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Specificity and Strain-Typing Capabilities of Nanorod Array-Surface Enhanced Raman Spectroscopy for Mycoplasma pneumoniae Detection.

Henderson KC, Benitez AJ, Ratliff AE, Crabb DM, Sheppard ES, Winchell JM, Dluhy RA, Waites KB, Atkinson TP, Krause DC - PLoS ONE (2015)

Bottom Line: At present the most effective means for detection and strain-typing is quantitative polymerase chain reaction (qPCR), which can exhibit excellent sensitivity and specificity but requires separate tests for detection and genotyping, lacks standardization between available tests and between labs, and has limited practicality for widespread, point-of-care use.Here we demonstrate using partial least squares- discriminatory analysis (PLS-DA) of sample spectra that NA-SERS correctly identified M. pneumoniae clinical isolates from globally diverse origins and distinguished these from a panel of 12 other human commensal and pathogenic mycoplasma species with 100% cross-validated statistical accuracy.Furthermore, PLS-DA correctly classified by strain type all 30 clinical isolates with 96% cross-validated accuracy for type 1 strains, 98% cross-validated accuracy for type 2 strains, and 90% cross-validated accuracy for type 2V strains.

View Article: PubMed Central - PubMed

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

ABSTRACT
Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the human respiratory tract that accounts for > 20% of all community-acquired pneumonia (CAP). At present the most effective means for detection and strain-typing is quantitative polymerase chain reaction (qPCR), which can exhibit excellent sensitivity and specificity but requires separate tests for detection and genotyping, lacks standardization between available tests and between labs, and has limited practicality for widespread, point-of-care use. We have developed and previously described a silver nanorod array-surface enhanced Raman Spectroscopy (NA-SERS) biosensing platform capable of detecting M. pneumoniae with statistically significant specificity and sensitivity in simulated and true clinical throat swab samples, and the ability to distinguish between reference strains of the two main genotypes of M. pneumoniae. Furthermore, we have established a qualitative lower endpoint of detection for NA-SERS of < 1 genome equivalent (cell/μl) and a quantitative multivariate detection limit of 5.3 ± 1 cells/μl. Here we demonstrate using partial least squares- discriminatory analysis (PLS-DA) of sample spectra that NA-SERS correctly identified M. pneumoniae clinical isolates from globally diverse origins and distinguished these from a panel of 12 other human commensal and pathogenic mycoplasma species with 100% cross-validated statistical accuracy. Furthermore, PLS-DA correctly classified by strain type all 30 clinical isolates with 96% cross-validated accuracy for type 1 strains, 98% cross-validated accuracy for type 2 strains, and 90% cross-validated accuracy for type 2V strains.

No MeSH data available.


Related in: MedlinePlus

Comparison of averaged, baseline-corrected, and normalized SERS spectra for type 1, type 2, and type 2V genotypes.Raw spectra of all type 1 (n = 155), type 2 (n = 135), and type 2V (n = 60) clinical isolates and controls were averaged, baseline-corrected, and normalized using GRAMS32/A1 spectral software package (Galactic Industries, Nashua, NH). Red, average spectrum of all type 1 M. pneumoniae strains; green, average spectrum of all type 2 M. pneumoniae strains; blue, average spectrum of all type 2V M. pneumoniae strains. Peaks unique to a specific genotype of M. pneumoniae are indicated by arrows and identified above the spectral fingerprint. Type 1 peaks, red arrows; type 2 peaks, green arrows; and type 2V peaks, blue arrows. Inset at top right of image depicts zoomed-in view of the type 2 doublet at 767 and 778 cm-1.
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pone.0131831.g006: Comparison of averaged, baseline-corrected, and normalized SERS spectra for type 1, type 2, and type 2V genotypes.Raw spectra of all type 1 (n = 155), type 2 (n = 135), and type 2V (n = 60) clinical isolates and controls were averaged, baseline-corrected, and normalized using GRAMS32/A1 spectral software package (Galactic Industries, Nashua, NH). Red, average spectrum of all type 1 M. pneumoniae strains; green, average spectrum of all type 2 M. pneumoniae strains; blue, average spectrum of all type 2V M. pneumoniae strains. Peaks unique to a specific genotype of M. pneumoniae are indicated by arrows and identified above the spectral fingerprint. Type 1 peaks, red arrows; type 2 peaks, green arrows; and type 2V peaks, blue arrows. Inset at top right of image depicts zoomed-in view of the type 2 doublet at 767 and 778 cm-1.

Mentions: Additionally, we compared averaged, baseline-corrected, and normalized spectra of all three genotypes to look for any differences in band pattern between the three genotypes that could be contributing to the classification capabilities demonstrated in the PLS-DA modeling (Fig 6). The majority of the spectral fingerprint was identical for all three strain types, which is to be expected since they are all the same species and classify as such in the PLS-DA models shown in Figs 2 and 3. However, several visible differences in band pattern were present in the spectra for each genotype of M. pneumoniae, which could account for the ability of NA-SERS to distinguish between the three genotypes with statistically significant sensitivity and specificity. The averaged type 1 spectrum had two unique peaks, one at 1636 cm-1 that does not appear in the averaged type 2 or 2V spectra, and one at 959 cm-1 which appeared as more distinct and shifted slightly right in the type 1 spectrum when compared to the type 2 spectrum, and did not appear in the type 2V spectrum. The averaged type 2 strain spectrum was very similar to the type 1 strain spectrum aside from the differences mentioned above and the presence of a doublet at 767 and 778 cm-1, which appeared as more distinct than that present in the type 2V spectrum and as a broad singlet in the type 1 spectrum. The averaged type 2V spectrum appeared to be the most distinct of the three, with a doublet at 875 and 890 cm-1 that appeared as a single peak at 890 in type 1 and 2 spectra, and a small peak at 521 that was also absent in type 1 and 2 spectra. While these spectral differences were extremely subtle, chemometric analysis is highly capable of discerning differences such as these with substantial discriminatory classification power [43].


Specificity and Strain-Typing Capabilities of Nanorod Array-Surface Enhanced Raman Spectroscopy for Mycoplasma pneumoniae Detection.

Henderson KC, Benitez AJ, Ratliff AE, Crabb DM, Sheppard ES, Winchell JM, Dluhy RA, Waites KB, Atkinson TP, Krause DC - PLoS ONE (2015)

Comparison of averaged, baseline-corrected, and normalized SERS spectra for type 1, type 2, and type 2V genotypes.Raw spectra of all type 1 (n = 155), type 2 (n = 135), and type 2V (n = 60) clinical isolates and controls were averaged, baseline-corrected, and normalized using GRAMS32/A1 spectral software package (Galactic Industries, Nashua, NH). Red, average spectrum of all type 1 M. pneumoniae strains; green, average spectrum of all type 2 M. pneumoniae strains; blue, average spectrum of all type 2V M. pneumoniae strains. Peaks unique to a specific genotype of M. pneumoniae are indicated by arrows and identified above the spectral fingerprint. Type 1 peaks, red arrows; type 2 peaks, green arrows; and type 2V peaks, blue arrows. Inset at top right of image depicts zoomed-in view of the type 2 doublet at 767 and 778 cm-1.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4487258&req=5

pone.0131831.g006: Comparison of averaged, baseline-corrected, and normalized SERS spectra for type 1, type 2, and type 2V genotypes.Raw spectra of all type 1 (n = 155), type 2 (n = 135), and type 2V (n = 60) clinical isolates and controls were averaged, baseline-corrected, and normalized using GRAMS32/A1 spectral software package (Galactic Industries, Nashua, NH). Red, average spectrum of all type 1 M. pneumoniae strains; green, average spectrum of all type 2 M. pneumoniae strains; blue, average spectrum of all type 2V M. pneumoniae strains. Peaks unique to a specific genotype of M. pneumoniae are indicated by arrows and identified above the spectral fingerprint. Type 1 peaks, red arrows; type 2 peaks, green arrows; and type 2V peaks, blue arrows. Inset at top right of image depicts zoomed-in view of the type 2 doublet at 767 and 778 cm-1.
Mentions: Additionally, we compared averaged, baseline-corrected, and normalized spectra of all three genotypes to look for any differences in band pattern between the three genotypes that could be contributing to the classification capabilities demonstrated in the PLS-DA modeling (Fig 6). The majority of the spectral fingerprint was identical for all three strain types, which is to be expected since they are all the same species and classify as such in the PLS-DA models shown in Figs 2 and 3. However, several visible differences in band pattern were present in the spectra for each genotype of M. pneumoniae, which could account for the ability of NA-SERS to distinguish between the three genotypes with statistically significant sensitivity and specificity. The averaged type 1 spectrum had two unique peaks, one at 1636 cm-1 that does not appear in the averaged type 2 or 2V spectra, and one at 959 cm-1 which appeared as more distinct and shifted slightly right in the type 1 spectrum when compared to the type 2 spectrum, and did not appear in the type 2V spectrum. The averaged type 2 strain spectrum was very similar to the type 1 strain spectrum aside from the differences mentioned above and the presence of a doublet at 767 and 778 cm-1, which appeared as more distinct than that present in the type 2V spectrum and as a broad singlet in the type 1 spectrum. The averaged type 2V spectrum appeared to be the most distinct of the three, with a doublet at 875 and 890 cm-1 that appeared as a single peak at 890 in type 1 and 2 spectra, and a small peak at 521 that was also absent in type 1 and 2 spectra. While these spectral differences were extremely subtle, chemometric analysis is highly capable of discerning differences such as these with substantial discriminatory classification power [43].

Bottom Line: At present the most effective means for detection and strain-typing is quantitative polymerase chain reaction (qPCR), which can exhibit excellent sensitivity and specificity but requires separate tests for detection and genotyping, lacks standardization between available tests and between labs, and has limited practicality for widespread, point-of-care use.Here we demonstrate using partial least squares- discriminatory analysis (PLS-DA) of sample spectra that NA-SERS correctly identified M. pneumoniae clinical isolates from globally diverse origins and distinguished these from a panel of 12 other human commensal and pathogenic mycoplasma species with 100% cross-validated statistical accuracy.Furthermore, PLS-DA correctly classified by strain type all 30 clinical isolates with 96% cross-validated accuracy for type 1 strains, 98% cross-validated accuracy for type 2 strains, and 90% cross-validated accuracy for type 2V strains.

View Article: PubMed Central - PubMed

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

ABSTRACT
Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the human respiratory tract that accounts for > 20% of all community-acquired pneumonia (CAP). At present the most effective means for detection and strain-typing is quantitative polymerase chain reaction (qPCR), which can exhibit excellent sensitivity and specificity but requires separate tests for detection and genotyping, lacks standardization between available tests and between labs, and has limited practicality for widespread, point-of-care use. We have developed and previously described a silver nanorod array-surface enhanced Raman Spectroscopy (NA-SERS) biosensing platform capable of detecting M. pneumoniae with statistically significant specificity and sensitivity in simulated and true clinical throat swab samples, and the ability to distinguish between reference strains of the two main genotypes of M. pneumoniae. Furthermore, we have established a qualitative lower endpoint of detection for NA-SERS of < 1 genome equivalent (cell/μl) and a quantitative multivariate detection limit of 5.3 ± 1 cells/μl. Here we demonstrate using partial least squares- discriminatory analysis (PLS-DA) of sample spectra that NA-SERS correctly identified M. pneumoniae clinical isolates from globally diverse origins and distinguished these from a panel of 12 other human commensal and pathogenic mycoplasma species with 100% cross-validated statistical accuracy. Furthermore, PLS-DA correctly classified by strain type all 30 clinical isolates with 96% cross-validated accuracy for type 1 strains, 98% cross-validated accuracy for type 2 strains, and 90% cross-validated accuracy for type 2V strains.

No MeSH data available.


Related in: MedlinePlus