Large Community Outbreak of Legionnaires Disease Potentially Associated with a Cooling Tower - Napa County, California, 2022.

Legionnaires disease is a serious infection acquired by inhalation of water droplets from human-made building water systems that contain Legionella bacteria. On July 11 and 12, 2022, Napa County Public Health (NCPH) in California received reports of three positive urinary antigen tests for Legionella pneumophila serogroup 1 in the town of Napa. By July 21, six Legionnaires disease cases had been confirmed among Napa County residents, compared with a baseline of one or two cases per year. NCPH requested assistance from the California Department of Public Health (CDPH) and CDC to aid in the investigations. Close temporal and geospatial clustering permitted a focused environmental sampling strategy of high-risk facilities which, coupled with whole genome sequencing results from samples and investigation of water system maintenance, facilitated potential linking of the outbreak with an environmental source. NCPH, with technical support from CDC and CDPH, instructed and monitored remediation practices for all environmental locations that tested positive for Legionella. The investigation response to this community outbreak illustrates the importance of interdisciplinary collaboration by public health agencies, laboratory support, timely communication with the public, and cooperation of managers of potentially implicated water systems. Timely identification of possible sources, sampling, and remediation of any facility testing positive for Legionella is crucial to interrupting further transmission.

VARIABLE SELECTION AND BIOMARKER CORRELATION IN THE ANALYSIS OF MYCOPLASMA PNEUMONIAE STRAINS BY SURFACE-ENHANCED RAMAN SPECTROSCOPY.

Mycoplasma pneumoniae is a human respiratory tract pathogen causing chronic bronchitis and atypical or "walking" pneumonia. The major surface protein P1 must form complexes with proteins P30 and P40/P90 in order to function in receptor binding and gliding motility, and variability in P1 and P40/P90 distinguishes the two major M. pneumoniae genotypes. Strains belonging to each genotype can be differentiated with high sensitivity and specificity by utilizing surface-enhanced Raman spectroscopy on silver nanorod arrays. Here we used the variable selection method of Variable Importance in Projection (VIP) to identify Raman bands important in M. pneumoniae strain classification. Furthermore, VIP analysis of mutants lacking P40/P90, or P1and P40/P90, correlated certain Raman bands important in distinguishing genotypes, with specific mycoplasma surface protein composition and presentation. Variable selection, and its correlation with specific mycoplasma surface components, is an important next step in developing this platform for M. pneumoniae detection and genotyping.

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

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.

The multivariate detection limit for Mycoplasma pneumoniae as determined by nanorod array-surface enhanced Raman spectroscopy and comparison with limit of detection by qPCR.

Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the human respiratory tract that accounts for up to 20% of community-acquired pneumonia. At present, the standard for detection and genotyping is quantitative polymerase chain reaction (qPCR), which can exhibit excellent sensitivity but lacks standardization and has limited practicality for widespread, point-of-care use. We previously described a Ag nanorod array-surface enhanced Raman spectroscopy (NA-SERS) biosensing platform capable of detecting M. pneumoniae in simulated and true clinical throat swab samples with statistically significant specificity and sensitivity. We report here that differences in sample preparation influence the integrity of mycoplasma cells for NA-SERS analysis, which in turn impacts the resulting spectra. We have established a multivariate detection limit (MDL) using NA-SERS for M. pneumoniae intact-cell sample preparations. Using an adaptation of International Union of Pure and Applied Chemistry (IUPAC)-recommended methods for analyzing multivariate data sets, we found that qPCR had roughly 10× better detection limits than NA-SERS when expressed in CFU ml(-1) and DNA concentration (fg). However, the NA-SERS MDL for intact M. pneumoniae was 5.3 ± 1.0 genome equivalents (cells per µl). By comparison, qPCR of a parallel set of samples yielded a limit of detection of 2.5 ± 0.25 cells per µl. Therefore, for certain standard metrics NA-SERS provides a multivariate detection limit for M. pneumoniae that is essentially identical to that determined via qPCR.