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Detection of Mycoplasma pneumoniae in simulated and true clinical throat swab specimens by nanorod array-surface-enhanced Raman spectroscopy.

Hennigan SL, Driskell JD, Dluhy RA, Zhao Y, Tripp RA, Waites KB, Krause DC - PLoS ONE (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
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.

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PLS regression analysis of serial dilutions of M. pneumoniae.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.
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pone-0013633-g004: PLS regression analysis of serial dilutions of M. pneumoniae.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.

Mentions: To assess NA-SERS sensitivity further we analyzed the spectra from ten serial ten-fold dilutions (10−1 to 10−10) per strain by PLS-DA in a single model (Table 2), detecting and differentiating the three strains with >94% sensitivity and specificity, an outcome especially meaningful given the range in bacterial loads likely in clinical samples. Spectra from the serial dilutions of strain II-3 were also assessed by PLS regression analysis, revealing a correlation between true mycoplasma concentration and that predicted for cross-validated samples (Figure 4). This correlation suggests that spectral intensity reflects concentration, although signal quenching was apparent with the most concentrated mycoplasma suspensions. High SERS enhancement is achieved through the local electric field between nanorods and requires that the sample penetrate within the array itself [39], likely accounting for the quenching effect at high sample concentrations. A similar linear relationship was observed in the quantitative detection of rotavirus by NA-SERS [11]. Finally, M. pneumoniae strain II-3 spectra were correctly differentiated at dilutions as high as 10−9, corresponding to a lower limit of detection of 0.02 CFU/sample analyzed on the substrate; a comparable limit was observed with strain M129 (data not shown). By comparison, standard PCR performed on parallel samples yielded the expected 277-bp product from primers specific for the M. pneumoniae 16S rRNA gene through the 10−7 dilution (Figure 5), which corresponds to a lower detection limit by standard PCR of 3.6 CFU/reaction, or 0.7 CFU analyzed on the gel, consistent with the range reported for M. pneumoniae by diagnostic PCR [40]. It is difficult to extrapolate these results to the single-cell level for mycoplasmas, which tend to clump and for which CFU and plating efficiency are poorly defined [3]. Furthermore, we also cannot rule out the possibility that intact cells are not essential, and the ability to detect M. pneumoniae spectra may fall below the single-cell level due to recognition by SERS of cell components in cell lysates, as was reported recently for mycobacteria [41].


Detection of Mycoplasma pneumoniae in simulated and true clinical throat swab specimens by nanorod array-surface-enhanced Raman spectroscopy.

Hennigan SL, Driskell JD, Dluhy RA, Zhao Y, Tripp RA, Waites KB, Krause DC - PLoS ONE (2010)

PLS regression analysis of serial dilutions of M. pneumoniae.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.
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Related In: Results  -  Collection

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pone-0013633-g004: PLS regression analysis of serial dilutions of M. pneumoniae.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.
Mentions: To assess NA-SERS sensitivity further we analyzed the spectra from ten serial ten-fold dilutions (10−1 to 10−10) per strain by PLS-DA in a single model (Table 2), detecting and differentiating the three strains with >94% sensitivity and specificity, an outcome especially meaningful given the range in bacterial loads likely in clinical samples. Spectra from the serial dilutions of strain II-3 were also assessed by PLS regression analysis, revealing a correlation between true mycoplasma concentration and that predicted for cross-validated samples (Figure 4). This correlation suggests that spectral intensity reflects concentration, although signal quenching was apparent with the most concentrated mycoplasma suspensions. High SERS enhancement is achieved through the local electric field between nanorods and requires that the sample penetrate within the array itself [39], likely accounting for the quenching effect at high sample concentrations. A similar linear relationship was observed in the quantitative detection of rotavirus by NA-SERS [11]. Finally, M. pneumoniae strain II-3 spectra were correctly differentiated at dilutions as high as 10−9, corresponding to a lower limit of detection of 0.02 CFU/sample analyzed on the substrate; a comparable limit was observed with strain M129 (data not shown). By comparison, standard PCR performed on parallel samples yielded the expected 277-bp product from primers specific for the M. pneumoniae 16S rRNA gene through the 10−7 dilution (Figure 5), which corresponds to a lower detection limit by standard PCR of 3.6 CFU/reaction, or 0.7 CFU analyzed on the gel, consistent with the range reported for M. pneumoniae by diagnostic PCR [40]. It is difficult to extrapolate these results to the single-cell level for mycoplasmas, which tend to clump and for which CFU and plating efficiency are poorly defined [3]. Furthermore, we also cannot rule out the possibility that intact cells are not essential, and the ability to detect M. pneumoniae spectra may fall below the single-cell level due to recognition by SERS of cell components in cell lysates, as was reported recently for mycobacteria [41].

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus