Detection of Mycoplasma pneumoniae in Simulated and True Clinical Throat Swab Specimens by Nanorod Array-Surface-Enhanced Raman Spectroscopy

The prokaryote Mycoplasma pneumoniae is a major cause of respiratory disease in humans, accounting for 20% of all community-acquired pneumonia and the leading cause of pneumonia in older children and young adults. The limitations of existing options for mycoplasma diagnosis highlight a critical need for a new detection platform with high sensitivity, specificity, and expediency. Here we evaluated silver nanorod arrays (NA) as a biosensing platform for detection and differentiation of M. pneumoniae in culture and in spiked and true clinical throat swab samples by surface-enhanced Raman spectroscopy (SERS). Three M. pneumoniae strains were reproducibly differentiated by NA-SERS with 95%–100% specificity and 94–100% sensitivity, and with a lower detection limit exceeding standard PCR. Analysis of throat swab samples spiked with M. pneumoniae yielded detection in a complex, clinically relevant background with >90% accuracy and high sensitivity. In addition, NA-SERS correctly classified with >97% accuracy, ten true clinical throat swab samples previously established by real-time PCR and culture to be positive or negative for M. pneumoniae. Our findings suggest that the unique biochemical specificity of Raman spectroscopy, combined with reproducible spectral enhancement by silver NA, holds great promise as a superior platform for rapid and sensitive detection and identification of M. pneumoniae, with potential for point-of-care application

Principal Component and Hierarchal Cluster Analyses.

<p>Chemometric analysis was conducted on the spectral data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013633#pone-0013633-g002" target="_blank">Figure 2</a>. (<b>A</b>) PC scores plot 1vs. 3 of <i>M. pneumoniae</i> strains FH, M129, and II-3, as indicated. 77% of the variance was captured in PC1 and 3% in PC3 to distinguish the strains. (<b>B</b>) HCA of pre-processed spectra of <i>M. pneumoniae</i> strains FH (dashed lines), M129 (solid lines), and II-3 (bold lines). Four spectra from strain II-3 were misclassified with FH (at left).</p

Differentiation of <i>M. pneumoniae</i> strains.

<p>(<b>A</b>) Average spectra (n = 15) of <i>M. pneumoniae</i> strains with formalin background, baseline corrected and offset; and (<b>B</b>) first derivative spectra from panel A demonstrating strong similarities but also clear differences (boxes) among the strains.</p

Reproducibility of spectra.

<p>Raw spectra (<b>A</b>) of <i>M. pneumoniae</i> strain FH collected from five random spots from three separate NA substrate wells, baseline corrected and offset. (<b>B</b>) First derivative spectra for strain FH from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013633#pone-0013633-g001" target="_blank">Figure 1A</a>.</p

Differentiation of spiked and control throat swab samples.

<p>Representative difference spectra of spiked, pooled throat swab samples after subtraction of the spectrum of the un-spiked, pooled throat swab control. Bold and thin solid lines, spiked samples (8.2×10⁴ and 8.2×10³ CFU <i>M. pneumoniae</i> M129/µl, respectively); dashed line, representative spectrum of <i>M. pneumoniae</i> M129.</p

PCR analysis of serial dilutions of <i>M. pneumoniae</i> strain II-3.

<p>Dilutions 10⁻¹⁰ to 10⁰ are indicated; std, DNA size standard (350 kbp); +, positive control (277 kbp). Starting concentration, 1.8×10⁹ CFU/ml.</p

PLS-DA of throat swabs spiked with serial dilutions of <i>M. pneumoniae</i>.

a<p>abbreviations same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013633#pone-0013633-t001" target="_blank">Table 1</a>.</p

PLS regression analysis of serial dilutions of <i>M. pneumoniae.</i>

<p>Serial 10-fold dilutions of strain II-3 spanning 10 logs of concentration were assessed by PLS regression for degree of linearity in actual intensity of the measured spectra plotted against the predicted intensity. Starting concentration, 1.8×10⁹ CFU/ml; R² = 0.810.</p

PLS-DA of NA-SERS specificity and sensitivity in discriminating three <i>M. pneumoniae</i> strains and two negative controls (formalin and water).

a<p>abbreviations the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013633#pone-0013633-t001" target="_blank">Table 1</a>; single model generated using 12 latent variables accounting for 88% of the total variance for all serial dilutions of each strain.</p