Mycoplasma Infections (Mycoplasma pneumoniae) Workup

Updated: Dec 13, 2022
  • Author: Ken B Waites, MD; Chief Editor: Michael Stuart Bronze, MD  more...
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Approach Considerations

Consider the possibility of infection with M pneumoniae in patients of any age who present with respiratory tract infections. Laboratory investigation should focus on both the clinical illness (eg, tracheobronchitis vs pneumonia) and the many possible infectious etiologies that can cause clinically similar manifestations. The extent of laboratory investigation also should reflect the severity of the illness and whether the illness warrants hospitalization. [11, 12]

In as many as half of all cases of community-acquired pneumonias, the microbiological etiology is never determined, despite appropriate laboratory testing. The typical mild illness caused by M pneumoniae in otherwise healthy persons may not warrant a comprehensive microbiological investigation because empiric treatment with oral antimicrobials can cover M pneumoniae and most other bacterial agents that produce similar illnesses.


Laboratory Studies

Laboratory analysis

Twenty-five percent of patients develop leukocytosis; the rest have leukocyte counts within the reference range.

Patients may have an elevated erythrocyte sedimentation rate (ESR).

Cellular response of sputum is mononuclear, with no bacteria visible with Gram staining.

About 75% of patients have a cold agglutinin titer of at least 1:32 by the second week of illness, disappearing by 6-8 weeks. This is not a specific test for M pneumoniae infection but the greater the cold agglutinin titer is (>1:64) in a patient with community-acquired pneumonia, the more likely the cold agglutinins are due to M pneumoniae. No specific abnormalities of hepatic or renal function are likely to occur. [12, 13, 14]

To confirm mycoplasmal respiratory tract infection, culture, molecular-based tests, and/or serological tests are necessary.


Respiratory tract specimens suitable for culture include throat swabs, sputum, tracheal aspirates, nasal swabs, bronchial lavage fluid, pleural fluid, or lung biopsy tissue, depending on the patient's clinical condition. Other body fluids such as CSF or synovial fluid, and tissues obtained by biopsy, including skin lesions or heart valve tissue also may be cultured as necessary in the presence of extrapulmonary manifestations. 

Mycoplasmal organisms have fastidious growth requirements and often are difficult and slow to grow in vitro. Take care during specimen collection to inoculate into a suitable transport medium (eg, SP4 broth or universal transport medium), at the bedside whenever possible, and to not allow desiccation. Clinicians advise freezing at -70°C if specimens cannot be transported to the diagnostic laboratory immediately after collection.

Growth in culture is slow, requiring 3 weeks or longer in some cases, and the culture is not extremely sensitive for detecting M pneumoniae infection. The culture medium often is unavailable except from specialized reference laboratories. If culture is attempted, alternative procedures including serology and/or molecular-based nucleic acid amplification tests also should be performed. [10]

Serologic testing

Physicians can use serology to confirm M pneumoniae infection even though these tests suffer from significant problems.

Because primary infection does not guarantee protective immunity against future infections, and residual immunoglobulin G (IgG) may remain from earlier encounters with the organism, experts have launched a great impetus to develop sensitive and specific tests that can differentiate between acute and remote infection.

Definitive diagnosis requires seroconversion documented by paired specimens obtained 2-4 weeks apart. Although some researchers purport that single-titer immunoglobulin M (IgM) or immunoglobulin A (IgA) assays reveal current infection, IgM may persist for up to several months in some people, and many adults may not mount a detectable IgM response. Therefore, relying on a single serologic test can be clinically misleading, and experts recommend basing diagnosis of acute infection on seroconversion measured simultaneously in assays for both IgM and IgG. Use of serology for diagnosis of mycoplasmal infection is valid only if the patient has a satisfactory capacity of the humoral immune system to mount an antibody response.

Rapid diagnostic enzyme-linked immunosorbent assays

Qualitative, rapid, single-specimen, membrane-based enzyme-linked immunosorbent assays readily adaptable to the primary care physician's office laboratory have been available for several years. [15]

The Remel IgG/IgM Antibody Test System (Thermo-Fisher) measures both IgG and IgM simultaneously.

The Meridian ImmunoCard (Meridian Bioscience, Inc) measures only IgM.

Physicians can perform both tests without special expertise or equipment, and they can interpret the results in approximately 10 minutes, eliminating the need for collection of paired sera for later antibody measurement, although erroneous results sometimes are obtained when only single serum samples are analyzed. [15, 16]

Both tests have a moderate complexity classification under the Clinical Laboratory Improvement Amendment (CLIA), allowing many physicians to offer serologic assays for M pneumoniae antibodies as point-of-care tests so that they can be used to direct patient management.

Such single-specimen assays have limitations as described above; perhaps the most practical use for the IgM ImmunoCard is when an acute infection with M pneumoniae is suspected in children and young adults.

Molecular analysis

Researchers have developed molecular-based systems for detection of M pneumoniae using polymerase chain reaction (PCR) or other technologies. A variety of gene targets have been described for PCR assays to detect M pneumoniae in clinical specimens. Traditional PCR is gradually being replaced by quantitative real-time PCR assays. Publications indicate that the CARDS toxin gene is more sensitive for M pneumoniae detection than assays targeting the P1 protein or ATPase genes. [17]

Some reference laboratories offer PCR assays that they developed themselves for detection of current mycoplasmal infection. Several molecular-based multiplex PCR tests for detection of M pneumoniae and other viral and bacterial respiratory pathogens are now FDA-approved for use in the United States, and their accuracy in detecting M pneumoniae in nasal swabs has been compared. [18]  These integrated sample-to-answer tests can provide results in about an hour.

A singleplex test that detects only M pneumoniae in throat swabs is the Alethia Mycoplasma Direct Assay (Meridian Bioscience, Inc). This loop-mediated isothermal amplification (LAMP) assay enables detection of M pneumoniae in up to 10 clinical specimens that can be tested simultaneously within one hour without the need for expensive equipment. It has a moderate complexity CLIA classification so it can be offered through physician office laboratories as well as hospital laoratories, making it the closest thing to a point of care test that is currently available for direct detection of M pneumoniae in clinical specimens. [19]  

Carriage of mycoplasmas in the upper respiratory tract for variable periods following prior infection may confound the interpretation of a single positive PCR assay result. Furthermore, a PCR assay may reveal very small numbers of organisms that may not be of etiologic significance.

A specific threshold of quantity of mycoplasmas in the respiratory tract that can differentiate colonization from infection has not been established, so a highly sensitive detection method such as PCR performed in a nonquantitative manner may overestimate the clinical importance of M pneumoniae as a pathogen since it often cocirculates with other bacterial and viral respiratory pathogens. For these reasons, molecular-based assays can be accompanied by serologic assays for maximum diagnostic accuracy, unless testing a normally sterile body fluid in which the presence of any number of mycoplasmas would be considered evidence of disease. [1]

Given the significant limitations of serology, prolonged turnaround time, insensitivity of culture, and the growing availability of rapid diagnosis of mycoplasmal infection in symptomatic patients by molecular-based assays, molecular-based tests are now the preferred method for diagnosis of M pneumoniae infection when available to primary care physicians through a hospital or reference laboratory. The automated multiplex PCR platforms are designed to test nasal swabs and provide their own specimen collection kits and transport systems. 

Some reference laboratories can perform lab-developed PCR assays on non-respiratory specimens obtained to evaluate direct invasion of extrapulmonary sites such as CSF, heart valves, kidney biopsies, skin lesions, and synovial fluids. None of the FDA-cleared commercial PCRs have been validated to test non-respiratory specimens.


Imaging Studies

Abnormalities on chest radiographs often appear more severe than predicted based on the clinical condition of the patient.

Lobar consolidation is unusual.

Diffuse or interstitial infiltrates that involve the lower lobes are the most common radiographic abnormalities.

Small pleural effusions may develop in approximately 20% of cases.

Lung involvement tends to be unilateral but can be bilateral.


Histologic Findings

The histologal appearance of mycoplasmal pneumonia typically shows a patchy interstitial pneumonia, especially involving the lower lobes of the lungs, with swollen alveolar lining cells, bronchiolar walls thickened by congestion and edema, and an intraluminal exudate of neutrophils, epithelial cells, and proteinaceous debris.