Session Type: ePosters
Session Title: ePosters
Authors(s): V. De Sario (1), S. Morris-Jones (1), P. Grant (2), A. Ward (2), S. De (1)
Authors Affiliations(s): (1) University College London Hospital - London (United Kingdom) - London (United Kingdom), United Kingdom, (2) Health Services Laboratories, United Kingdom
Background:
Broad–range 16S ribosomal RNA gene polymerase chain reaction (16S-PCR) is a routinely-utilised assay. As part of a retrospective validation of novel species-specific real-time PCR (RT-PCR) assays, we evaluated samples tested in our laboratory over 3 years.
Methods:
Samples undergoing routine culture and referred for molecular investigations were tested on an in-house assay, utilising conventional PCR targeting the V1-V2 regions of the 16S-rRNA gene. Positive amplicons were sequenced using Sanger methodology. Species-specific assays utilising RT-PCR probes targeting S. aureus femA/mecA, S. pneumoniae, S. pyogenes, S. agalactiae, L. monocytogenes, H. influenzae, N. meningitidis and Mycobacterium spp., were performed at the discretion of the duty microbiologist; targets were selected according to the clinical context.
Results:
Between September 2017 and September 2020, 955 samples from UCLH NHS Trust underwent 16S-PCR, including 183 for species-specific PCR.
Samples were from a variety of sterile sites, including 301 neurosurgical and 197 orthopaedic specimens.
16S-PCR was positive in 330 (34.5%). Amongst the positive samples, 60 (20%) had positive, concordant microscopy, 104 (10.9%) had positive, concordant culture with 22 (2.3%) yielding a different culture result and 188 (19.7%) were culture-negative. 49 specimens were 16S-PCR-positive without species identification (probably due to mixed bacterial populations). Of the 16S-PCR-negative samples 73 (7.6%) were ultimately culture-positive. A wide-range of organisms were detected (Table 1).
Species-specific PCR was tested in 183 samples and was positive in 33 (18%) and detected 8 further positive results where 16S-PCR was negative. Concordance with 16S-PCR-positives was almost universal; one positive Mycobacterium sp. RT-PCR coinciding with Fusobacterium nucleatum on 16S-PCR, which could have been undetected otherwise (Table 2a and 2b).
Conclusions:Molecular methods identified a significant number of organisms in culture-negative specimens. Positive 16S-PCR was strongly associated with positive microscopy. Mixed cultures were problematic for 16S-PCR species identification. However combining culture with 16S-PCR and more sensitive species-specific-PCR assays maximised detection of significant pathogens.
Keyword(s): 16S, PCR, rRNASession Type: ePosters
Session Title: ePosters
Authors(s): V. De Sario (1), S. Morris-Jones (1), P. Grant (2), A. Ward (2), S. De (1)
Authors Affiliations(s): (1) University College London Hospital - London (United Kingdom) - London (United Kingdom), United Kingdom, (2) Health Services Laboratories, United Kingdom
Background:
Broad–range 16S ribosomal RNA gene polymerase chain reaction (16S-PCR) is a routinely-utilised assay. As part of a retrospective validation of novel species-specific real-time PCR (RT-PCR) assays, we evaluated samples tested in our laboratory over 3 years.
Methods:
Samples undergoing routine culture and referred for molecular investigations were tested on an in-house assay, utilising conventional PCR targeting the V1-V2 regions of the 16S-rRNA gene. Positive amplicons were sequenced using Sanger methodology. Species-specific assays utilising RT-PCR probes targeting S. aureus femA/mecA, S. pneumoniae, S. pyogenes, S. agalactiae, L. monocytogenes, H. influenzae, N. meningitidis and Mycobacterium spp., were performed at the discretion of the duty microbiologist; targets were selected according to the clinical context.
Results:
Between September 2017 and September 2020, 955 samples from UCLH NHS Trust underwent 16S-PCR, including 183 for species-specific PCR.
Samples were from a variety of sterile sites, including 301 neurosurgical and 197 orthopaedic specimens.
16S-PCR was positive in 330 (34.5%). Amongst the positive samples, 60 (20%) had positive, concordant microscopy, 104 (10.9%) had positive, concordant culture with 22 (2.3%) yielding a different culture result and 188 (19.7%) were culture-negative. 49 specimens were 16S-PCR-positive without species identification (probably due to mixed bacterial populations). Of the 16S-PCR-negative samples 73 (7.6%) were ultimately culture-positive. A wide-range of organisms were detected (Table 1).
Species-specific PCR was tested in 183 samples and was positive in 33 (18%) and detected 8 further positive results where 16S-PCR was negative. Concordance with 16S-PCR-positives was almost universal; one positive Mycobacterium sp. RT-PCR coinciding with Fusobacterium nucleatum on 16S-PCR, which could have been undetected otherwise (Table 2a and 2b).
Conclusions:Molecular methods identified a significant number of organisms in culture-negative specimens. Positive 16S-PCR was strongly associated with positive microscopy. Mixed cultures were problematic for 16S-PCR species identification. However combining culture with 16S-PCR and more sensitive species-specific-PCR assays maximised detection of significant pathogens.
Keyword(s): 16S, PCR, rRNA
Session Type: ePosters
Session Title: ePosters
Authors(s): V. De Sario (1), S. Morris-Jones (1), P. Grant (2), A. Ward (2), S. De (1)
Authors Affiliations(s): (1) University College London Hospital - London (United Kingdom) - London (United Kingdom), United Kingdom, (2) Health Services Laboratories, United Kingdom
Background:
Broad–range 16S ribosomal RNA gene polymerase chain reaction (16S-PCR) is a routinely-utilised assay. As part of a retrospective validation of novel species-specific real-time PCR (RT-PCR) assays, we evaluated samples tested in our laboratory over 3 years.
Methods:
Samples undergoing routine culture and referred for molecular investigations were tested on an in-house assay, utilising conventional PCR targeting the V1-V2 regions of the 16S-rRNA gene. Positive amplicons were sequenced using Sanger methodology. Species-specific assays utilising RT-PCR probes targeting S. aureus femA/mecA, S. pneumoniae, S. pyogenes, S. agalactiae, L. monocytogenes, H. influenzae, N. meningitidis and Mycobacterium spp., were performed at the discretion of the duty microbiologist; targets were selected according to the clinical context.
Results:
Between September 2017 and September 2020, 955 samples from UCLH NHS Trust underwent 16S-PCR, including 183 for species-specific PCR.
Samples were from a variety of sterile sites, including 301 neurosurgical and 197 orthopaedic specimens.
16S-PCR was positive in 330 (34.5%). Amongst the positive samples, 60 (20%) had positive, concordant microscopy, 104 (10.9%) had positive, concordant culture with 22 (2.3%) yielding a different culture result and 188 (19.7%) were culture-negative. 49 specimens were 16S-PCR-positive without species identification (probably due to mixed bacterial populations). Of the 16S-PCR-negative samples 73 (7.6%) were ultimately culture-positive. A wide-range of organisms were detected (Table 1).
Species-specific PCR was tested in 183 samples and was positive in 33 (18%) and detected 8 further positive results where 16S-PCR was negative. Concordance with 16S-PCR-positives was almost universal; one positive Mycobacterium sp. RT-PCR coinciding with Fusobacterium nucleatum on 16S-PCR, which could have been undetected otherwise (Table 2a and 2b).
Conclusions:Molecular methods identified a significant number of organisms in culture-negative specimens. Positive 16S-PCR was strongly associated with positive microscopy. Mixed cultures were problematic for 16S-PCR species identification. However combining culture with 16S-PCR and more sensitive species-specific-PCR assays maximised detection of significant pathogens.
Keyword(s): 16S, PCR, rRNASession Type: ePosters
Session Title: ePosters
Authors(s): V. De Sario (1), S. Morris-Jones (1), P. Grant (2), A. Ward (2), S. De (1)
Authors Affiliations(s): (1) University College London Hospital - London (United Kingdom) - London (United Kingdom), United Kingdom, (2) Health Services Laboratories, United Kingdom
Background:
Broad–range 16S ribosomal RNA gene polymerase chain reaction (16S-PCR) is a routinely-utilised assay. As part of a retrospective validation of novel species-specific real-time PCR (RT-PCR) assays, we evaluated samples tested in our laboratory over 3 years.
Methods:
Samples undergoing routine culture and referred for molecular investigations were tested on an in-house assay, utilising conventional PCR targeting the V1-V2 regions of the 16S-rRNA gene. Positive amplicons were sequenced using Sanger methodology. Species-specific assays utilising RT-PCR probes targeting S. aureus femA/mecA, S. pneumoniae, S. pyogenes, S. agalactiae, L. monocytogenes, H. influenzae, N. meningitidis and Mycobacterium spp., were performed at the discretion of the duty microbiologist; targets were selected according to the clinical context.
Results:
Between September 2017 and September 2020, 955 samples from UCLH NHS Trust underwent 16S-PCR, including 183 for species-specific PCR.
Samples were from a variety of sterile sites, including 301 neurosurgical and 197 orthopaedic specimens.
16S-PCR was positive in 330 (34.5%). Amongst the positive samples, 60 (20%) had positive, concordant microscopy, 104 (10.9%) had positive, concordant culture with 22 (2.3%) yielding a different culture result and 188 (19.7%) were culture-negative. 49 specimens were 16S-PCR-positive without species identification (probably due to mixed bacterial populations). Of the 16S-PCR-negative samples 73 (7.6%) were ultimately culture-positive. A wide-range of organisms were detected (Table 1).
Species-specific PCR was tested in 183 samples and was positive in 33 (18%) and detected 8 further positive results where 16S-PCR was negative. Concordance with 16S-PCR-positives was almost universal; one positive Mycobacterium sp. RT-PCR coinciding with Fusobacterium nucleatum on 16S-PCR, which could have been undetected otherwise (Table 2a and 2b).
Conclusions:Molecular methods identified a significant number of organisms in culture-negative specimens. Positive 16S-PCR was strongly associated with positive microscopy. Mixed cultures were problematic for 16S-PCR species identification. However combining culture with 16S-PCR and more sensitive species-specific-PCR assays maximised detection of significant pathogens.
Keyword(s): 16S, PCR, rRNA