Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Improving the sensitivity of real-time PCR detection of group B Streptococcus using consensus sequence-derived oligonucleotides

Improving the sensitivity of real-time PCR detection of group B Streptococcus using consensus... Improving the sensitivity of real-time PCR detection of group B Streptococcus using consensus sequence-derived oligonucleotides 1 1 1 2 1 Ameneh Khatami , Tara M. Randis , Anna Chamby , Thomas A. Hooven , Margaret Gegick , 1 3 4,5 1# Evan Suzman , Brady A'Hearn-Thomas , Andrew P. Steenhoff , Adam J. Ratner Departments of Pediatrics and Microbiology, New York University School of Medicine, New York, NY, USA. Department of Pediatrics, Columbia University College of Physicians & Surgeons, New York, NY, USA. Department of Pediatrics, University of Iowa Carver College of Medicine, Iowa City, IA, USA. Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. Division of Pediatric Infectious Diseases, Children's Hospital of Philadelphia, Philadelphia, PA, USA. Address for Correspondence: Adam J. Ratner Departments of Pediatrics and Microbiology Division of Pediatric Infectious Diseases New York University School of Medicine 430 East 29th Street, Room 505 New York, NY 10016 Tel: (646) 501-0044 Adam.Ratner@nyumc.org Running Title: Consensus sip PCR for GBS Article Type: Brief Report ABSTRACT Group B Streptococcus (GBS) is a perinatal pathogen and an emerging cause of disease in adults. Culture-independent GBS detection relies on PCR of conserved genes, including sip. We demonstrate © The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution- NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofy164/5050461 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Accepted Manuscript suboptimal sensitivity of the existing sip PCR strategy and validate an improved method based on consensus sequences from >100 GBS genomes. BRIEF REPORT Group B Streptococcus (GBS) colonizes the gastrointestinal tract and vagina of healthy individuals. It is an important pathogen in neonates and pregnant women and is emerging as a cause of disease in non- pregnant adult populations. Laboratory detection of GBS involves culture using selective and enrichment media or, increasingly, molecular methods such as polymerase chain reaction (PCR). GBS surface immunogenic protein (Sip) is a conserved GBS-specific protein encoded by a 1311 base pair (bp) gene (sip) that is used as a target for real-time (RT) PCR-based detection of GBS. A primer-probe set targeting 4-9 a 78 bp region within the sip gene has been described and used in human studies for GBS detection. In the course of laboratory investigations, we observed that the published sip RT-PCR method did not detect the A909 (serotype Ia) GBS strain. On examination of the A909 genome sequence (Genbank NC_007432.1), we noted a single nucleotide polymorphism (SNP) (sip g.312C>T) in the predicted region of probe binding that we hypothesized was responsible for detection failure. Here we report the design and validation of a new primer-probe set targeting a more highly conserved region of the sip gene, leading to improved sensitivity of detection. METHODS Sequence alignment and oligonucleotide design We identified the open reading frame (ORF) encoding Sip (GenBank AIX03825.1) from GBS strain CNCTC 10/84. We aligned the short DNA reads from a set of 113 previously reported GBS draft genomes (NCBI BioProject PRJEB4456) to this ORF, identified SNPs, and mapped existing and candidate primer-probe sets using Geneious (ver. 11.0.3; Biomatters, Ltd.). Serotype information was obtained from the available metadata. Based on the alignment, we designed a new primer-probe set (F: 5’- CAG CAA CAA CGA TTG TTT CGC C-3’; R: 5’-CTT CCT CTT TAG CTG CTG GAA C-3’; Probe: 5’-AGA CAT ATT CTT CTG CGC CAG CTT TG-3’) targeting a conserved 171 bp region as a candidate for validation. For comparison, we used the previously described primer-probe set (F: 5’-ATC CTG AGA CAA CAC TGA CA-3’; R: 5’-TTG CTG GTG TTT CTA TTT TCA-3’; Probe: 5’-ATC AGA AGA GTC ATA CTG CCA CTT C-3’). In addition, we designed primers to amplify and sequence the target area (F: 5’-GTC AAT TGA TAT GAA TGT CTT AGC-3’; R: 5’-GTA TTG AGA GAA ACT TTT TGG TCT-3’) and full-length sip gene (F: 5’-ATG AAA ATG AAT AAA AAG GTA CTA TTG-3’; R: 5’-TTA TTT GTT AAA TGA TAC GTG AAC-3’). All oligonucleotides were synthesized by Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofy164/5050461 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Accepted Manuscript Integrated DNA Technologies. Probes were labeled with a 5' 6xFAM fluorescent tag and contained internal ZEN and 3' Iowa Black fluorescent quenchers. PCR methods Real-time PCR reactions consisted of 10 µl TaqMan Universal Mastermix II, 0.2 µl per primer and probe (primer stocks: 40 µM; probes: 20 µM), and 9.4 µl of DNA/water (10 ng DNA for rectovaginal samples, 2 ng DNA for vaginal lavages, with water to make reaction volume 20 µl.) Amplification and detection were performed in an ABI StepOne Plus cycler with StepOne software. A positive sip PCR was defined as a cycle threshold less than 36 with confirmation by a serotype-specific PCR. For amplification of sip regions for sequencing, we used Q5 DNA polymerase (New England BioLabs) according to the manufacturer's instructions. Bands were sized on 1% agarose gels, extracted using the QIAquick Gel Extraction kit (Qiagen, Inc.), and Sanger sequenced by Genewiz. Validation sample sets We used a set of 274 rectovaginal swabs from pregnant women as well as a GBS-positive subset of 91 vaginal lavage samples from non-pregnant women. Samples were grown in Lim broth overnight prior to DNA extraction for PCR. For the rectovaginal samples, Lim broth growth was cultured on chromogenic media and colonies confirmed using latex agglutination with ImmuLex Streptococcus Group B (SSI Diagnostica). For the vaginal lavage samples, culture and latex agglutination were performed only in the setting of discordance between the sip PCR assays. Serotype was determined using real-time PCR. Sensitivity and specificity were calculated using OpenEpi. RESULTS We aligned reads from 113 publicly available GBS genomes, including both invasive and carriage isolates from European and African nations over several decades (NCBI BioProject PRJEB4456) to the sip sequence from GBS CNCTC 10/84. We noted that the same g.312C>T polymorphism that occurred in strain A909 was also present in a minority of those genomes (6/113, 5.3%), distributed among three serotypes (Ia, N=1; Ib, N=4; II, N=1). We performed validation of sip PCR techniques in a set of rectovaginal swabs from women in late pregnancy (Table). The new set detected GBS in 53/274 (19%) of samples. Among these PCR-positive samples, 3/53 (5.7%) were not detected by the previously published primer-probe set. Two discordant samples were serotype Ib, and one was serotype IV. Among the GBS-negative samples, there was 100% Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofy164/5050461 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Accepted Manuscript concordance between the two assays. Thus, within the rectovaginal sample set, the sensitivity of the previously published primer-probe set was 94.3% and specificity was 100% using the new set as the gold standard. Using culture with latex agglutination as an alternative standard, the new sip PCR assay had a sensitivity of 95.8% and a specificity of 96.9%, whereas the previous assay had a sensitivity of 89.6% and a specificity of 96.9%. For further investigation of discordance, we used Lim broth cultures from vaginal lavage samples that tested GBS-positive with the new primer-probe set. Of these, 5/91 (5.5%) were negative with the previous primer-probe set. Three of those samples were serotype Ib, one was serotype II, and one had both serotypes Ia and Ib. To determine genetic changes in the 8 samples (3 rectovaginal and 5 vaginal lavage) with discordance between the two PCR methods, we amplified a 220 bp segment of the sip ORF spanning the binding sites for the prior primer-probe set for sequencing. All but one sample (serotype IV) produced a band of the predicted size and had the same SNP as A909 revealed by sequencing. As a control, we amplified the same region from 3 serotype Ib strains that were detected by both primer-probe sets – none had the g.312C>T SNP. The serotype IV sample that did not yield a band also did not amplify with primers targeting full-length sip, suggesting a deletion at one end of the ORF. CONCLUSIONS Current guidelines for GBS screening in pregnancy suggest testing in late gestation and provision of intrapartum antibiotic prophylaxis to colonized women. Reliable point-of-care culture-independent detection of GBS could add flexibility to screening and might minimize missed opportunities for treatment. Because of the conserved nature of the GBS sip gene, real-time PCR strategies for its detection have been used in clinical studies. More recently, additional culture-independent sip detection strategies, 5,14 including loop-mediated isothermal amplification and immunochromatography have been described. Here we report that the widely used sip primer-probe set is suboptimal for detection of a subset of GBS isolates, likely due to a mutation in the probe-binding site that is present in ~5% of strains. Using a set of >100 GBS genomes, we designed a new primer-probe set that detected an additional 8/144 (5.6%) of GBS-positive samples missed by the old primer-probe set. This new strategy requires validation in larger clinical sample sets, and it is notable that one GBS strain that was detected by the new but not the old PCR strategy did not have the g.312C>T SNP but appeared to have a larger deletion of a portion of sip containing the target of the prior primer-probe set. Although not the case for that particular isolate, deletions and as yet unrecognized polymorphisms in the sip sequence could lead to imperfect sensitivity Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofy164/5050461 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Accepted Manuscript of the new primer-probe set as well, emphasizing the importance of refinement of culture-independent detection strategies as new genomic information becomes available. FOOTNOTES PRESENTATION The results of this study were presented in part at the 1st International Symposium on Streptococcus agalactiae Disease; Cape Town, South Africa; February 20-23, 2018. FUNDING This work was supported by the Doris Duke Charitable Foundation [DDCF CSDA 2009-039 to A.J.R.] and the National Institutes of Health [R33 AI098654 to A.J.R.; K23 HD065844 and R21 AI127957 to T.M.R.] M.G. and E.S. were supported by the Jack Cary Eichenbaum Neonatology Scholars Program at New York University School of Medicine. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding bodies. CONFLICT OF INTEREST STATEMENT Potential conflicts: A.J.R. has served as a consultant to Pfizer. All other authors report no conflicts of interest. REFERENCES 1. Randis TM, Baker JA, Ratner AJ. Group B Streptococcal Infections. Pediatr Rev. Jun 2017;38(6):254-262. 2. Verani JR, McGee L, Schrag SJ. Prevention of perinatal group B streptococcal disease--revised guidelines from CDC, 2010. MMWR Recomm Rep. Nov 19 2010;59(RR-10):1-36. 3. Brodeur BR, Boyer M, Charlebois I, et al. Identification of group B streptococcal Sip protein, which elicits cross-protective immunity. Infect Immun. Oct 2000;68(10):5610-5618. 4. Bergh K, Stoelhaug A, Loeseth K, Bevanger L. Detection of group B streptococci (GBS) in vaginal swabs using real-time PCR with TaqMan probe hybridization. Indian J Med Res. May 2004;119 Suppl:221-223. 5. McKenna JP, Cox C, Fairley DJ, et al. Loop-mediated isothermal amplification assay for rapid detection of Streptococcus agalactiae (group B streptococcus) in vaginal swabs - a proof of concept study. J Med Microbiol. Mar 2017;66(3):294-300. 6. Bergseng H, Bevanger L, Rygg M, Bergh K. Real-time PCR targeting the sip gene for detection of group B Streptococcus colonization in pregnant women at delivery. J Med Microbiol. Feb 2007;56(Pt 2):223-228. 7. Feuerschuette OM, Serratine AC, Bazzo ML, Martins TR, Silveira SK, da Silva RM. Performance of RT-PCR in the detection of Streptococcus agalactiae in the anogenital tract of pregnant women. Arch Gynecol Obstet. Dec 2012;286(6):1437-1442. Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofy164/5050461 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Accepted Manuscript 8. Cools P, Jespers V, Hardy L, et al. A Multi-Country Cross-Sectional Study of Vaginal Carriage of Group B Streptococci (GBS) and Escherichia coli in Resource-Poor Settings: Prevalences and Risk Factors. PLoS One. 2016;11(1):e0148052. 9. El Aila NA, Tency I, Claeys G, et al. Comparison of culture with two different qPCR assays for detection of rectovaginal carriage of Streptococcus agalactiae (group B streptococci) in pregnant women. Res Microbiol. Jun 2011;162(5):499-505. 10. Hooven TA, Randis TM, Daugherty SC, et al. Complete Genome Sequence of Streptococcus agalactiae CNCTC 10/84, a Hypervirulent Sequence Type 26 Strain. Genome Announc. Dec 24 2014;2(6). 11. Da Cunha V, Davies MR, Douarre PE, et al. Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline. Nat Commun. Aug 4 2014;5:4544. 12. Breeding KM, Ragipani B, Lee KD, Malik M, Randis TM, Ratner AJ. Real-time PCR-based serotyping of Streptococcus agalactiae. Sci Rep. Dec 2 2016;6:38523. 13. Dean AG, Sullivan KM, Soe MM. OpenEpi: Open Source Epidemiologic Statistics for Public Health, Version 3.01. Accessed 05/10/2018. 14. Takayama Y, Matsui H, Adachi Y, et al. Detection of Streptococcus agalactiae by immunochromatography with group B streptococcus-specific surface immunogenic protein in pregnant women. J Infect Chemother. Oct 2017;23(10):678-682. Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofy164/5050461 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Accepted Manuscript TABLE Table: Test characteristics of sip PCR testing on rectovaginal samples from late pregnancy. New PCR Positive Negative Previous PCR Positive 50 0 Sensitivity 94.3% Negative 3 221 Specificity 100% Culture Positive Negative New PCR Positive 46 7 Sensitivity 95.8% Negative 2 219 Specificity 96.9% Culture Positive Negative Previous PCR Positive 43 7 Sensitivity 89.6% Negative 5 219 Specificity 96.9% Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofy164/5050461 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Accepted Manuscript http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Open Forum Infectious Diseases Oxford University Press

Improving the sensitivity of real-time PCR detection of group B Streptococcus using consensus sequence-derived oligonucleotides

Download PDF
7 pages

Loading next page...
 
/lp/oxford-university-press/improving-the-sensitivity-of-real-time-pcr-detection-of-group-b-j0BlrsJLoE

References (28)

Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases Society of America.
eISSN
2328-8957
DOI
10.1093/ofid/ofy164
Publisher site
See Article on Publisher Site

Abstract

Improving the sensitivity of real-time PCR detection of group B Streptococcus using consensus sequence-derived oligonucleotides 1 1 1 2 1 Ameneh Khatami , Tara M. Randis , Anna Chamby , Thomas A. Hooven , Margaret Gegick , 1 3 4,5 1# Evan Suzman , Brady A'Hearn-Thomas , Andrew P. Steenhoff , Adam J. Ratner Departments of Pediatrics and Microbiology, New York University School of Medicine, New York, NY, USA. Department of Pediatrics, Columbia University College of Physicians & Surgeons, New York, NY, USA. Department of Pediatrics, University of Iowa Carver College of Medicine, Iowa City, IA, USA. Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. Division of Pediatric Infectious Diseases, Children's Hospital of Philadelphia, Philadelphia, PA, USA. Address for Correspondence: Adam J. Ratner Departments of Pediatrics and Microbiology Division of Pediatric Infectious Diseases New York University School of Medicine 430 East 29th Street, Room 505 New York, NY 10016 Tel: (646) 501-0044 Adam.Ratner@nyumc.org Running Title: Consensus sip PCR for GBS Article Type: Brief Report ABSTRACT Group B Streptococcus (GBS) is a perinatal pathogen and an emerging cause of disease in adults. Culture-independent GBS detection relies on PCR of conserved genes, including sip. We demonstrate © The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution- NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofy164/5050461 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Accepted Manuscript suboptimal sensitivity of the existing sip PCR strategy and validate an improved method based on consensus sequences from >100 GBS genomes. BRIEF REPORT Group B Streptococcus (GBS) colonizes the gastrointestinal tract and vagina of healthy individuals. It is an important pathogen in neonates and pregnant women and is emerging as a cause of disease in non- pregnant adult populations. Laboratory detection of GBS involves culture using selective and enrichment media or, increasingly, molecular methods such as polymerase chain reaction (PCR). GBS surface immunogenic protein (Sip) is a conserved GBS-specific protein encoded by a 1311 base pair (bp) gene (sip) that is used as a target for real-time (RT) PCR-based detection of GBS. A primer-probe set targeting 4-9 a 78 bp region within the sip gene has been described and used in human studies for GBS detection. In the course of laboratory investigations, we observed that the published sip RT-PCR method did not detect the A909 (serotype Ia) GBS strain. On examination of the A909 genome sequence (Genbank NC_007432.1), we noted a single nucleotide polymorphism (SNP) (sip g.312C>T) in the predicted region of probe binding that we hypothesized was responsible for detection failure. Here we report the design and validation of a new primer-probe set targeting a more highly conserved region of the sip gene, leading to improved sensitivity of detection. METHODS Sequence alignment and oligonucleotide design We identified the open reading frame (ORF) encoding Sip (GenBank AIX03825.1) from GBS strain CNCTC 10/84. We aligned the short DNA reads from a set of 113 previously reported GBS draft genomes (NCBI BioProject PRJEB4456) to this ORF, identified SNPs, and mapped existing and candidate primer-probe sets using Geneious (ver. 11.0.3; Biomatters, Ltd.). Serotype information was obtained from the available metadata. Based on the alignment, we designed a new primer-probe set (F: 5’- CAG CAA CAA CGA TTG TTT CGC C-3’; R: 5’-CTT CCT CTT TAG CTG CTG GAA C-3’; Probe: 5’-AGA CAT ATT CTT CTG CGC CAG CTT TG-3’) targeting a conserved 171 bp region as a candidate for validation. For comparison, we used the previously described primer-probe set (F: 5’-ATC CTG AGA CAA CAC TGA CA-3’; R: 5’-TTG CTG GTG TTT CTA TTT TCA-3’; Probe: 5’-ATC AGA AGA GTC ATA CTG CCA CTT C-3’). In addition, we designed primers to amplify and sequence the target area (F: 5’-GTC AAT TGA TAT GAA TGT CTT AGC-3’; R: 5’-GTA TTG AGA GAA ACT TTT TGG TCT-3’) and full-length sip gene (F: 5’-ATG AAA ATG AAT AAA AAG GTA CTA TTG-3’; R: 5’-TTA TTT GTT AAA TGA TAC GTG AAC-3’). All oligonucleotides were synthesized by Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofy164/5050461 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Accepted Manuscript Integrated DNA Technologies. Probes were labeled with a 5' 6xFAM fluorescent tag and contained internal ZEN and 3' Iowa Black fluorescent quenchers. PCR methods Real-time PCR reactions consisted of 10 µl TaqMan Universal Mastermix II, 0.2 µl per primer and probe (primer stocks: 40 µM; probes: 20 µM), and 9.4 µl of DNA/water (10 ng DNA for rectovaginal samples, 2 ng DNA for vaginal lavages, with water to make reaction volume 20 µl.) Amplification and detection were performed in an ABI StepOne Plus cycler with StepOne software. A positive sip PCR was defined as a cycle threshold less than 36 with confirmation by a serotype-specific PCR. For amplification of sip regions for sequencing, we used Q5 DNA polymerase (New England BioLabs) according to the manufacturer's instructions. Bands were sized on 1% agarose gels, extracted using the QIAquick Gel Extraction kit (Qiagen, Inc.), and Sanger sequenced by Genewiz. Validation sample sets We used a set of 274 rectovaginal swabs from pregnant women as well as a GBS-positive subset of 91 vaginal lavage samples from non-pregnant women. Samples were grown in Lim broth overnight prior to DNA extraction for PCR. For the rectovaginal samples, Lim broth growth was cultured on chromogenic media and colonies confirmed using latex agglutination with ImmuLex Streptococcus Group B (SSI Diagnostica). For the vaginal lavage samples, culture and latex agglutination were performed only in the setting of discordance between the sip PCR assays. Serotype was determined using real-time PCR. Sensitivity and specificity were calculated using OpenEpi. RESULTS We aligned reads from 113 publicly available GBS genomes, including both invasive and carriage isolates from European and African nations over several decades (NCBI BioProject PRJEB4456) to the sip sequence from GBS CNCTC 10/84. We noted that the same g.312C>T polymorphism that occurred in strain A909 was also present in a minority of those genomes (6/113, 5.3%), distributed among three serotypes (Ia, N=1; Ib, N=4; II, N=1). We performed validation of sip PCR techniques in a set of rectovaginal swabs from women in late pregnancy (Table). The new set detected GBS in 53/274 (19%) of samples. Among these PCR-positive samples, 3/53 (5.7%) were not detected by the previously published primer-probe set. Two discordant samples were serotype Ib, and one was serotype IV. Among the GBS-negative samples, there was 100% Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofy164/5050461 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Accepted Manuscript concordance between the two assays. Thus, within the rectovaginal sample set, the sensitivity of the previously published primer-probe set was 94.3% and specificity was 100% using the new set as the gold standard. Using culture with latex agglutination as an alternative standard, the new sip PCR assay had a sensitivity of 95.8% and a specificity of 96.9%, whereas the previous assay had a sensitivity of 89.6% and a specificity of 96.9%. For further investigation of discordance, we used Lim broth cultures from vaginal lavage samples that tested GBS-positive with the new primer-probe set. Of these, 5/91 (5.5%) were negative with the previous primer-probe set. Three of those samples were serotype Ib, one was serotype II, and one had both serotypes Ia and Ib. To determine genetic changes in the 8 samples (3 rectovaginal and 5 vaginal lavage) with discordance between the two PCR methods, we amplified a 220 bp segment of the sip ORF spanning the binding sites for the prior primer-probe set for sequencing. All but one sample (serotype IV) produced a band of the predicted size and had the same SNP as A909 revealed by sequencing. As a control, we amplified the same region from 3 serotype Ib strains that were detected by both primer-probe sets – none had the g.312C>T SNP. The serotype IV sample that did not yield a band also did not amplify with primers targeting full-length sip, suggesting a deletion at one end of the ORF. CONCLUSIONS Current guidelines for GBS screening in pregnancy suggest testing in late gestation and provision of intrapartum antibiotic prophylaxis to colonized women. Reliable point-of-care culture-independent detection of GBS could add flexibility to screening and might minimize missed opportunities for treatment. Because of the conserved nature of the GBS sip gene, real-time PCR strategies for its detection have been used in clinical studies. More recently, additional culture-independent sip detection strategies, 5,14 including loop-mediated isothermal amplification and immunochromatography have been described. Here we report that the widely used sip primer-probe set is suboptimal for detection of a subset of GBS isolates, likely due to a mutation in the probe-binding site that is present in ~5% of strains. Using a set of >100 GBS genomes, we designed a new primer-probe set that detected an additional 8/144 (5.6%) of GBS-positive samples missed by the old primer-probe set. This new strategy requires validation in larger clinical sample sets, and it is notable that one GBS strain that was detected by the new but not the old PCR strategy did not have the g.312C>T SNP but appeared to have a larger deletion of a portion of sip containing the target of the prior primer-probe set. Although not the case for that particular isolate, deletions and as yet unrecognized polymorphisms in the sip sequence could lead to imperfect sensitivity Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofy164/5050461 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Accepted Manuscript of the new primer-probe set as well, emphasizing the importance of refinement of culture-independent detection strategies as new genomic information becomes available. FOOTNOTES PRESENTATION The results of this study were presented in part at the 1st International Symposium on Streptococcus agalactiae Disease; Cape Town, South Africa; February 20-23, 2018. FUNDING This work was supported by the Doris Duke Charitable Foundation [DDCF CSDA 2009-039 to A.J.R.] and the National Institutes of Health [R33 AI098654 to A.J.R.; K23 HD065844 and R21 AI127957 to T.M.R.] M.G. and E.S. were supported by the Jack Cary Eichenbaum Neonatology Scholars Program at New York University School of Medicine. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding bodies. CONFLICT OF INTEREST STATEMENT Potential conflicts: A.J.R. has served as a consultant to Pfizer. All other authors report no conflicts of interest. REFERENCES 1. Randis TM, Baker JA, Ratner AJ. Group B Streptococcal Infections. Pediatr Rev. Jun 2017;38(6):254-262. 2. Verani JR, McGee L, Schrag SJ. Prevention of perinatal group B streptococcal disease--revised guidelines from CDC, 2010. MMWR Recomm Rep. Nov 19 2010;59(RR-10):1-36. 3. Brodeur BR, Boyer M, Charlebois I, et al. Identification of group B streptococcal Sip protein, which elicits cross-protective immunity. Infect Immun. Oct 2000;68(10):5610-5618. 4. Bergh K, Stoelhaug A, Loeseth K, Bevanger L. Detection of group B streptococci (GBS) in vaginal swabs using real-time PCR with TaqMan probe hybridization. Indian J Med Res. May 2004;119 Suppl:221-223. 5. McKenna JP, Cox C, Fairley DJ, et al. Loop-mediated isothermal amplification assay for rapid detection of Streptococcus agalactiae (group B streptococcus) in vaginal swabs - a proof of concept study. J Med Microbiol. Mar 2017;66(3):294-300. 6. Bergseng H, Bevanger L, Rygg M, Bergh K. Real-time PCR targeting the sip gene for detection of group B Streptococcus colonization in pregnant women at delivery. J Med Microbiol. Feb 2007;56(Pt 2):223-228. 7. Feuerschuette OM, Serratine AC, Bazzo ML, Martins TR, Silveira SK, da Silva RM. Performance of RT-PCR in the detection of Streptococcus agalactiae in the anogenital tract of pregnant women. Arch Gynecol Obstet. Dec 2012;286(6):1437-1442. Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofy164/5050461 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Accepted Manuscript 8. Cools P, Jespers V, Hardy L, et al. A Multi-Country Cross-Sectional Study of Vaginal Carriage of Group B Streptococci (GBS) and Escherichia coli in Resource-Poor Settings: Prevalences and Risk Factors. PLoS One. 2016;11(1):e0148052. 9. El Aila NA, Tency I, Claeys G, et al. Comparison of culture with two different qPCR assays for detection of rectovaginal carriage of Streptococcus agalactiae (group B streptococci) in pregnant women. Res Microbiol. Jun 2011;162(5):499-505. 10. Hooven TA, Randis TM, Daugherty SC, et al. Complete Genome Sequence of Streptococcus agalactiae CNCTC 10/84, a Hypervirulent Sequence Type 26 Strain. Genome Announc. Dec 24 2014;2(6). 11. Da Cunha V, Davies MR, Douarre PE, et al. Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline. Nat Commun. Aug 4 2014;5:4544. 12. Breeding KM, Ragipani B, Lee KD, Malik M, Randis TM, Ratner AJ. Real-time PCR-based serotyping of Streptococcus agalactiae. Sci Rep. Dec 2 2016;6:38523. 13. Dean AG, Sullivan KM, Soe MM. OpenEpi: Open Source Epidemiologic Statistics for Public Health, Version 3.01. Accessed 05/10/2018. 14. Takayama Y, Matsui H, Adachi Y, et al. Detection of Streptococcus agalactiae by immunochromatography with group B streptococcus-specific surface immunogenic protein in pregnant women. J Infect Chemother. Oct 2017;23(10):678-682. Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofy164/5050461 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Accepted Manuscript TABLE Table: Test characteristics of sip PCR testing on rectovaginal samples from late pregnancy. New PCR Positive Negative Previous PCR Positive 50 0 Sensitivity 94.3% Negative 3 221 Specificity 100% Culture Positive Negative New PCR Positive 46 7 Sensitivity 95.8% Negative 2 219 Specificity 96.9% Culture Positive Negative Previous PCR Positive 43 7 Sensitivity 89.6% Negative 5 219 Specificity 96.9% Downloaded from https://academic.oup.com/ofid/advance-article-abstract/doi/10.1093/ofid/ofy164/5050461 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Accepted Manuscript

Journal

Open Forum Infectious Diseases – Oxford University Press

Published: Jul 7, 2018

There are no references for this article.