Mycoplasma pneumoniae: A significant but underrated pathogen in paediatric community-acquired lower respiratory tract infections

Abstract

Lower respiratory tract infections are considered a common cause responsible for morbidity and mortality among children, and Mycoplasma pneumoniae is identified to be responsible for up to 40 per cent of community-acquired pneumonia in children greater than five years of age. Extrapulmonary manifestations have been reported either due to spread of infection or autoimmune mechanisms. Infection by M. pneumoniae has high incidence and clinical importance but is still an underrated disease. Most widely used serologic methods are enzyme immunoassays for detection of immunoglobulin M (IgM), IgG and IgA antibodies to M. pneumoniae, though other methods such as particle agglutination assays and immunofluorescence methods are also used. Detection of M. pneumoniae by nucleic acid amplification techniques provides fast, sensitive and specific results. Utilization of polymerase chain reaction (PCR) has improved the diagnosis of M. pneumoniae infections. Besides PCR, other alternative amplification techniques include (i) nucleic acid sequence-based amplification, (ii) Qβ replicase amplification, (iii) strand displacement amplification, (iv) transcription-mediated amplification, and (v) ligase chain reaction. Macrolides are used as the first-line treatment in childhood for M. pneumoniae infections; however, emergence of macrolide-resistant M. pneumoniae is a cause of concern. Development of a safe vaccine is important that gives protective immunity and would be a major step in reducing M. pneumoniae infections.

Lower respiratory tract infections (LRTIs) are considered a common cause liable for morbidity and mortality among children1. Many studies have addressed the problems of diagnosis of LRTIs and its management. In children over five years of age, 40 per cent community-acquired pneumonia (CAP) cases are caused by Mycoplasma pneumoniae2. Increase in CAP attributable to M. pneumoniae may occur many times during epidemics which occur at an interval of 4-7 yr because of waning of herd immunity and introduction of new subtypes into the population3.

The correct identification of M. pneumoniae infections is vital for prescription of the appropriate therapy since, based on clinical signs and symptoms, detection of M. pneumoniae infection is not possible. In only a minority of cases, specific aetiologic diagnosis of M. pneumoniae infection is established4. Important reasons for underreporting are scarcity of clinical and chest X-ray features, relative unavailability of quick and specific laboratory techniques and the difficulty in culture of this infective agent in the laboratory. In most such cases, empirical therapy is given. Treatment of infections due to this atypical pathogen with beta-lactam antibiotic is ineffective, so a specific diagnosis is important. The duration of the illness will be markedly reduced using antibiotics such as macrolides. M. pneumoniae should be considered in differential diagnosis of CAP and additionally it should also be considered in co-infections which are unresponsive to commonly administered beta-lactams5,6.

History

Mycoplasmas are small prokaryotic cells without a rigid cell wall. Nocard and Roux7 in 1898 isolated the first mycoplasma in culture which was the bovine pleuropneumonia agent because of similarities to Mycoplasma mycoides subsp. mycoides. Isolation of the first human mycoplasma was done in 1937 by Dienes and Edstall8 from a Bartholin's gland abscess9,10. Another mycoplasma was isolated in 1944 by Eaton et al11 from the sputum sample of a patient having primary atypical pneumonia and it was called Eaton agent. Chanok et al12 proposed taxonomic designation to Eaton agent as M. pneumoniae in 19629.

Biology of Mycoplasma pneumoniae and Pathogenesis of Infection

Mycoplasmas are the smallest free-living prokaryotes known, having an extremely small genome size of 580-2200 kilobase pair. Mycoplasmas are classified in the family Mycoplasmataceae and order Mycoplasmatales9,10,13,14. Six of the 16 species of human mycoplasma cause diseases, the most important and the most predominant pathogen is M. pneumoniae14. Mycoplasmas cannot be detected by light microscopy due to their small size, and also, visible turbidity is not produced in liquid growth medium due to their small cellular mass9,10,13. The absence of a cell wall barrier in mycoplasmas is unique among the prokaryotes, and so, these organisms are not sensitive to cell wall antimicrobial agents such as beta-lactams, are not stained by Gram staining, are very susceptible to effect of drying and also influence their pleomorphic appearance15.

M. pneumoniae adheres to the ciliated cells of the epithelium lining of the respiratory tract with an attachment organelle after inhalation. At the tip of this polarized attachment organelle, a 170 kDa protein called P1 is cluttered and adherence of M. pneumoniae to host cells is mediated through its several additional accessory proteins (HMW1, HMW2, HMW3, P90, P40 and P30)15. The lack of cell wall in M. pneumoniae facilitates close contact of the membrane with the host cell, facilitating the exchange of compounds which are important for its growth as well as proliferation. Similar to pertussis toxin, there is an ADP-ribosyl transferase known as the community-acquired respiratory distress syndrome toxin (CARDS toxin), which is responsible for the binding to surfactant protein A and for entering host cells by clathrin-mediated endocytosis16.

CARDS toxin causes ciliostasis as well as nuclear fragmentation and stimulates proinflammatory cytokines production and acute cellular inflammatory reaction causing airway damage. Intracellular localization of this organism may protect it from antibodies and antibiotics; furthermore, it may help in establishing persistent infections shown in tissue culture models17. Lack of protective immunity may be due to important factors such as variation and rearrangement of the surface antigens which may allow repeated M. pneumoniae infections over time2.

Establishment of persistent infections and development of autoimmune phenomenon in this organism are due to immunomodulation of the host immune response. Clinical manifestations due to acute infection and extrapulmonary manifestations of M pneumoniae are the results of immunopathologic and inflammatory effects made by the host but not due to the organism itself. Interleukin-6, tumour necrosis factor alpha (TNFα) as well as neutrophil infiltration production is stimulated by various surface lipoproteins. Macrophages get activated and undergo chemotactic migration to the site of infection after opsonization of M. pneumoniae by complement or antibody and then followed by infiltration of neutrophils, T-lymphocytes (CD4+), B-lymphocytes and plasma cells in the lung2.

Autoimmune reactions with M. pneumoniae infections occur due to amino acid sequence similarity of mycoplasmal adhesins with several human tissues, I antigen on human red cells, CD4 lymphocyte and class II major histocompatibility complex antigens and by immune complexes development. B- and T-lymphocytes are also stimulated by M. pneumoniae which induce autoantibodies formation and have reaction with a range of host tissues. Autoimmunity has an important role to play in the extrapulmonary involvement of M. pneumoniae disease2.

Many immunogenic M. pneumoniae proteins and lipids produce antibodies after infection due to a strong humoral immune response. After about one week of illness, immunoglobulin M (IgM) may be detected with a peak at 3-6 wk which gradually declines in children more than six months of age2. Two weeks later, IgM is followed by IgG response. Sometimes, IgM persists for weeks to months, or it may not occur at all. If the patient is immunocompromised, then the antibody production may be absent. Lack of protective immunity may be due to surface antigen variation and rearrangement which appears to be an important factor, leading to repeated M. pneumoniae infections over time18.

Epidemiology of Mycoplasma pneumoniae infections

The main bacterial aetiological agent in all age groups is Streptococcus pneumoniae, but varied prevalence of M. pneumoniae depends on the population studied and numerous methods used for its diagnosis. M. pneumoniae is known as a common cause of CAP throughout the world and causes up to 40 per cent or more of cases of CAP, and 18 per cent paediatric cases require hospitalization15. In an earlier study9, pneumonia due to M. pneumoniae infection was reported as somewhat uncommon in children under five years of age and highest incidence was shown among school children from 5 to 15 yr of age. However, M. pneumoniae disease may occur both endemically and epidemically in older adults and in younger children under five years of age3,18. Climate and geography do not seem to be of major significance. Children who may also represent an asymptomatic reservoir of infection may cause outbreaks in families. Immunity due to mycoplasma infection is short lived, and recurrent infections may develop.

Generally, M. pneumoniae is not known as a neonatal pathogen, but Kumar et al19 reported persistent pneumonia in a three week old neonate because of M. pneumoniae infection. Ursi et al20 showed probable transplacental transmission of M. pneumoniae by the use of polymerase chain reaction (PCR) assay in the nasopharyngeal aspirate in a neonate who had congenital pneumonia. There is evidence that M. pneumoniae may play a significant role in chronic asthma as compared to a typical explanation for common cause of acute exacerbations2.

Various studies used nucleic acid amplification techniques (NAATs) and antigen capture assays for the CARDS toxin detection and proved the role of M. pneumoniae in chronic asthma. Peters et al21 observed a high prevalence of M. pneumoniae (52%) in 64 adults with treatment-resistant asthma in a study which used PCR for detection of the gene for the CARDS toxin in mostly serological-negative population. In a paediatric group, a study by the same group included non-asthmatic controls and detected M. pneumoniae in acute asthma (64%), in refractory asthma (65%) and in healthy controls (56%). M. pneumoniae antibody was detected in lower levels in asthmatic children as compared to healthy controls22.

The spread of infection is from infected persons by contact with droplets discharged from upper and lower respiratory tracts. Epidemics are known to occur in the community or in closed or semi-closed settings such as hospitals, military personnel, schools, religious communities and facilities for mentally or developmentally disabled. The incubation period is from one to three weeks. The mean incubation time is 20-23 days when infections occur in families and other close groups due to slow spread of the organism2. Older children and adolescents tend to have increased severity of symptoms. However, the European epidemic report in 2010-2011 has shown high infection rates in children less than four years of age3.

Clinical manifestations of Mycoplasma pneumoniae in children

M. pneumoniae infections may involve either upper or lower respiratory tract or both of them. The most common clinical symptoms are cough (non-productive at the start and non-bloody sputum small to moderate amounts later on), fever, chills, sore throat, headache, hoarseness, myalgias and general malaise10. Up to one-fifth of infections are actually asymptomatic and may represent reinfection. Dyspnoea may be present in more severe cases, and cough presentation may be like a pertussis-like character9. Coryza and wheezing are manifested in children below five years of age; however, progression to pneumonia is not common, whereas bronchopneumonia (with one or more lobes involvement) may develop in children aged 5-15 yr which sometimes needs hospitalization15.

The clinical entity of pneumonia which was subsequently proved to be due to M. pneumoniae was known several years before the actual identification and establishment of the aetiological agent. Atypical pneumonia historically was used for primary pneumonia which was not demonstrated due to an accepted pathogen such as Pneumococcus. Antimicrobial therapy response was lacking and considered as ‘atypical', so it was thought that it was a primary form of lung disease with uncertain aetiology, and therefore, the term ‘atypical pneumonia’ was used for it8. With the onset of pneumonic symptoms, characteristically M. pneumoniae is mild, non-debilitating and patients yet will continue to function relatively normally, so termed ‘walking pneumonia'9. The terms ‘primary atypical pneumonia' and ‘walking pneumonia' have been used to denote mycoplasmal respiratory disease by physicians as well as by the lay public.

Clinical presentation due to M. pneumoniae respiratory disease is indistinguishable from other atypical pathogens notably numerous respiratory viruses, Chlamydophila pneumoniae and S. pneumoniae. M. pneumoniae causes cough, fever and unilateral crackles. On chest auscultation, rales, rhonchi (scattered or localized) and expiratory wheezes may be shown. The acute febrile period continues for about a week; however, cough and lassitude may continue for two weeks or more in uncomplicated cases9. Antimicrobial treatment if started early within the course of illness will generally shorten period of signs and symptoms of the disease. It is often difficult to differentiate M. pneumoniae pneumonia and viral pneumonia clinically, so it influenced the recommendations for the use of antibiotics in the management of childhood pneumonia23.

M. pneumoniae can also be observed in the respiratory tract along with other pathogens. Human and animal models indicate that M. pneumoniae infection may precede and intensify resulting infections with bacteria such as Streptococcus pyogenes and Neisseria meningitidis and respiratory viruses9. This type of synergistic effect may be due to immunosuppression or respiratory tract flora alteration in the presence of M. pneumoniae. A risk of developing more fulminant pneumonia owing to M. pneumoniae occurs in children who presented with functional asplenia and immune system impairment which are due to immunosuppression, Down syndrome and sickle cell disease15.

Respiratory tract is the main site of M. pneumoniae infection; however, any organ system may be involved. Host responses after M. pneumoniae infection may contribute to autoimmunity, and cardiovascular, gastrointestinal, renal and musculoskeletal complications may occur in about 25 per cent of M. pneumoniae- infected cases9. Extrapulmonary complications caused by M. pneumoniae infection may involve every organ system, and it may be the result of spread of infection or autoimmune mechanisms2. The I blood group antigen (cold agglutinins) generates an autoantibody producing a rapidly evolvinghaemolytic anaemia which is perhaps the most common. Neurologic complications such as Guillain-Barré syndrome and acute demyelinating encephalomyelitis are also prominent2. Gorthi et al24 reported M. pneumoniae infection in 50 per cent of patients with Guillain-Barré syndrome as compared to controls (25% of household and 15% of hospital controls) in India. In some individuals, a syndrome of severe mucocutaneous involvement such as Stevens–Johnson syndrome is seen. M. pneumoniae can cause septic arthritis especially in persons with hypogammaglobulinaemia and also some cases of chronic arthritis in children2.

Mycoplasma pneumoniae with community-acquired lower respiratory tract infections (LRTIs) in children in India

M. pnemoniae infection has high incidence and clinical importance, but still, it is an underrated disease. M. pneumoniae is well acknowledged as a pulmonary pathogen within the West, but in the developing countries, there is little information of disease prevalence because reliable and rapid diagnostic laboratory tests are not available. Kashyap et al25 reported M. pneumoniae infection in 24 per cent children with CAP by culture, serology and PCR assay. Maheshwari et al18 used the criteria of serology and PCR assay on throat swab with LRTIs documented M. pneumoniae infection in 23 (30.7%) of 75 children, while Kumar et al4 observed M. pneumoniae infection in 71 (35.5%) out of 200 children with community-acquired LRTIs by employment of serology and PCR assay. Shenoy et al26 reported M. pneumoniae infection in 24 per cent pneumonia cases in hospitalized children. Chaudhry et al27 observed M. pneumoniae positive in six (16%) of 37 paediatric patients using any test (serology, PCR and real-time PCR) in clinical samples consisting of blood and respiratory fluids, nasopharyngeal aspirates throat swabs and bronchoalveolar lavage.

Mycoplasmas tend to cause more severe and prolonged infections in the human immunodeficiency virus (HIV)-infected cases and other immunodeficient subjects. An Indian study by Nadagir et al28 reported 32.2 per cent M. pneumoniae infection among HIV-seropositive children with respiratory tract infection. In these patients, early diagnosis and prompt initiation of treatment of M. pneumoniae infection may prevent CD4 cells depletion further and speedy progression to AIDS28. Seroprevalence study among HIV-positive patients with pulmonary symptoms by Shankar et al29 reported 21 per cent prevalence of M. pneumoniae IgM antibody by ELISA, and among 34 per cent of the cases screened, non-specific diagnosis was confirmed. In another study, Shankar et al30 detected mycoplasmas in 36 per cent of the AIDS patients and in only 16.6 per cent of the non-HIV control individuals with underlying pulmonary symptoms using culture on pleuropneumonia-like organisms glucose agar.

Diagnosis

There are scarce specific findings of clinical laboratory results for the diagnosis of M. pneumoniae infection. Physicians usually depend on their clinical suspicion and adopt empiric treatment in most infections due to M. pneumoniae in children, and these children are managed on an outpatient basis. Microbiologic diagnosis is needed if illness is adequate to justify hospitalization, if initial antimicrobial therapy has unsatisfactory clinical response, if there are important underlying comorbidities or immunosuppression that may lead to severe and disseminated disease and if important extrapulmonary manifestations are present31.

Radiological diagnosis

Radiographic findings can be variable and mimic different lung diseases (a viral or bacterial pneumonia). Inflammatory response due to M. pneumoniae in lungs causes interstitial mononuclear inflammation, and the manifestation may be in the form of bronchopneumonia which is of the perihilar regions or of lower lobes radiographically often having unilateral distribution and hilar adenopathy. The findings of lobar consolidation and bilateral involvement are also seen. The degree of consolidation may be more than the expectation depending on the severity of clinical manifestations. Pleural effusions and diffuse alveolar damage may occur which are in association with more severe cases. In patients with sickle cell disease, massive and bilateral effusion is reported32,33.

Non-specific laboratory diagnosis

Leucocytosis and/or a high erythrocyte sedimentation rate are found roughly in about a third of patients who have upper respiratory tract infection due to M. pneumoniae. Haemolytic anaemia occurs in many patients. Mononuclear cells or neutrophils as well as normal flora can be shown in Gram staining of the sputum9,15.

Microbiological tests

Fast and correct diagnostic laboratory tests are lacking for the detection of M. pneumoniae directly, or serological response produced by M. pneumoniae creates hindrance for understanding of the epidemiology. Currently, the following tests are available with their limitations:

Antigen detection: Immunological methods are used for M. pneumoniae antigen detection which do not depend on infective agent viability. Several tests such as immunoblotting assay, immunofluorescence assay (IFA), counter-immunoelectrophoresis assay and antigen-capture enzyme immunoassay (EIA) can be used. The limits of detection are in the range of 103-105 colony forming units/ml with a limited sensitivity and specificity9,13.

Culture: Culture of M. pneumoniae is laborious, expensive and time-consuming due to slow growth in vitro and colonies become visible in 2-5 weeks34,35,36. Culture sensitivity is approximately 61 per cent compared to PCR37. The advantage of positive culture is that it is 100 per cent specific if appropriate procedures are used for the identification of the organism isolated to species level. Culture is rarely performed and not recommended for routine diagnosis because of the prolonged turnaround time, with limited availability, requirement of specialized expertise and low sensitivity.

Serology: Serological tests are more sensitive for the detection of acute M. pneumoniae infection than culture. Cold agglutinins production is the first humoral response by the second week in approximately 50 per cent of M. pneumoniae infections which disappear after a gap of 6-8 wk. Antibiotic therapy also influences cold agglutinin levels, resulting in lower titres37. False-positive results are also frequent.

The development of antibody to M. pneumoniae infection is performed by a range of serological methods which include IFA, EIAs and particle agglutination (PA) assay. These tests are easy to use; their sensitivities and specificities are also improved and have largely replaced the older complement fixation test (CFT) which was popular in the past as the primary method for detection of M. pneumoniae antibodies.

The PA assays are simple to perform, quick and can provide qualitative or semi-quantitative results. Interpretation of IFAs is more subjective and a fluorescent microscope is required, but they have favourable sensitivities and specificities in comparison to CFTs. EIAs can be performed with serum in very small volumes to test isotype-specific IgM, IgG and IgA. Rapid EIAs for IgM detection of acute infection are available where a single serum specimen is employed2. Detection of M. pneumoniae infection in children by employing a combination of the PCR assay and IgM detection has been recommended by some experts with the advantage of improved early detection of infection31. Evaluation of EIAs and PA assays has shown problems with sensitivity and specificity when employing PCR as a reference and only a single specimen is used for analysis.

M. pneumoniae is a mucosal pathogen, so IgA is produced at an early stage of the infection and may have rapid rise and decline than IgM or IgG. Detection of IgM or IgA and PCR in a combination may be an optimum diagnostic approach for M. pnemoniae infection2 but adding considerable cost to laboratory testing.

Molecular assay

DNA probes: DNA probes may be used for M. pneumoniae detection with 16S rRNA genes as the target. These probes use a ¹²⁵I-radioactive label to generate a detection signal. These have low sensitivity and specificity; other methods have replaced them10.

Nucleic acid amplification techniques (NAATs): NAATs are used for detecting M. pneumoniae infection earlier than serology because antibodies development requires many days. NAATs have the potential to give results which are rapid, sensitive and specific and which may help for early appropriate antibiotic therapy. Many PCR systems for the detection of M. pneumoniae have been described, employing many targets36. Major gene targets utilized in PCR assays for M. pneumoniae detection are P1 adhesin gene, 16S rRNA gene, ATPase operon gene, the tuf gene (codes for elongation factor 2) and the repetitive element repMP117. Interpretative guidance may also be provided by combining serology with PCR for differentiation and colonization from active disease.

M. pneumoniae and other respiratory pathogens can be detected using multiplex PCR assays, but monoplex assays have higher sensitivity and specificity as compared to multiplex assays. Traditional PCR assays with further refinements to real-time PCR detection will be important15. In many studies, comparison of NAATs was done with culture or serology as a reference method which gave disparate results predictably as a result of more sensitive NAATs inherently2.

Besides PCR, other alternative amplification techniques include (i) nucleic acid sequence-based amplification (NASBA), (ii) Qβ replicase amplification, (iii) strand displacement amplification, (iv) transcription-mediated amplification, and (v) ligase chain reaction. Both viable and non-viable organisms can be detected by NAATs targeting DNA, while RNA detection using reverse-transcriptase PCR (RT-PCR) or NASBA is also a helpful technique for the identification of productive M. pneumoniae infections. Initial studies38 showed that NASBA and PCR performance in terms of sensitivity was comparable. A multiplex NASBA assay38 and different techniques as multiplex RT-PCR are also described39 as other techniques. NAATs is not recommended for children who do not have typical manifestations of mycoplasmal infection2.

Interpretation of various tests for diagnosis of Mycoplasma pneumoniae infections

It is essential to have correct and rapid diagnosis of M. pneumoniae infections for the initiation of applicable antibiotic treatment. Laboratory diagnosis for M. pneumoniae detection is especially important since this disease cannot be diagnosed solely on clinical signs and symptoms. Culture is time-consuming because the organism grows slowly and, therefore, for routine diagnosis is not recommended. EIAs are the most widely used serologic methods for the detection of IgM, IgG and IgA antibodies to M. pneumoniae, although other methods such as PA assays and IF methods are also used.

NAATs can detect M. pneumoniae earlier than serology and generate rapid, sensitive and specific results, especially real-time PCR into routine diagnosis. Although these tests are superior in diagnosing M. pneumoniae infections than other tests, still serology cannot be replaced40. M. pneumoniae detection with real-time PCR with P1 gene target in respiratory sample has shown 60 per cent sensitivity and 96.7 per cent specificity when compared with serology41. The sensitivity of NAATs is always superior than traditional procedures and they are more and more thought of as ‘new gold standard'42. PCR in combination with serology can be used as good screening tests which give reliable and correct diagnosis of M. pneumoniae.

Amplification-Free and Other New Technologies

Microfluidics and application of nanotechnology offer the potential to an even more rapid detection of important pathogens with near-patient testing. Colloidal gold-based immune chromatographic assay has been developed by employing a pair of monoclonal antibodies which target a region of the P1 gene43. Other amplification-free detection methodologies are being made available as biosensing detection strategies: a prototype of an enzyme-free electrochemical genosensor on nanostructured screen-printed gold electrodes44; a silver nanorod array-surface enhanced Raman Spectroscopy biosensing platform was used successfully in simulated and clinical throat swabs for the detection of M. pneumoniae45.

Antimicrobial Susceptibility and Treatment of Mycoplasma pneumoniae Infections

Macrolides are used for M. pneumoniae infections in children as the first-line treatment9. Quinolones are still not recommended in children, although ciprofloxacin has been reported safe in the paediatric population14. Before 2000, resistance was not common, but since then, emergence of macrolide-resistant M. pneumoniae (MRMP) has been noticed with spread into Europe and North America caused by point mutations in domain V of 23S rRNA2. Recent surveillance studies conducted in paediatric populations have documented high resistance rates of >90 per cent in China46 and 87.1 per cent in Japan47. In Europe, macrolide resistance has been reported 3 per cent in Germany to 9.8 per cent in France48,49 and 8.2 per cent in the United States50. Minocycline, doxycycline, tigecycline or fluoroquinolones can be used for successful treatment of MRMP. These drugs are not used normally in children, but there are no other realistic alternatives in the case of MRMP. In Asia, MRMP resistance rates are very high2, so alternative to macrolides for suspected or confirmed M. pneumoniae infection as initial treatment must be considered by clinicians.

Vaccines

A safe vaccine development offers protecting immunity and would be a significant step for reducing the extent of M. pneumoniae infections. Various vaccine trials have shown disappointing results so far16,51,52, indicating that vaccine development may be some time away. There are not any new strategies on the horizon, but the recent discovery of the CARDS TX of M. pneumoniae has yet to be explored by a new vaccine target16.

Conclusions

The role of M. pneumoniae is underlined in children (usually less than five years of age) with community-acquired LRTIs and more significantly in those aged less than one year. Although M. pneumoniae infection has high incidence and clinical importance, yet it is still an underrated disease. Most mycoplasmal respiratory infections do not have a microbiological diagnosis because affordable and reasonably priced methods are not readily available for its direct detection. Therefore, detection assays need further improvement with focus on serology and PCR that may finally give some much-needed diagnostic tools. Macrolides are useful in the effective management of M. pneumoniae infections in children; however, the emergence of MRMP is of concern for macrolide treatment failures when managing these infections. A safe vaccine development is needed which would offer protective immunity and would be a significant step towards reducing M. pneumoniae infections.