Supportive Data

Accuracy:

During laboratory verification studies, 129 respiratory specimens (86 respiratory swabs, 16 bronchoalveolar lavage, 1 pleural/pericardial, 25 sputum, and 1 cerebrospinal fluid) previously tested via Mayo Clinic's Mycoplasma (Mycoplasmoides) pneumoniae PCR test were reflexed for macrolide prediction. Upon reflex, this assay detected M pneumoniae 23S ribosomal RNA (rRNA) DNA in 114/129 samples (89.4% overall percent agreement).

Assessment of macrolide resistance prediction was made by performing bidirectional Sanger sequencing on all macrolide (Azithromycin) resistant samples. All 103 samples with predicted macrolide susceptible M pneumoniae, demonstrated wildtype 23S rRNA gene sequence at positions 2063 and 2064. All 11 samples with predicted clarithromycin resistant M pneumoniae, demonstrated single nucleotide polymorphisms of A2063G or A2064G with the M pneumoniae 23S rRNA gene.

Limit of detection:

The limit of detection of the assay is less than 10 target copies/mcL for all validated specimen types and gene targets.

Analytical specificity:

The assay was tested against a panel of 87 organisms consisting of bacteria and viruses representing normal respiratory microbiota and/or respiratory pathogens (including 26 Mollicute species). Among the 23 other species of Mycoplasma (Mycoplasmoides) tested, there was cross-reactivity noted among Mycoplasma (Mycoplasmoides) testudinis (likely not found in humans and has not been reported to cause disease in humans) and Mycoplasma (Mycoplasmoides) pirum. This assay is intended to be used as reflex for previously identified M pneumoniae positive samples. Therefore, this cross reactivity with other Mycoplasma (Mycoplasmoides) species is not a major concern.