923 M rP1-C 38 9 70% (58%-83%) 90% (83%-96%) 0.897 M rAtpD-rP1-C 40 5 74% (60%-80%) 94% (89%-99%) 0.925 M Ani Labsystems 39 7 72% (60%-84%) 92% (81%-97%) 0.824 A rAtpD 30 Crenolanib in vivo 5 56% (42%-69%) 94% (89%-99%) 0.842 A rP1-C 27 7 50% (37%-63%) 92% (86%-98%) 0.775 A rAtpD-rP1-C 31 8 57% (44%-71%) 91% (89%-99%) 0.842 A Ani Labsystems 46 38 85% (77%-95%) 56% (45%-66%) 0.801 G rAtpD 42 3 78% (67%-89%) 97% (93%-100%) 0.943 G rP1-C 37 9 69% (56%-81%) 90% (83%-96%) 0.869 G rAtpD-rP1-C 43 5 80% (69%-90%) 94% (89%-99%)

0.925 G Ani Labsystems 52 61 96% (91%-100%) 29% (19%-39%) 0.663 aChildren PF-02341066 cost infected by M. pneumoniae. bHealthy blood donors. Table 3 Performance of the rAtpD, rP1-C ELISAs and the Ani Labsystems kit in adults Ig class Type of test No. of positive sera in Sensitivity (95% CI) Specificity (95% CI) AUC     Patients a (49) Controls b (86)       M rAtpD 33 8 67% (54%-80%) 91% (85%-97%) 0.877 M rP1-C 22 9 45% (31%-59%) 90% (83%-96%) 0.708 M rAtpD-rP1-C 39 7 80% (68%-91%) 92% (86%-98%) 0.891

M Ani Labsystems 24 7 49% (35%-61%) 92% (81%-97%) 0.685 A rAtpD 32 5 65% (52%-78%) 94% (89%-99%) 0.894 A rP1-C 27 9 55% (41%-69%) 90% (83%-96%) 0.779 A rAtpD-rP1-C 36 9 73% (61%-86%) 90% BAY 73-4506 (83%-96%) 0.841 A Ani Labsystems 48 38 98% (94%-100%) 56% (45%-66%) 0.803 G rAtpD 30 3 61% (48%-75%) 97% (93%-100%) 0.877 G rP1-C 22 9 45% (31%-59%) 90% (83%-96%) 0.708 G rAtpD-rP1-C 33 1 67% (54%-80%)

99% (97%-100%) 0.891 G Ani Labsystems 48 61 98% (94%-100%) 29% (19%-39%) 0.734 aAdults infected by FAD M. pneumoniae. bHealthy blood donors. Serum samples from 39 (72%) children and 24 (49%) adults were IgM-positive based on the Ani Labsystems ELISA. The IgA and IgG Ani Labsystems EIA assays showed the best sensitivity for serum samples from both children and adult patients, with IgA being detected in 46 (85%) children and 48 (98%) adults and IgG being detected in 52 (96%) children and 48 (98%) adults (Tables 2 and 3). It should be noted that although the IgM Ani Labsystems showed good specificity for children and adults (92%), its specificity for IgA and IgG were much lower, at 56% and 29%, respectively (Tables 2 and 3). Indeed, 44% (38/86) and 71% (61/86) of the blood donor serum samples were found to be positive by the IgA and IgG Ani Labsystems commercial kits, respectively (Tables 2 and 3). For the three ELISA tests, a significant increase in IgM, between two- and three-fold, was detected between the first (acute-phase serum) and second of the six paired serum samples. A two-fold increase in the IgA and IgG responses was also seen between the first and second samples (data not shown).