Table 1. Overview of diagnostic tests for M. pneumoniae.
| Method | Test | Target/Antigen | Antibodies | Specimen | Performance1 | Value | Comments |
| Direct identification of M. pneumoniae | PCR | Different target genes (e.g., P1 gene, 16S rDNA, 16S rRNA, RepMP elements, etc.) | - | Respiratory specimen (nasopharyngeal secretion, pharyngeal swab, sputum, bronchoalveolar lavage), CSF, and other bodily fluids or tissues | High sensitivity, high specificity | RD2 | NAATs provide fast results (in less than a day) and may be earlier than serology (because antibody production requires several days); validation and standardization required for routine diagnostic |
| Culture | - | - | Respiratory specimen (see above) | Low sensitivity, high specificity | AD | Special enriched broth or agar media; isolation takes up to 21 days | |
| Nonspecific serological tests for M. pneumoniae | Cold-agglutinin test (“bedside test”) | Erythrocytes (I antigen) | Cold agglutinins (IgM) | Serum | Low sensitivity, low specificity | -3 | Cold agglutinins target the I antigen of erythrocytes (alternative theory: cold agglutinins target directly M. pneumoniae adhered to erythrocytes); positive in only about 50% and in the first week of symptoms; less well studied in children; cross-reactivity with other pathogens and noninfectious diseases |
| Specific serological tests for M. pneumoniae | CFT | Crude antigen extract with glycolipids and/or proteins | Igs (no discrimination between isotypes) | Serum | Sensitivity and specificity comparable to EIA | -3 | Positive criteria: 4-fold titer increase between acute and convalescent sera or single titer ≥1∶32; cross-reactivity with other pathogens and noninfectious diseases |
| PA | IgM and IgG simultaneously | -3 | See above | ||||
| EIA | Proteins (e.g., adhesion protein P1) and/or glycolipids | IgM, IgG,4 , 5 (IgA)6 | Serum4, CSF5 , 7, other bodily fluids7 | Moderate-high sensitivity, moderate-high specificity | RD | The sensitivity depends on the time point of the first serum and on the availability of paired sera (for seroconversion and/or rise in titer); “gold standard”: 4-fold titer increase as measured in paired sera | |
| Immunoblotting | High sensitivity, high specificity8 | AD | Confirmatory assay | ||||
| IFA | Less sensitive and less specific than EIA | AD | Subjective interpretation |
Abbreviations: AD, advanced diagnostic test; CFT, complement fixation test; CNS, central nervous system; CSF, cerebrospinal fluid; EIA, enzyme immunoassay; IFA, immunofluorescent assay; Ig, immunoglobulin; NAATs, nucleic acid amplification tests; PA, particle agglutination assay; PCR, polymerase chain reaction; RD, routine diagnostic test; RepMP, repeated M. pneumoniae DNA. References: [13], [15]–[24].
Qualitative statements included because of the wide range of test performances, which depend on the assay, the patient cohort (children and/or adults), the reference standard (PCR, culture, and/or serology), the respiratory specimen (for PCR), and the time point of the sample collection after disease onset (for EIA)—e.g., sensitivities and specificities for PCR [17], [18]: 79%–100% and 96%–99%; IgM EIA (in relation to PCR) [19]: 35%–77% and 49%–100%; and for IgG EIA [17], [19]: 37%–100% (no indication on specificity because of missing information on previous M. pneumoniae infections).
Epidemiological differentiation of clinical strains on the basis of differences in the P1 gene by PCR or in the number of repetitive sequences at a given genomic locus by multilocus variable-number tandem-repeat analysis (MLVA) [23].
Largely replaced by EIA.
Kinetics of antibody responses in blood. IgM: onset: within 1 week after the onset of symptoms; peak: 3–6 weeks; persistence: months (to years). IgG: onset and peak: 2 weeks after IgM; persistence: years (to lifelong); reinfection in adults may lead directly to an IgG response in the absence of an IgM response. IgA: onset, peak, and decrease earlier than IgM.
Antibody responses in the CNS differ from blood. There is no switch from an IgM to an IgG response, the pattern of IgM, IgG, and IgA synthesis remains rather constant and depends on the cause, and there is a long-lasting and slow decay of intrathecal antibody synthesis [22]. In M. pneumoniae encephalitis, a dominant IgM response has been observed [29].
The prevalence of serum IgA determined by EIA has been shown to be very low in PCR-positive children with symptomatic respiratory tract infection (2.0%) [13].
To our knowledge, no validated test is available.
Immunoblotting with a combination of at least five specific M. pneumoniae proteins showed sensitivities (in relation to PCR) of 83% (IgM), 51% (IgG), and 64% (IgA), and specificities of 94%–100% (IgM), 98%–100% (IgG), and 93%–97% (IgA) [24].