae infection, including encephalopathy and hemolytic anemia.[158,159] IVIG may also be useful for the treatment of severe M. pneumoniae infections in patients for whom steroids remain a contraindication. We experienced an interesting case, showing a direct IVIG effect on pulmonary lesions. A 5-year-old girl who had macrolide-nonresponsive progressive pneumonia and suspected carditis was treated with IVIG at a dose of 1 g/kg for 2 days. After the first-dose infusion, she became afebrile and pulmonary lesions halted. After the second-dose infusion, the improvement in pulmonary lesions and clinical signs of carditis were noted (Lee K-Y, Hwang J-Y, Yim J-W, Yoon Y-S, Unpublished Data). It also suggested that the progression of M. pneumoniae pneumonia is associated with a hyperimmune reaction of the host but this anecdotal therapy could not be used for patients with M. pneumoniae pneumonia alone.

The use of corticosteroid therapy for acute respiratory distress syndrome, especially in adult patients, is controversial.[160] Since immunological pathogenesis of patients with acute respiratory distress syndrome may be different according to underlying diseases, an early, short-term empirical use of corticosteroids or IVIG may help to determine further treatment modality. Severe M. pneumoniae pneumonia is occasionally associated with not only functional lung changes[72,112,113] but also anatomical complications, such as Swyer-James syndrome.[161,162] Trials to reduce the severity and morbidity of the disease will be mandatory.



Abstract and Introduction
Bacteriology
Epidemiology
Pathogenesis
Clinical Manifestations
Diagnosis
Treatment
Expert Commentary
Five-year View

References
Sidebar: Key Issues

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Expert Commentary

Since M. pneumoniae infection is usually a mild disease, healthcare administration has paid this disease less attention. The prevalence of infection and the age of acquisition of M. pneumoniae, as well as other infectious diseases might change with living circumstances and socio-economic development. In addition, the severity of infectious diseases seems to be changing with environmental factors. Since clinical manifestations of M. pneumoniae infection may be associated with immune maturation of the host, physicians should pay more attention to older children and young adults, independent of whether severe or progressive pulmonary lesions develop. Long-term epidemiologic and clinical studies in a given population, including subtype identification, will help to improve our understanding of the clinical and pathogenic characteristics of M. pneumoniae infection. Although diagnosis of M. pneumoniae infection at the acute stage is limited, the paired-sample serologic and PCR assays are needed for definitive and rapid diagnosis. The pathogenesis of M. pneumoniae pneumonia may be associated with cell-mediated immunity, similar to some viral infections, such as measles and SARS. Immune modulators, including corticosteroids, may help to reduce the severity and morbidity of M. pneumoniae infection in severe antibiotic-nonresponsive patients. Comprehensive approaches, including the innate immune system with normal flora, adaptive immune system and virology, may be needed to resolve the pathogenesis of M. pneumoniae infection.

Five-year View

Basic research into the pathogenesis of M. pneumoniae and the accuracy of diagnostic tools that use PCR and serology, augmented by knowledge obtained from the complete genome sequence, will progress. In addition, new subtypes, including macrolide-resistant strains, will be introduced into epidemiologic and clinical studies. Study of the effects of new antibiotics and immune modulators on pulmonary and extrapulmonary manifestations due to M. pneumoniae, as well as new approaches for vaccine development, will be introduced. This article is expected to aid future studies into the pathogenesis of M. pneumoniae infections.