<p><b>(A)</b> VGR-1<b>A</b> is conserved in strain Philadelphia-1 (... more <p><b>(A)</b> VGR-1<b>A</b> is conserved in strain Philadelphia-1 (CDC) and in 19 of 22 additional ST36 strains, including strains C2-S, C3-O, C4-S, C5-P, C6-S, C7-O, C8-S, C9-S, C10-S, C11-O, E3-N, E4-N, E5-N, E6-N, E7-O, E8-O, E9-O, E10-P, E11-U, as well as <i>L</i>. <i>pneumophila</i> str. LPE509 and a sg12 strain (ATCC 43290), while VGR-1<b>B</b> is conserved in strains Philadelphia-2, -3, -4, E1-P, C1-S, and Paris. <b>(B)</b> VGR-2<b>A</b> is conserved in strain Philadelphia-1 (CDC) and in 21 of 22 additional ST36 strains, including C1-S, C2-S, C3-O, C4-S, C5-P, C6-S, C7-O, C8-S, C9-S, C10-S, C11-O, E2-N, E3-N, E4-N, E5-N, E6-N, E7-O, E8-O, E9-O, E10-P, and E11-U, while VGR-2<b>B</b> is conserved in strains Philadelphia-2, -3, -4, E1-P, and Paris. Solid grey strips, vertical, and horizontal black lines represent conserved sequence, SNPs, and sequence gaps, respectively, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164074#pone.0164074.g003" target="_blank">Fig 3</a>. A Solid blue or green rectangle above the sequence delineates the boundaries of the VGR. Nucleotide boundaries and neighboring genes (in red) are relative to the genome immediately below these descriptions.</p
<p>Analysis conducted with 799 core, non-recombinant SNPs obtained from 187 isolate sequenc... more <p>Analysis conducted with 799 core, non-recombinant SNPs obtained from 187 isolate sequences after applying the Gubbins algorithm (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0206110#sec010" target="_blank">Methods</a>). Colors were generated automatically in GenGIS to distinguish isolates based on the 9 NOAA climate regions. Regions and matching colors lines and branches include: Northwest, bright green; West North Central, grey; East North Central, dark green; Northeast, blue; West, red; Southwest, orange; South, salmon; Southeast, maroon; Central, bright pink. Isolates from Hawaii and Alaska are not represented in this figure.</p
<p>ProgressiveMauve was used to compare the fully assembled sequences of the Philadelphia h... more <p>ProgressiveMauve was used to compare the fully assembled sequences of the Philadelphia historical <i>Legionella</i> strains as well as isolate E1-P. The minimum weight for pairwise LCBs (locally collinear blocks), which share common colors across genomes, was set to 100, otherwise, the program was run using default parameters as described in the Methods. The general clade organization, as well as the identity and location of the ~40-kb pP36-Ph and the ~45-kb pLP45 elements are shown. The general, expanded Philadelphia clade organization from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164074#pone.0164074.g001" target="_blank">Fig 1</a> is shown, therefore the phylogenetic distances are not to scale.</p
<p><b>A</b>) Unrooted tree of 28 different <i>L</i>. <i>pneum... more <p><b>A</b>) Unrooted tree of 28 different <i>L</i>. <i>pneumophila</i> sequence types, including the ST1 clade (collapsed red triangle) alongside an international ST752 double locus variant of ST1. Clade numbers are discussed in the text. Isolate ID’s for each ST are defined in the ‘Comments’ column of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0206110#pone.0206110.s002" target="_blank">S1 Table</a>. <b>B</b>) All-SNP-based phylogenetic tree of 502 ST1 and ST-like isolates rooted on the ST752 international sequence, ‘SZ2012007’. Isolate names highlighted by outbreak or potential outbreak. Clade letter designations are discussed in the text. Additional metadata displayed on the outer rings labeled according to the included legends.</p
Here, we report the complete genome sequences of Legionella pneumophila serogroup 1 strains OLDA ... more Here, we report the complete genome sequences of Legionella pneumophila serogroup 1 strains OLDA and Pontiac, which predate the 1976 Philadelphia Legionnaires' disease outbreak. Strain OLDA was isolated in 1947 from an apparent sporadic case, and strain Pontiac caused an explosive outbreak at a Michigan health department in 1968.
This event is hosted by Sigma Iota Rho, the International Studies Honor Society.Presented on Octo... more This event is hosted by Sigma Iota Rho, the International Studies Honor Society.Presented on October 6, 2014 at 6:00 p.m. in the Student Center Theater on the Georgia Tech Campus.Runtime: 80:41 minutesThis interdisciplinary panel will feature Dr. Margaret Kosal of the Nunn School, Dr. Alberto Fuentes of the Nunn School, Dr. Dave Bull of the Centers for Disease Control, and Dr. Jonas Winchell of the School of Biology. These four professionals will explain the microbiology and epidemiology of the ebola virus, the recent U.S. military response, and the implications that this epidemic poses for economic development in Western Africa
New diagnostic platforms often use nasopharyngeal or oropharyngeal (NP/OP) swabs for pathogen det... more New diagnostic platforms often use nasopharyngeal or oropharyngeal (NP/OP) swabs for pathogen detection for patients hospitalized with community-acquired pneumonia (CAP). We applied multipathogen testing to high-quality sputum specimens to determine if more pathogens can be identified relative to NP/OP swabs. Children (<18 years old) and adults hospitalized with CAP were enrolled over 2.5 years through the Etiology of Pneumonia in the Community (EPIC) study. NP/OP specimens with matching high-quality sputum (defined as ≤10 epithelial cells/low-power field [lpf] and ≥25 white blood cells/lpf or a quality score [q-score] definition of 2+) were tested by TaqMan array card (TAC), a multipathogen real-time PCR detection platform. Among 236 patients with matched specimens, a higher proportion of sputum specimens had ≥1 pathogen detected compared with NP/OP specimens in children (93% versus 68%; P < 0.0001) and adults (88% versus 61%; P < 0.0001); for each pathogen targeted, cross...
Members of the Gram-negative genusLegionellaare typically found in freshwater environments, with ... more Members of the Gram-negative genusLegionellaare typically found in freshwater environments, with the exception ofL. longbeachae, which is present in composts and potting mixes. When contaminated aerosols are inhaled, legionellosis may result, typically as either the more serious pneumonia Legionnaires’ disease or the less severe flu-like illness Pontiac fever. It is presumed that all species of the genusLegionellaare capable of causing disease in humans. As a followup to a prior clinical study of legionellosis in rural Thailand, indigenous soil samples were collected proximal to cases’ homes and workplaces and tested for the presence of legionellae by culture. We obtained 115 isolates from 22/39 soil samples and used sequence-based methods to identify 12 known species ofLegionellarepresented by 87 isolates.
Recently we evaluated a custom TaqMan array card (TAC) detection system, formerly known as a TaqM... more Recently we evaluated a custom TaqMan array card (TAC) detection system, formerly known as a TaqMan low-density array (TLDA) card, for simultaneous real-time PCR identification of 21 pathogens and three control targets in duplicate from respiratory specimens (M. Kodani et al., J. Clin. Microbiol. 49:2175–2182, 2011). We engineered an adaptable and expandable system of in vitro RNA transcripts to serve as a combined positive control for both DNA and RNA targets in multiple-pathogen-detection technologies based on real-time reverse transcription-PCR.
We report here the complete genome sequences of three Legionella pneumophila isolates that are as... more We report here the complete genome sequences of three Legionella pneumophila isolates that are associated with a Legionnaires’ disease outbreak in New York in 2012. Two clinical isolates (D7630 and D7632) and one environmental isolate (D7631) were recovered from this outbreak. A single isolate-specific virulence gene was found in D7632. These isolates were included in a large study evaluating the genomic resolution of various bioinformatics approaches for L. pneumophila serogroup 1 isolates.
Background The causal attribution of bacterial pathogens to severe acute respiratory infections (... more Background The causal attribution of bacterial pathogens to severe acute respiratory infections (SARI) is challenging because many bacteria are frequently detected in the upper respiratory tract of asymptomatic persons. Quantification of pathogen load may help differentiate asymptomatic pathogen carriage from clinically significant infection. We aimed to determine whether real-time PCR (rt-PCR) cycle threshold (Ct) values, as a proxy for bacterial load, differ between adults with SARI and asymptomatic adults. Methods Adults with SARI (acute onset of fever and cough, requiring hospitalization) were frequency matched to asymptomatic adults (enrolled from trauma and orthopedic inpatient wards) by age group, catchment area, and enrollment date at three surveillance sites in Guatemala. Nasopharyngeal and oropharyngeal specimens were collected from all participants and tested for pathogens using rt-PCR. Using the Wilcoxon rank sum test, we compared the distributions and median Ct values b...
Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-pro... more Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-producing strains of Corynebacterium diphtheriae, with similar illness produced occasionally by toxigenic Corynebacterium ulcerans or, rarely, Corynebacterium pseudotuberculosis. While diphtheria laboratory confirmation requires culture methods to determine toxigenicity, real-time PCR (RT-PCR) provides a faster method to detect the toxin gene (tox). Nontoxigenic tox-bearing (NTTB) Corynebacterium isolates have been described, but impact of these isolates on the accuracy of molecular diagnostics is not well characterized. Here, we describe a new triplex RT-PCR assay to detect tox and distinguish C. diphtheriae from the closely related species C. ulcerans and C. pseudotuberculosis. Analytical sensitivity and specificity of the assay were assessed in comparison to culture using 690 previously characterized microbial isolates. The new triplex assay characterized Corynebacterium isolates accurat...
Background Psittacosis is a respiratory illness caused by Chlamydia psittaci. The most commonly a... more Background Psittacosis is a respiratory illness caused by Chlamydia psittaci. The most commonly available diagnostic tests are serologic tests, which have low sensitivity and can cross-react with other chlamydial species. Serologic tests also require paired sera collected weeks apart, which is impractical for patient management. Real-time polymerase chain reaction (rtPCR) testing for C. psittaci is rapid, sensitive, and specific. However, rtRCR testing is only available at the CDC Respiratory Diseases Branch laboratory, and appropriate clinical specimen types need to be validated since psittacosis case detection is infrequent. In 2018, the first large psittacosis outbreak in the United States in 30 years occurred, allowing assessment of rtPCR performance among multiple clinical specimen types. Methods rtPCR test positivity rate and turnaround time were determined among 89 specimens tested at CDC from 54 outbreak patients with suspected psittacosis. rtPCR testing was performed on nuc...
Between 2000 and 2017, a total of 236 Legionella species isolates from Arizona were submitted to ... more Between 2000 and 2017, a total of 236 Legionella species isolates from Arizona were submitted to the CDC for reference testing. Most of these isolates were recovered from bronchoalveolar lavage specimens. Although the incidence of legionellosis in Arizona is less than the overall U.S. incidence, Arizona submits the largest number of isolates to the CDC for testing compared to those from other states. In addition to a higher proportion of culture confirmation of legionellosis cases in Arizona than in other states, all Legionellapneumophila isolates are forwarded to the CDC for confirmatory testing. Compared to that from other states, a higher proportion of isolates from Arizona were identified as belonging to L. pneumophila serogroups 6 (28.2%) and 8 (8.9%). Genome sequencing was conducted on 113 L. pneumophila clinical isolates not known to be associated with outbreaks in order to understand the genomic diversity of strains causing legionellosis in Arizona. Whole-genome multilocus s...
<p><b>(A)</b> VGR-1<b>A</b> is conserved in strain Philadelphia-1 (... more <p><b>(A)</b> VGR-1<b>A</b> is conserved in strain Philadelphia-1 (CDC) and in 19 of 22 additional ST36 strains, including strains C2-S, C3-O, C4-S, C5-P, C6-S, C7-O, C8-S, C9-S, C10-S, C11-O, E3-N, E4-N, E5-N, E6-N, E7-O, E8-O, E9-O, E10-P, E11-U, as well as <i>L</i>. <i>pneumophila</i> str. LPE509 and a sg12 strain (ATCC 43290), while VGR-1<b>B</b> is conserved in strains Philadelphia-2, -3, -4, E1-P, C1-S, and Paris. <b>(B)</b> VGR-2<b>A</b> is conserved in strain Philadelphia-1 (CDC) and in 21 of 22 additional ST36 strains, including C1-S, C2-S, C3-O, C4-S, C5-P, C6-S, C7-O, C8-S, C9-S, C10-S, C11-O, E2-N, E3-N, E4-N, E5-N, E6-N, E7-O, E8-O, E9-O, E10-P, and E11-U, while VGR-2<b>B</b> is conserved in strains Philadelphia-2, -3, -4, E1-P, and Paris. Solid grey strips, vertical, and horizontal black lines represent conserved sequence, SNPs, and sequence gaps, respectively, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164074#pone.0164074.g003" target="_blank">Fig 3</a>. A Solid blue or green rectangle above the sequence delineates the boundaries of the VGR. Nucleotide boundaries and neighboring genes (in red) are relative to the genome immediately below these descriptions.</p
<p>Analysis conducted with 799 core, non-recombinant SNPs obtained from 187 isolate sequenc... more <p>Analysis conducted with 799 core, non-recombinant SNPs obtained from 187 isolate sequences after applying the Gubbins algorithm (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0206110#sec010" target="_blank">Methods</a>). Colors were generated automatically in GenGIS to distinguish isolates based on the 9 NOAA climate regions. Regions and matching colors lines and branches include: Northwest, bright green; West North Central, grey; East North Central, dark green; Northeast, blue; West, red; Southwest, orange; South, salmon; Southeast, maroon; Central, bright pink. Isolates from Hawaii and Alaska are not represented in this figure.</p
<p>ProgressiveMauve was used to compare the fully assembled sequences of the Philadelphia h... more <p>ProgressiveMauve was used to compare the fully assembled sequences of the Philadelphia historical <i>Legionella</i> strains as well as isolate E1-P. The minimum weight for pairwise LCBs (locally collinear blocks), which share common colors across genomes, was set to 100, otherwise, the program was run using default parameters as described in the Methods. The general clade organization, as well as the identity and location of the ~40-kb pP36-Ph and the ~45-kb pLP45 elements are shown. The general, expanded Philadelphia clade organization from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164074#pone.0164074.g001" target="_blank">Fig 1</a> is shown, therefore the phylogenetic distances are not to scale.</p
<p><b>A</b>) Unrooted tree of 28 different <i>L</i>. <i>pneum... more <p><b>A</b>) Unrooted tree of 28 different <i>L</i>. <i>pneumophila</i> sequence types, including the ST1 clade (collapsed red triangle) alongside an international ST752 double locus variant of ST1. Clade numbers are discussed in the text. Isolate ID’s for each ST are defined in the ‘Comments’ column of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0206110#pone.0206110.s002" target="_blank">S1 Table</a>. <b>B</b>) All-SNP-based phylogenetic tree of 502 ST1 and ST-like isolates rooted on the ST752 international sequence, ‘SZ2012007’. Isolate names highlighted by outbreak or potential outbreak. Clade letter designations are discussed in the text. Additional metadata displayed on the outer rings labeled according to the included legends.</p
Here, we report the complete genome sequences of Legionella pneumophila serogroup 1 strains OLDA ... more Here, we report the complete genome sequences of Legionella pneumophila serogroup 1 strains OLDA and Pontiac, which predate the 1976 Philadelphia Legionnaires' disease outbreak. Strain OLDA was isolated in 1947 from an apparent sporadic case, and strain Pontiac caused an explosive outbreak at a Michigan health department in 1968.
This event is hosted by Sigma Iota Rho, the International Studies Honor Society.Presented on Octo... more This event is hosted by Sigma Iota Rho, the International Studies Honor Society.Presented on October 6, 2014 at 6:00 p.m. in the Student Center Theater on the Georgia Tech Campus.Runtime: 80:41 minutesThis interdisciplinary panel will feature Dr. Margaret Kosal of the Nunn School, Dr. Alberto Fuentes of the Nunn School, Dr. Dave Bull of the Centers for Disease Control, and Dr. Jonas Winchell of the School of Biology. These four professionals will explain the microbiology and epidemiology of the ebola virus, the recent U.S. military response, and the implications that this epidemic poses for economic development in Western Africa
New diagnostic platforms often use nasopharyngeal or oropharyngeal (NP/OP) swabs for pathogen det... more New diagnostic platforms often use nasopharyngeal or oropharyngeal (NP/OP) swabs for pathogen detection for patients hospitalized with community-acquired pneumonia (CAP). We applied multipathogen testing to high-quality sputum specimens to determine if more pathogens can be identified relative to NP/OP swabs. Children (<18 years old) and adults hospitalized with CAP were enrolled over 2.5 years through the Etiology of Pneumonia in the Community (EPIC) study. NP/OP specimens with matching high-quality sputum (defined as ≤10 epithelial cells/low-power field [lpf] and ≥25 white blood cells/lpf or a quality score [q-score] definition of 2+) were tested by TaqMan array card (TAC), a multipathogen real-time PCR detection platform. Among 236 patients with matched specimens, a higher proportion of sputum specimens had ≥1 pathogen detected compared with NP/OP specimens in children (93% versus 68%; P < 0.0001) and adults (88% versus 61%; P < 0.0001); for each pathogen targeted, cross...
Members of the Gram-negative genusLegionellaare typically found in freshwater environments, with ... more Members of the Gram-negative genusLegionellaare typically found in freshwater environments, with the exception ofL. longbeachae, which is present in composts and potting mixes. When contaminated aerosols are inhaled, legionellosis may result, typically as either the more serious pneumonia Legionnaires’ disease or the less severe flu-like illness Pontiac fever. It is presumed that all species of the genusLegionellaare capable of causing disease in humans. As a followup to a prior clinical study of legionellosis in rural Thailand, indigenous soil samples were collected proximal to cases’ homes and workplaces and tested for the presence of legionellae by culture. We obtained 115 isolates from 22/39 soil samples and used sequence-based methods to identify 12 known species ofLegionellarepresented by 87 isolates.
Recently we evaluated a custom TaqMan array card (TAC) detection system, formerly known as a TaqM... more Recently we evaluated a custom TaqMan array card (TAC) detection system, formerly known as a TaqMan low-density array (TLDA) card, for simultaneous real-time PCR identification of 21 pathogens and three control targets in duplicate from respiratory specimens (M. Kodani et al., J. Clin. Microbiol. 49:2175–2182, 2011). We engineered an adaptable and expandable system of in vitro RNA transcripts to serve as a combined positive control for both DNA and RNA targets in multiple-pathogen-detection technologies based on real-time reverse transcription-PCR.
We report here the complete genome sequences of three Legionella pneumophila isolates that are as... more We report here the complete genome sequences of three Legionella pneumophila isolates that are associated with a Legionnaires’ disease outbreak in New York in 2012. Two clinical isolates (D7630 and D7632) and one environmental isolate (D7631) were recovered from this outbreak. A single isolate-specific virulence gene was found in D7632. These isolates were included in a large study evaluating the genomic resolution of various bioinformatics approaches for L. pneumophila serogroup 1 isolates.
Background The causal attribution of bacterial pathogens to severe acute respiratory infections (... more Background The causal attribution of bacterial pathogens to severe acute respiratory infections (SARI) is challenging because many bacteria are frequently detected in the upper respiratory tract of asymptomatic persons. Quantification of pathogen load may help differentiate asymptomatic pathogen carriage from clinically significant infection. We aimed to determine whether real-time PCR (rt-PCR) cycle threshold (Ct) values, as a proxy for bacterial load, differ between adults with SARI and asymptomatic adults. Methods Adults with SARI (acute onset of fever and cough, requiring hospitalization) were frequency matched to asymptomatic adults (enrolled from trauma and orthopedic inpatient wards) by age group, catchment area, and enrollment date at three surveillance sites in Guatemala. Nasopharyngeal and oropharyngeal specimens were collected from all participants and tested for pathogens using rt-PCR. Using the Wilcoxon rank sum test, we compared the distributions and median Ct values b...
Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-pro... more Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-producing strains of Corynebacterium diphtheriae, with similar illness produced occasionally by toxigenic Corynebacterium ulcerans or, rarely, Corynebacterium pseudotuberculosis. While diphtheria laboratory confirmation requires culture methods to determine toxigenicity, real-time PCR (RT-PCR) provides a faster method to detect the toxin gene (tox). Nontoxigenic tox-bearing (NTTB) Corynebacterium isolates have been described, but impact of these isolates on the accuracy of molecular diagnostics is not well characterized. Here, we describe a new triplex RT-PCR assay to detect tox and distinguish C. diphtheriae from the closely related species C. ulcerans and C. pseudotuberculosis. Analytical sensitivity and specificity of the assay were assessed in comparison to culture using 690 previously characterized microbial isolates. The new triplex assay characterized Corynebacterium isolates accurat...
Background Psittacosis is a respiratory illness caused by Chlamydia psittaci. The most commonly a... more Background Psittacosis is a respiratory illness caused by Chlamydia psittaci. The most commonly available diagnostic tests are serologic tests, which have low sensitivity and can cross-react with other chlamydial species. Serologic tests also require paired sera collected weeks apart, which is impractical for patient management. Real-time polymerase chain reaction (rtPCR) testing for C. psittaci is rapid, sensitive, and specific. However, rtRCR testing is only available at the CDC Respiratory Diseases Branch laboratory, and appropriate clinical specimen types need to be validated since psittacosis case detection is infrequent. In 2018, the first large psittacosis outbreak in the United States in 30 years occurred, allowing assessment of rtPCR performance among multiple clinical specimen types. Methods rtPCR test positivity rate and turnaround time were determined among 89 specimens tested at CDC from 54 outbreak patients with suspected psittacosis. rtPCR testing was performed on nuc...
Between 2000 and 2017, a total of 236 Legionella species isolates from Arizona were submitted to ... more Between 2000 and 2017, a total of 236 Legionella species isolates from Arizona were submitted to the CDC for reference testing. Most of these isolates were recovered from bronchoalveolar lavage specimens. Although the incidence of legionellosis in Arizona is less than the overall U.S. incidence, Arizona submits the largest number of isolates to the CDC for testing compared to those from other states. In addition to a higher proportion of culture confirmation of legionellosis cases in Arizona than in other states, all Legionellapneumophila isolates are forwarded to the CDC for confirmatory testing. Compared to that from other states, a higher proportion of isolates from Arizona were identified as belonging to L. pneumophila serogroups 6 (28.2%) and 8 (8.9%). Genome sequencing was conducted on 113 L. pneumophila clinical isolates not known to be associated with outbreaks in order to understand the genomic diversity of strains causing legionellosis in Arizona. Whole-genome multilocus s...
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