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Table 2 Diagnostic approach for M. pneumoniae with test result constellations. ASC antibody-secreting cell, CAP community-acquired pneumonia, ELISpot enzyme-linked immunospot, FAS full analysis set, FUP follow-up, Ig immunoglobulin, LFA lateral flow assay, NA not available, NPS nasopharyngeal swab, PCR polymerase chain reaction, PPS per protocol set

From: A randomized controlled non-inferiority trial of placebo versus macrolide antibiotics for Mycoplasma pneumoniae infection in children with community-acquired pneumonia: trial protocol for the MYTHIC Study

Test

Method

Turn-around time

Specimen

Test result constellationa

1. Screening test:

IgM LFA

10 min

Capillary blood

–

+

 Expected resultsa

   

67.2%

(n = 84/125)

32.8%

(n = 41/125)

 Study procedure:

  

Randomization

2. Reference test:

PCRb

After close-out visit

NPSb

–

+

–

+

 Expected resultsa

   

92.9%

(n = 78/84)

7.1%

(n = 6/84)

9.8%

(n = 4/41)

90.2%

(n = 37/41)

 Interpretation of the IgM LFA result (screening test):

Negative

False-negative

False-positive

Detection

3. Confirmatory test:

IgM ASC ELISpotd

After close-out visit

Venous bloodd,e

NAc

NAc

–

–

+

 Expected resultsa

  

9.8%

(n = 4/41)

14.6%

(n = 6/41)

75.6%

(n = 31/41)

 Final interpretation:

Negative

False-negative

False-positive

Carriage and/or persistence

Infectiond

 Study procedure:

No randomization

No randomization

FUP until final visit on day 28b

FUP until final visit on day 28

FUP until final visit on day 28

 Statistical analysis:

Diagnostic

accuracy

Diagnostic

accuracy

Intention-to-treat

(FAS)

Per protocol

(PPS)

Strict per protocol

(strict PPS)

  1. aExpected results as proportion (number) based on results using the myCAP cohort [42] (n = 94) and KIDS–STEP cohort [43] (n = 31, unpublished results)
  2. bAll screened and enrolled participants will provide a NPS sample, either performed as part of the clinical routine diagnostic workup or exclusively for the use in this study. This NPS will be tested with an M. pneumoniae-specific PCR as reference test to verify the M. pneumoniae-specific IgM LFA test result
  3. If a NPS is performed as part of clinical routine diagnostics, the sample will be frozen and stored locally so that no more than one swab will be performed on patients on day 1. The stored NPS sample will be transferred to and analyzed by M. pneumoniae-specific PCR at University Children’s Hospital Zurich. The results of the M. pneumoniae-specific PCR will not be available before the close-out visit on day 28
  4. In case a M. pneumoniae-specific PCR (single or multiplex) is performed for clinical reasons and indicates a false-positive M. pneumoniae-specific IgM LFA result, participants will be followed up until the close-out visit on day 28, but they will be excluded from per protocol analyses
  5. cNo venous blood sampling because not enrolled
  6. dThe M. pneumoniae-specific IgM ASC ELISpot assay may not be available in all patients (refusal to draw blood) and/or peripheral blood mononuclear cell viability can be decreased in very few instances (pre-analytical processing) and result in poor assay performance
  7. eIf venous blood is available also M. pneumoniae-specific IgM enzyme-linked immunosorbent assay (ELISA) will additionally be performed but these results will not be used to guide study procedures and statistical analyses
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