MIMET-04511; No of Pages 5 Journal of Microbiological Methods xxx (2014) xxx–xxx
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Anne Karin Brigtsen a,⁎, Lumnije Dedi b, Kjetil K. Melby a,b, Mona Holberg-Petersen b, Andreas Radtke c,d, Randi Valsø Lyng c, Lise Lima Andresen b, Anne Flem Jacobsen a,e, Drude Fugelseth a,f, Andrew Whitelaw a
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Article history: Received 23 July 2014 Received in revised form 3 November 2014 Accepted 4 November 2014 Available online xxxx
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Keywords: Group B Streptococcus Streptococcus agalactiae Serotype Serotyping Genotyping Pregnancy
Institute of Clinical Medicine, University of Oslo, Oslo, Norway Department of Microbiology, Oslo University Hospital Ullevaal, Oslo, Norway Department of Medical Microbiology, St. Olavs University Hospital, Trondheim, Norway d Department of Laboratory Medicine, Children's and Women's Health, Norwegian University of Science and Technology, Trondheim, Norway e Department of Obstetrics and Gynaecology, Oslo University Hospital Ullevaal, Oslo, Norway f Department of Neonatal Intensive Care, Oslo University Hospital Ullevaal, Oslo, Norway
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Streptococcus agalactiae (GBS) is a leading cause of invasive neonatal infection. Serotyping of GBS is important in following epidemiological trends and vaccine development. Capsular serotyping of GBS by latex agglutination has been the predominant typing method, but more recently capsular genotyping has been introduced as an alternative method. The purpose of this study was to compare the relative performance of these methods in a contemporary population of pregnant women. We typed isolates from an unselected population of 426 colonized women at delivery using latex agglutination and a combination of four PCR methods. Antibiotic resistance was tested in 449 isolates. Capsular genotyping gave a result in all except three of 426 isolates. Fifty-nine of 426 isolates could not be typed by latex agglutination. Agreement between serotyping and genotyping was shown in 303 (71.1%) of the isolates. 10.2% of the isolates were resistant to erythromycin, 9.6% to clindamycin, 76.6% to tetracycline and none to penicillin. In conclusion, a substantial proportion of the colonizing strains were non-typeable by serotyping, but typeable by genotyping. This suggests that a diagnostic genotyping strategy is preferable to serotyping of the GBS polysaccharide capsule in colonized, pregnant women. © 2014 Published by Elsevier B.V.
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Streptococcus agalactiae (group B Streptococcus, GBS) is a leading cause of serious neonatal infection. In addition, GBS is an increasingly important cause of infections in older persons and immunocompromised patients (Skoff et al., 2009). Maternal vaginal–rectal colonization is considered the main route for neonatal infection. The capsular polysaccharide is a major virulence factor of this organism, and serotyping of the capsular polysaccharide is used to characterize strains and investigate GBS epidemiology in humans (Rubens et al., 1987). To date, ten capsular serotypes have been characterized, Ia, Ib and II to IX (Slotved et al., 2007). Serotypes Ia, Ib, II, III and V are predominant among both neonatal and adult invasive strains and maternal colonizing strains in the Western hemisphere (Edmond et al., 2012; Remington et al., 2011; Skoff et al., 2009). Serotyping of GBS is important in following epidemiological trends and in vaccine development (Johri et al., 2006). The most common phenotypic methods for serotyping GBS are latex agglutination tests
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1. Introduction
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Comparison of PCR and serotyping of Group B Streptococcus in pregnant women: The Oslo GBS-study
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⁎ Corresponding author at: Institute of Clinical Medicine, University of Oslo, Kirkeveien 166, N-0450 Oslo, Norway. Tel.: +47 22118765; fax: +47 22118763. E-mail address: [email protected] (A.K. Brigtsen).
(Afshar et al., 2011), which are based on polyclonal antibodies specific for the capsular polysaccharides. Latex agglutination tests are commercially available and in general considered reliable and easy to perform. In addition, several PCR-based methods for GBS capsular typing have been developed. The initial assays for genotyping were published by Kong in 2002 and Borchardt in 2004 using single PCRs for the different capsular polysaccharide types (Borchardt et al., 2004; Kong et al., 2002). Later Poyart and Imperi developed multiplex-PCR approaches (Imperi et al., 2010; Poyart et al., 2007). The two-set multiplex PCR by Poyart et al. was developed before the discovery of serotype IX. Strengths of the assay by Imperi are the inclusion of the most recently discovered serotype IX and the possibility to distinguish all the 10 known serotypes in a single PCR reaction. The serotype-specific assay described by Kong et al. does not include specific PCRs for serotypes II, VII and VIII, but these are included in Borchardt's assay. Although PCR assays are commonly considered modern, sophisticated techniques, molecular methods also have limitations (Yao et al., 2013). For instance, molecular methods do not reveal if the capsular polysaccharide gene locus detected is expressed as a polysaccharide capsule. Sparse data exist on the relative performance of these methods in a contemporary population of pregnant women. Accordingly, in this
http://dx.doi.org/10.1016/j.mimet.2014.11.001 0167-7012/© 2014 Published by Elsevier B.V.
Please cite this article as: Brigtsen, A.K., et al., Comparison of PCR and serotyping of Group B Streptococcus in pregnant women: The Oslo GBSstudy, J. Microbiol. Methods (2014), http://dx.doi.org/10.1016/j.mimet.2014.11.001
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The Oslo GBS Study is a prospective cohort study of pregnant women admitted to the Delivery Department at Oslo University Hospital, Ullevaal, Oslo, Norway. This hospital serves a population of approximately 600 000 in the metropolitan Oslo area. During the period from June 2009 to September 2011, 16 000 pregnant women were invited to participate at the time of routine ultrasound screening in the second trimester. 4450 women consented to participate in the study, and 1739 women had one vaginal–rectal sample obtained (Fig. 1). The study was approved by the Regional Committees for Medical and Health Research Ethics and is registered in the Norwegian Biobank Registry. Written informed consent was obtained from the participants.
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2.2. Specimen collection and identification
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The S. agalactiae isolates were collected and processed in accordance with the CDC guideline (Verani et al., 2010). Combined vaginal–rectal swabs were obtained during the vaginal examination at the onset of labor and prior to antibiotic administration. The swabs were placed in transport tubes containing Amies medium (Copan, Brescia, Italy) and transported to the laboratory within 24 h. Each strain was cultured to both Columbia agar (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA) and inoculated into Lim broth (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA). After incubation at 35 °C in ambient air for 24 h, turbid broths were subcultured onto blood agar plates and incubated at 35 °C in 5% CO2 for 24 h. Columbia agar plates were reincubated at 35 °C for 18–24 h if GBS was not identified after incubation. Isolates were identified based on colony morphology, β-hemolysis, catalase test, and latex agglutination. The Lancefield group B antigen was determined using a latex grouping kit (Prolex, Prolab Diagnostics, Richmond Hill, Ontario, Canada), according to the manufacturer's recommendations.
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2.3. Serotyping by latex agglutination
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From Lim broth cultures showing visible growth, a 10 μl aliquot was applied to a reaction card and mixed with 10 μl of latex suspension
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2.4. Molecular capsular typing
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All primers used in this study were published previously by four different groups (Borchardt et al., 2004; Imperi et al., 2010; Kong et al., 2002; Poyart et al., 2007). PCR conditions for the multiplex PCR by Imperi et al. were set according to the initial publication. Using the primers described by Borchardt, Kong and Poyart, the samples were amplified by a denaturation step for 10 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 60 s and a final extension of 72 °C for 5 min. The PCR mix qPCR Core kit (Eurogentec, Seraing, Belgium) was used for amplification and PCR products were visualized by by electrophoresis through a 1.5% agarose gel (SeaKem LE Agarose, Lonza, Rockland, ME, USA) containing ethidium bromide. All isolates were initially examined by the one-set multiplex PCR involving 19 primers developed by Imperi et al. (2010). This assay was adopted by the pan-European program DEVANI (DEsign of a Vaccine Against Neonatal Infections) as the standard method for molecular GBS capsular gene typing (Afshar et al., 2011). The initial multiplexPCR testing was performed blinded to the results of the serotyping. If serotyping and initial genotyping results were discordant, the Imperi assay was repeated. Isolates non-typeable by the method developed by Imperi et al. and isolates with discordant results for PCR and latex agglutination were in addition classified by a two-set multiplex PCR developed by Poyart et al. If this did not resolve the ambiguities, the serotype-specific PCRs developed by Kong et al. or Borchardt et al. were used (Borchardt et al., 2004; Kong et al., 2002).
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2.5. Antimicrobial susceptibility testing
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Isolates were tested against penicillin, erythromycin, clindamycin, tetracycline and trimethoprim-sulfamethoxazole by disk diffusion. Antimicrobial susceptibility disks from Oxoid (Cheshire, UK) were exchanged with disks from Becton, Dickinson and Company (Franklin Lakes, New Jersey, USA) January 2011. Norwegian Working Group on
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1739 Women had one vaginal-rectal sample obtained
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2.1. Study population
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(reagents Ia, Ib, and II to IX; Strep-B-Latex kit, Statens Serum Institut, Copenhagen, Denmark). The two drops were mixed and the card was rotated slowly for up to 30 s and observed for agglutination. A distinct agglutination was scored as a positive reaction. A distinct agglutination reaction with one antigen occuring simultaneously with a weak agglutination reaction with one or more antigens resulted in repeated testing. If the same pattern of reaction occurred, the serotype with the distinct reaction was scored as positive. If a pattern of equally stong reactions with two ore more serotype antigens appeared, the isolate was scored as non-typeable.
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report we share our experience with the commonly used procedures for capsular serotyping and genotyping to characterize GBS isolates from an unselected population of pregnant women. In addition, we report antimicrobial susceptibility data from the isolates.
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452 Women were GBS culture positive
26 Samples were not GBS typed
426 Samples were available for serotyping/genotyping
3 Samples were not susceptibility tested
449 Samples were available for susceptibility testing
Fig. 1. Flowchart illustrating the selection of included samples.
Please cite this article as: Brigtsen, A.K., et al., Comparison of PCR and serotyping of Group B Streptococcus in pregnant women: The Oslo GBSstudy, J. Microbiol. Methods (2014), http://dx.doi.org/10.1016/j.mimet.2014.11.001
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2.6. Statistical analysis
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The Mann–Whitney U test was used to compare the difference in age between colonized and non-colonized women. Fisher's exact or chi-square tests were used, as appropriate, to compare colonization by parity and antimicrobial resistance between GBS capsular polysaccharide genotypes. Two-sided p-values b0.05 were considered statistically significant.
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3. Results
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3.1. Carriage study
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Of the 4450 women who consented to partcipate in the Oslo GBSstudy, 1739 had one vaginal–rectal sample obtained at delivery. Four hundred and fifty-two (26.0%) of these samples were culture positive for GBS. The median age of the women in the study was 32.0 years, ranging from 19.7 to 46.2 years, and 63.2% were giving birth to their first child. There was no significant difference between the age of the 452 colonized women and the 1287 non-colonized women (median age (25–75 percentile): 31.8 (29.6–34.0) vs. 32.0 (29.5–34.9) years; p = 0.34). No significant association between colonization rate and parity was observed (nulliparous (25.9%) vs. primi- or multiparous (26.2%); p = 0.89).
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3.2. Serotyping by latex agglutination
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Four hundred and twenty-six isolates were serotyped by latex agglutination of which 367 (86.2%) were assigned to one of the ten serotypes (Table 1). Of 59 isolates scored as non-typeable, 28 isolates reacted equally well with reagents to two serotypes, of which one serotype was identical to the genotype (Table 2). Furthermore, 3 isolates reacted equally well with reagents to two serotypes, neither of which corresponded to the genotype. Thirty-eight isolates reacted with serotype IX latex antiserum. Of these, 20 isolates also tested positive for serotypes Ia (4), Ib (2), II (3), III (6), IV (2) or V (3).
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Table 2 Isolates reacting to 2 latex serotype reagents using genotyping as the reference. Serotype by latex agglutination Ia Ib II III IV V VI VII VIII IX Total a
Genotype Ia
Ib
II
III
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4 1
1
1 1
4 9
1 2
3 6
IV
V
VI
VII
VIII
IX
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NT, non-typeable.
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Total 3
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Antibiotics (NWGA) guidelines for performance and interpretation of susceptibility testing were applied until January 2011 (Bergan et al., 1997), after which the EUCAST guidelines were applied (http://www. eucast.org/antimicrobial_susceptibility_testing).
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Table 1 Comparison of results of 426 GBS isolates serotyped by latex agglutination and a combination of PCR methods. Serotype by latex agglutination
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Ia Ib II III IV V VI VII VIII IX NTa Subtotal Ac Bd Total
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Genotype Ia 45
Ib
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III
IV
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30
3 4
II
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2 87 1
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36
1 58
VI
VII
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1
2 0 2 3 57 9 1
7 2 40 2
10 7 51 6 2
1 2 6 101 5
3 1 3 57 3
3 7 70 2
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0
3
11
2
11 1
3
Total 49 48 33 88 42 63 2 0 4 38 28 395 28 3 426
NT, non-typeable. Boldface: Serotyping concordant to genotyping. c A: Isolates reacting to 2 latex antigens using genotyping as a reference. Please see Table 2 for details. d B: Isolates reacting to 2 latex antigens, none concordant to the genotype. b
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The 426 isolates were examined using the molecular capsular typing 195 methods, as described above. By combining the multiplex PCR devel- 196 oped by Imperi et al. with three additional PCR assays for molecular cap- 197 sular typing, we were able to assign a capsular gene type to 423 (99.3%) 198 of the strains. Based on results from the genotyping, serotype III was 199 most frequent, making up 24.9% of the isolates. Serotypes V (16.9%) 200 and Ia (15.7%) were second and third in frequency, respectively (Fig. 2). 201 Using the multiplex PCR by Imperi et al., Yao et al. found that some 202 strains of serotypes Ib and IV were incorrectly typed as Ia because the 203 middle band was very weak or not detectable. A total of 67 isolates ini- 204 tially serotyped as Ia were therefore reexamined using a singleplex PCR 205 assay (Kong et al., 2002). Two of the Ia strains showed disagreement 206 with the results from the Imperi assay and were classified as non- 207 typeable. 208
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3.4. Comparison of latex agglutination and molecular capsular typing
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A total of 303 (71.1%) isolates showed agreement between the two methods, as indicated by the numbers marked in bold in Table 1. By capsular genotyping, we identified 11 serotype IX isolates, compared to 38 by latex agglutination. Serotype VII strains were not detected by latex agglutination or PCR methods.
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3.5. Susceptibility
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Four hundred and forty-nine isolates were available for susceptibility testing (Fig. 1). All isolates were susceptible to penicillin. Resistance to erythromycin and clindamycin was oberved in 10.2% and 9.6% of the isolates, respectively (Table 3). Of the erythromycin-resistant isolates, 40/46 (87.0%) showed cross-resistance to clindamycin. A high frequency of resistance to tetracycline was observed, in particular among serotypes Ia (88.1%), III (86.8%) and IV (83.3%). All isolates belonging to the most recently described serotype IX were susceptible to the antibiotics tested. There was substantial heterogeneity in antibiotic susceptibility between genotypes (Table 3). For instance, the resistance to clindamycin differed from 3/67 for genotype Ia to 12/72 for genotype V (p = 0.028); the resistance to erythromycin from 3/67 (Ia) to 13/72 (V) (p = 0.016), and the resistance to tetracycline from 59/67 (Ia) to 24/42 (Ib) (p b 0.001).
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4. Discussion
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In this large unselected group of pregnant women colonized with GBS, we found that 13.8% of isolates could not be satisfactorily typed by latex agglutination, an unacceptably high failure rate. In contrast, using a combination of capsular genotyping methods, only 0.7% of isolates were not identified. Previous studies applying capsular latex
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Please cite this article as: Brigtsen, A.K., et al., Comparison of PCR and serotyping of Group B Streptococcus in pregnant women: The Oslo GBSstudy, J. Microbiol. Methods (2014), http://dx.doi.org/10.1016/j.mimet.2014.11.001
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Percentage of isolates
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0 Ib
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Fig. 2. Distribution of GBS capsular genotypes among 426 isolates using a combination of 4 PCR assays. a NT, non-typeable.
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Genotype
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Ia Ib II III IV V VI VII VIII IX NT Sum genotyped Not genotyped Total (%)
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Table 3 Distribution of resistance among GBS genotypes.
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distribution in pregnant women, identifying serotypes Ia, II and III as predominant in a subset of 26 culture-positive women (Hordnes et al., 1996). In the present study, serotypes III, V, Ia, IV and Ib were the most common isolates using the molecular gene typing results. The predominance of serotype III is in accordance with results from other authors (Davies et al., 2001; Hakansson et al., 2008; Lu et al., 2013; Savoia et al., 2008). A trivalent GBS capsular polysaccharide conjugate vaccine prepared with capsular polysaccharides to serotypes Ia, Ib and III is undergoing phase-II evaluation among pregnant women (Heath, 2011; Madhi et al., 2013). Based on the current observations, this vaccine may not cover more than half of the colonizing strains in the general population of pregnant women. We observed resistance rates of 10.2% to erythromycin and 9.6% to clindamycin. This is relatively low compared to rates as high as 66% and 54% for erythromycin, and 56% and 33% for clindamycin reported for colonizing strains in China and USA, respectively (DiPersio and DiPersio, 2006; Lu et al., 2013). A restrictive policy for administering antibiotics in Norway may partially explain this. A risk-based rather than a screening-based approach for identifying women who should receive intrapartum antibiotic prophylaxis to prevent invasive neonatal GBS disease keeps the use of antibiotics low in women giving birth (Verani et al., 2010). Erythromycin is the treatment of choice in
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agglutination methods have in most cases been able to serotype more than 90% of the isolates (Savoia et al., 2008; Turner et al., 2012). An exception to this is the study by Yao et al. in which 27% of 281 human isolates could not be typed by latex agglutination (Yao et al., 2013). Some of their isolates were selected because they were problematic when serotyped by latex agglutination. Our results demonstrate that difficulties with serotyping are common in the clinical setting of an unselected population of pregnant women. The GBS colonization rate of 26.0% was lower than the 34.7% reported in a Norwegian study of perinatal samples from 251 women (Bergseng et al., 2007). However, it is in the range of previous reports from North America and several European countries (Campbell et al., 2000; Davies et al., 2001; Hakansson et al., 2008; Jones et al., 2006; Kunze et al., 2011), but higher than recently reported rates from Asia (7%–12%) and Denmark (10%) (Lu et al., 2013; Stokholm et al., 2013; Turner et al., 2012). The proportions of different serotypes may vary considerably between geographical areas, between subgroups of the population, and over time. Knowing the serotype distribution is essential for finding the most suitable capsular polysaccharide based vaccine for a given country. Limited data are available on the distribution of GBS serotypes in Europe. In Norway, only one study has examined the serotype
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67 42 59 106 60 72 2 0 3 12 3 426 26 452
a
Resistance
n
Clindamycin No. (%)
Erythromycin No. (%)
Both clindamycin and erythromycin No. (%)
67 42 57 105 60 72 2 0 3 12 3 423 26 449
3 (4.5) 4 (9.5) 8 (13.6) 11 (10.4) 3 (5.0) 12 (16.7)
3 (4.5) 5 (11.9) 9 (15.3) 10 (9.4) 4 (6.7) 13 (18.1)
2 (3.0) 4 (9.5) 8 (13.6) 9 (8.5) 3 (5.0) 12 (16.7)
Tetracycline No. (%) 59 (88.1) 24 (57.1) 46 (78.0) 92 (86.8) 50 (83.3) 49 (68.1) 1 (50.0)
T-sb No. (%)
Penicillin No. (%)
2 2
3 (100.0) 2 (7.7) 43 (9.6)
2 (7.7) 46 (10.2)
2 (7.7) 40 (8.9)
20 (76.9) 344 (76.6)
4 (0.9)
0 (0)
Three samples were genotyped (2 genotype II and one genotype III), but were not available for susceptibility testing. T-s: Trimethoprim-sulfametoxazole.
Please cite this article as: Brigtsen, A.K., et al., Comparison of PCR and serotyping of Group B Streptococcus in pregnant women: The Oslo GBSstudy, J. Microbiol. Methods (2014), http://dx.doi.org/10.1016/j.mimet.2014.11.001
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We wish to thank everyone involved in enrolment, sampling and culturing the GBS strains at the Department of Obstetrics and Gynaecology and the Department of Microbiology at Oslo University Hospital Ullevaal. We are grateful to Cathrine Nygaard for expert technical assistance. This work was partially supported by funds from Renée and Bredo Grimsgaard's Foundation and Eckbo's Foundation. The sponsors were not involved in the design and execution of the study.
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Kong, F., Gowan, S., Martin, D., James, G., Gilbert, G.L., 2002. Serotype identification of group B streptococci by PCR and sequencing. J. Clin. Microbiol. 40, 216–226. Kunze, M., Ziegler, A., Fluegge, K., Hentschel, R., Proempeler, H., Berner, R., 2011. Colonization, serotypes and transmission rates of group B streptococci in pregnant women and their infants born at a single University Center in Germany. J. Perinat. Med. 39, 417–422. Lin, F.Y., Azimi, P.H., Weisman, L.E., Philips III, J.B., Regan, J., Clark, P., Rhoads, G.G., Clemens, J., Troendle, J., Pratt, E., Brenner, R.A., Gill, V., 2000. Antibiotic susceptibility profiles for group B streptococci isolated from neonates, 1995–1998. Clin. Infect. Dis. 31, 76–79. Lu, B., Li, D., Cui, Y., Sui, W., Huang, L., Lu, X., 2013. Epidemiology of Group B streptococcus isolated from pregnant women in Beijing, China. Clin. Microbiol. Infect. Madhi, S.A., Dangor, Z., Heath, P.T., Schrag, S., Izu, A., Sobanjo-Ter Meulen, A., Dull, P.M., 2013. Considerations for a phase-III trial to evaluate a group B Streptococcus polysaccharide-protein conjugate vaccine in pregnant women for the prevention of early- and late-onset invasive disease in young-infants. Vaccine 31 (Suppl. 4), D52–D57. Poyart, C., Tazi, A., Reglier-Poupet, H., Billoet, A., Tavares, N., Raymond, J., Trieu-Cuot, P., 2007. Multiplex PCR assay for rapid and accurate capsular typing of group B streptococci. J. Clin. Microbiol. 45, 1985–1988. Remington, J., Klein, J., Wilson, C., Nizet, V., Maldonado, Y., 2011. Infectious Diseases of the Fetus and Newborn Infant. Elsevier Saunders, Philadelphia. Rubens, C.E., Wessels, M.R., Heggen, L.M., Kasper, D.L., 1987. Transposon mutagenesis of type III group B Streptococcus: correlation of capsule expression with virulence. Proc. Natl. Acad. Sci. U. S. A. 84, 7208–7212. Savoia, D., Gottimer, C., Crocilla, C., Zucca, M., 2008. Streptococcus agalactiae in pregnant women: phenotypic and genotypic characters. J. Infect. 56, 120–125. Skoff, T.H., Farley, M.M., Petit, S., Craig, A.S., Schaffner, W., Gershman, K., Harrison, L.H., Lynfield, R., Mohle-Boetani, J., Zansky, S., Albanese, B.A., Stefonek, K., Zell, E.R., Jackson, D., Thompson, T., Schrag, S.J., 2009. Increasing burden of invasive group B streptococcal disease in nonpregnant adults, 1990–2007. Clin. Infect. Dis. 49, 85–92. Slotved, H.C., Kong, F., Lambertsen, L., Sauer, S., Gilbert, G.L., 2007. Serotype IX, a proposed new Streptococcus agalactiae serotype. J. Clin. Microbiol. 45, 2929–2936. Stokholm, J., Schjorring, S., Eskildsen, C.E., Pedersen, L., Bischoff, A.L., Folsgaard, N., Carson, C.G., Chawes, B.L., Bonnelykke, K., Molgaard, A., Jacobsson, B., Krogfelt, K.A., Bisgaard, H., 2013. Antibiotic use during pregnancy alters the commensal vaginal microbiota. Clin. Microbiol. Infect. Turner, C., Turner, P., Po, L., Maner, N., De Zoysa, A., Afshar, B., Efstratiou, A., Heath, P.T., Nosten, F., 2012. Group B streptococcal carriage, serotype distribution and antibiotic susceptibilities in pregnant women at the time of delivery in a refugee population on the Thai–Myanmar border. BMC Infect. Dis. 12, 34. Verani, J.R., McGee, L., Schrag, S.J., 2010. Prevention of perinatal group B streptococcal disease—revised guidelines from CDC, 2010. MMWR Recomm. Rep. 59, 1–36. Yao, K., Poulsen, K., Maione, D., Rinaudo, C.D., Baldassarri, L., Telford, J.L., Sorensen, U.B., Kilian, M., 2013. Capsular gene typing of Streptococcus agalactiae compared to serotyping by latex agglutination. J. Clin. Microbiol. 51, 503–507.
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pregnant women allergic to penicillin in Norway, and the choice of antibiotics to this group should be guided by contemporary information concerning antimicrobial resistance. We further observed an association between erythromycin and clindamycin resistance and capsular serotype V. Serotype V displayed the highest resistance to erythromycin (18.1%) and clindamycin (16.7%), which is in accordance with other studies (Borchardt et al., 2006; DiPersio and DiPersio, 2006; Lin et al., 2000). Strengths of our study include the prospective inclusion of patients in an unselected population as well as the size of the study, which to the best of our knowledge is the largest study investigating GBS colonization in pregnant women in Scandinavia (Hakansson et al., 2008; Hansen et al., 2004). Limitations of our study include the selection of consenting participants, who may not be fully representative of the whole population of pregnant women, and the fact that the midwives were not able to obtain vaginal–rectal samples from all the women participating in the study. This was mainly due to a high level of activity in the delivery ward during the study period and is thus unlikely to have introduced a systematic bias. In conclusion, GBS colonization rate and serotype distribution in the Oslo GBS-study are compatible with other Western countries. GBS capsular serotyping, in particular, but also GBS capsular genotyping has shortcomings. For epidemiological studies of GBS to be accurate, identification should include a combination of capsular gene typing methods in addition to traditional serotyping by latex agglutination.
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Please cite this article as: Brigtsen, A.K., et al., Comparison of PCR and serotyping of Group B Streptococcus in pregnant women: The Oslo GBSstudy, J. Microbiol. Methods (2014), http://dx.doi.org/10.1016/j.mimet.2014.11.001
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Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
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Anne Karin Brigtsen a,⁎, Lumnije Dedi b, Kjetil K. Melby a,b, Mona Holberg-Petersen b, Andreas Radtke c,d, Randi Valsø Lyng c, Lise Lima Andresen b, Anne Flem Jacobsen a,e, Drude Fugelseth a,f, Andrew Whitelaw a
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Article history: Received 23 July 2014 Received in revised form 3 November 2014 Accepted 4 November 2014 Available online xxxx
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Keywords: Group B Streptococcus Streptococcus agalactiae Serotype Serotyping Genotyping Pregnancy
Institute of Clinical Medicine, University of Oslo, Oslo, Norway Department of Microbiology, Oslo University Hospital Ullevaal, Oslo, Norway Department of Medical Microbiology, St. Olavs University Hospital, Trondheim, Norway d Department of Laboratory Medicine, Children's and Women's Health, Norwegian University of Science and Technology, Trondheim, Norway e Department of Obstetrics and Gynaecology, Oslo University Hospital Ullevaal, Oslo, Norway f Department of Neonatal Intensive Care, Oslo University Hospital Ullevaal, Oslo, Norway
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Streptococcus agalactiae (GBS) is a leading cause of invasive neonatal infection. Serotyping of GBS is important in following epidemiological trends and vaccine development. Capsular serotyping of GBS by latex agglutination has been the predominant typing method, but more recently capsular genotyping has been introduced as an alternative method. The purpose of this study was to compare the relative performance of these methods in a contemporary population of pregnant women. We typed isolates from an unselected population of 426 colonized women at delivery using latex agglutination and a combination of four PCR methods. Antibiotic resistance was tested in 449 isolates. Capsular genotyping gave a result in all except three of 426 isolates. Fifty-nine of 426 isolates could not be typed by latex agglutination. Agreement between serotyping and genotyping was shown in 303 (71.1%) of the isolates. 10.2% of the isolates were resistant to erythromycin, 9.6% to clindamycin, 76.6% to tetracycline and none to penicillin. In conclusion, a substantial proportion of the colonizing strains were non-typeable by serotyping, but typeable by genotyping. This suggests that a diagnostic genotyping strategy is preferable to serotyping of the GBS polysaccharide capsule in colonized, pregnant women. © 2014 Published by Elsevier B.V.
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Streptococcus agalactiae (group B Streptococcus, GBS) is a leading cause of serious neonatal infection. In addition, GBS is an increasingly important cause of infections in older persons and immunocompromised patients (Skoff et al., 2009). Maternal vaginal–rectal colonization is considered the main route for neonatal infection. The capsular polysaccharide is a major virulence factor of this organism, and serotyping of the capsular polysaccharide is used to characterize strains and investigate GBS epidemiology in humans (Rubens et al., 1987). To date, ten capsular serotypes have been characterized, Ia, Ib and II to IX (Slotved et al., 2007). Serotypes Ia, Ib, II, III and V are predominant among both neonatal and adult invasive strains and maternal colonizing strains in the Western hemisphere (Edmond et al., 2012; Remington et al., 2011; Skoff et al., 2009). Serotyping of GBS is important in following epidemiological trends and in vaccine development (Johri et al., 2006). The most common phenotypic methods for serotyping GBS are latex agglutination tests
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1. Introduction
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Comparison of PCR and serotyping of Group B Streptococcus in pregnant women: The Oslo GBS-study
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⁎ Corresponding author at: Institute of Clinical Medicine, University of Oslo, Kirkeveien 166, N-0450 Oslo, Norway. Tel.: +47 22118765; fax: +47 22118763. E-mail address: [email protected] (A.K. Brigtsen).
(Afshar et al., 2011), which are based on polyclonal antibodies specific for the capsular polysaccharides. Latex agglutination tests are commercially available and in general considered reliable and easy to perform. In addition, several PCR-based methods for GBS capsular typing have been developed. The initial assays for genotyping were published by Kong in 2002 and Borchardt in 2004 using single PCRs for the different capsular polysaccharide types (Borchardt et al., 2004; Kong et al., 2002). Later Poyart and Imperi developed multiplex-PCR approaches (Imperi et al., 2010; Poyart et al., 2007). The two-set multiplex PCR by Poyart et al. was developed before the discovery of serotype IX. Strengths of the assay by Imperi are the inclusion of the most recently discovered serotype IX and the possibility to distinguish all the 10 known serotypes in a single PCR reaction. The serotype-specific assay described by Kong et al. does not include specific PCRs for serotypes II, VII and VIII, but these are included in Borchardt's assay. Although PCR assays are commonly considered modern, sophisticated techniques, molecular methods also have limitations (Yao et al., 2013). For instance, molecular methods do not reveal if the capsular polysaccharide gene locus detected is expressed as a polysaccharide capsule. Sparse data exist on the relative performance of these methods in a contemporary population of pregnant women. Accordingly, in this
http://dx.doi.org/10.1016/j.mimet.2014.11.001 0167-7012/© 2014 Published by Elsevier B.V.
Please cite this article as: Brigtsen, A.K., et al., Comparison of PCR and serotyping of Group B Streptococcus in pregnant women: The Oslo GBSstudy, J. Microbiol. Methods (2014), http://dx.doi.org/10.1016/j.mimet.2014.11.001
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The Oslo GBS Study is a prospective cohort study of pregnant women admitted to the Delivery Department at Oslo University Hospital, Ullevaal, Oslo, Norway. This hospital serves a population of approximately 600 000 in the metropolitan Oslo area. During the period from June 2009 to September 2011, 16 000 pregnant women were invited to participate at the time of routine ultrasound screening in the second trimester. 4450 women consented to participate in the study, and 1739 women had one vaginal–rectal sample obtained (Fig. 1). The study was approved by the Regional Committees for Medical and Health Research Ethics and is registered in the Norwegian Biobank Registry. Written informed consent was obtained from the participants.
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2.2. Specimen collection and identification
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The S. agalactiae isolates were collected and processed in accordance with the CDC guideline (Verani et al., 2010). Combined vaginal–rectal swabs were obtained during the vaginal examination at the onset of labor and prior to antibiotic administration. The swabs were placed in transport tubes containing Amies medium (Copan, Brescia, Italy) and transported to the laboratory within 24 h. Each strain was cultured to both Columbia agar (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA) and inoculated into Lim broth (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA). After incubation at 35 °C in ambient air for 24 h, turbid broths were subcultured onto blood agar plates and incubated at 35 °C in 5% CO2 for 24 h. Columbia agar plates were reincubated at 35 °C for 18–24 h if GBS was not identified after incubation. Isolates were identified based on colony morphology, β-hemolysis, catalase test, and latex agglutination. The Lancefield group B antigen was determined using a latex grouping kit (Prolex, Prolab Diagnostics, Richmond Hill, Ontario, Canada), according to the manufacturer's recommendations.
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2.3. Serotyping by latex agglutination
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From Lim broth cultures showing visible growth, a 10 μl aliquot was applied to a reaction card and mixed with 10 μl of latex suspension
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2.4. Molecular capsular typing
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All primers used in this study were published previously by four different groups (Borchardt et al., 2004; Imperi et al., 2010; Kong et al., 2002; Poyart et al., 2007). PCR conditions for the multiplex PCR by Imperi et al. were set according to the initial publication. Using the primers described by Borchardt, Kong and Poyart, the samples were amplified by a denaturation step for 10 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 60 s and a final extension of 72 °C for 5 min. The PCR mix qPCR Core kit (Eurogentec, Seraing, Belgium) was used for amplification and PCR products were visualized by by electrophoresis through a 1.5% agarose gel (SeaKem LE Agarose, Lonza, Rockland, ME, USA) containing ethidium bromide. All isolates were initially examined by the one-set multiplex PCR involving 19 primers developed by Imperi et al. (2010). This assay was adopted by the pan-European program DEVANI (DEsign of a Vaccine Against Neonatal Infections) as the standard method for molecular GBS capsular gene typing (Afshar et al., 2011). The initial multiplexPCR testing was performed blinded to the results of the serotyping. If serotyping and initial genotyping results were discordant, the Imperi assay was repeated. Isolates non-typeable by the method developed by Imperi et al. and isolates with discordant results for PCR and latex agglutination were in addition classified by a two-set multiplex PCR developed by Poyart et al. If this did not resolve the ambiguities, the serotype-specific PCRs developed by Kong et al. or Borchardt et al. were used (Borchardt et al., 2004; Kong et al., 2002).
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2.5. Antimicrobial susceptibility testing
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Isolates were tested against penicillin, erythromycin, clindamycin, tetracycline and trimethoprim-sulfamethoxazole by disk diffusion. Antimicrobial susceptibility disks from Oxoid (Cheshire, UK) were exchanged with disks from Becton, Dickinson and Company (Franklin Lakes, New Jersey, USA) January 2011. Norwegian Working Group on
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1739 Women had one vaginal-rectal sample obtained
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(reagents Ia, Ib, and II to IX; Strep-B-Latex kit, Statens Serum Institut, Copenhagen, Denmark). The two drops were mixed and the card was rotated slowly for up to 30 s and observed for agglutination. A distinct agglutination was scored as a positive reaction. A distinct agglutination reaction with one antigen occuring simultaneously with a weak agglutination reaction with one or more antigens resulted in repeated testing. If the same pattern of reaction occurred, the serotype with the distinct reaction was scored as positive. If a pattern of equally stong reactions with two ore more serotype antigens appeared, the isolate was scored as non-typeable.
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report we share our experience with the commonly used procedures for capsular serotyping and genotyping to characterize GBS isolates from an unselected population of pregnant women. In addition, we report antimicrobial susceptibility data from the isolates.
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452 Women were GBS culture positive
26 Samples were not GBS typed
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449 Samples were available for susceptibility testing
Fig. 1. Flowchart illustrating the selection of included samples.
Please cite this article as: Brigtsen, A.K., et al., Comparison of PCR and serotyping of Group B Streptococcus in pregnant women: The Oslo GBSstudy, J. Microbiol. Methods (2014), http://dx.doi.org/10.1016/j.mimet.2014.11.001
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2.6. Statistical analysis
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The Mann–Whitney U test was used to compare the difference in age between colonized and non-colonized women. Fisher's exact or chi-square tests were used, as appropriate, to compare colonization by parity and antimicrobial resistance between GBS capsular polysaccharide genotypes. Two-sided p-values b0.05 were considered statistically significant.
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3. Results
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3.1. Carriage study
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Of the 4450 women who consented to partcipate in the Oslo GBSstudy, 1739 had one vaginal–rectal sample obtained at delivery. Four hundred and fifty-two (26.0%) of these samples were culture positive for GBS. The median age of the women in the study was 32.0 years, ranging from 19.7 to 46.2 years, and 63.2% were giving birth to their first child. There was no significant difference between the age of the 452 colonized women and the 1287 non-colonized women (median age (25–75 percentile): 31.8 (29.6–34.0) vs. 32.0 (29.5–34.9) years; p = 0.34). No significant association between colonization rate and parity was observed (nulliparous (25.9%) vs. primi- or multiparous (26.2%); p = 0.89).
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3.2. Serotyping by latex agglutination
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Four hundred and twenty-six isolates were serotyped by latex agglutination of which 367 (86.2%) were assigned to one of the ten serotypes (Table 1). Of 59 isolates scored as non-typeable, 28 isolates reacted equally well with reagents to two serotypes, of which one serotype was identical to the genotype (Table 2). Furthermore, 3 isolates reacted equally well with reagents to two serotypes, neither of which corresponded to the genotype. Thirty-eight isolates reacted with serotype IX latex antiserum. Of these, 20 isolates also tested positive for serotypes Ia (4), Ib (2), II (3), III (6), IV (2) or V (3).
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Table 2 Isolates reacting to 2 latex serotype reagents using genotyping as the reference. Serotype by latex agglutination Ia Ib II III IV V VI VII VIII IX Total a
Genotype Ia
Ib
II
III
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4 1
1
1 1
4 9
1 2
3 6
IV
V
VI
VII
VIII
IX
2
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NT, non-typeable.
1 3
NTa
Total 3
2 2
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Antibiotics (NWGA) guidelines for performance and interpretation of susceptibility testing were applied until January 2011 (Bergan et al., 1997), after which the EUCAST guidelines were applied (http://www. eucast.org/antimicrobial_susceptibility_testing).
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Table 1 Comparison of results of 426 GBS isolates serotyped by latex agglutination and a combination of PCR methods. Serotype by latex agglutination
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Ia Ib II III IV V VI VII VIII IX NTa Subtotal Ac Bd Total
t1:22 t1:23 t1:24 t1:25 t1:26
a
Genotype Ia 45
Ib
b
III
IV
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30
3 4
II
2 31 1
2 87 1
V
1 16
1
36
1 58
VI
VII
VIII
IX
NTa
1
1
2 0 2 3 57 9 1
7 2 40 2
10 7 51 6 2
1 2 6 101 5
3 1 3 57 3
3 7 70 2
2
0
3
11
2
11 1
3
Total 49 48 33 88 42 63 2 0 4 38 28 395 28 3 426
NT, non-typeable. Boldface: Serotyping concordant to genotyping. c A: Isolates reacting to 2 latex antigens using genotyping as a reference. Please see Table 2 for details. d B: Isolates reacting to 2 latex antigens, none concordant to the genotype. b
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The 426 isolates were examined using the molecular capsular typing 195 methods, as described above. By combining the multiplex PCR devel- 196 oped by Imperi et al. with three additional PCR assays for molecular cap- 197 sular typing, we were able to assign a capsular gene type to 423 (99.3%) 198 of the strains. Based on results from the genotyping, serotype III was 199 most frequent, making up 24.9% of the isolates. Serotypes V (16.9%) 200 and Ia (15.7%) were second and third in frequency, respectively (Fig. 2). 201 Using the multiplex PCR by Imperi et al., Yao et al. found that some 202 strains of serotypes Ib and IV were incorrectly typed as Ia because the 203 middle band was very weak or not detectable. A total of 67 isolates ini- 204 tially serotyped as Ia were therefore reexamined using a singleplex PCR 205 assay (Kong et al., 2002). Two of the Ia strains showed disagreement 206 with the results from the Imperi assay and were classified as non- 207 typeable. 208
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178 179
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A total of 303 (71.1%) isolates showed agreement between the two methods, as indicated by the numbers marked in bold in Table 1. By capsular genotyping, we identified 11 serotype IX isolates, compared to 38 by latex agglutination. Serotype VII strains were not detected by latex agglutination or PCR methods.
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Four hundred and forty-nine isolates were available for susceptibility testing (Fig. 1). All isolates were susceptible to penicillin. Resistance to erythromycin and clindamycin was oberved in 10.2% and 9.6% of the isolates, respectively (Table 3). Of the erythromycin-resistant isolates, 40/46 (87.0%) showed cross-resistance to clindamycin. A high frequency of resistance to tetracycline was observed, in particular among serotypes Ia (88.1%), III (86.8%) and IV (83.3%). All isolates belonging to the most recently described serotype IX were susceptible to the antibiotics tested. There was substantial heterogeneity in antibiotic susceptibility between genotypes (Table 3). For instance, the resistance to clindamycin differed from 3/67 for genotype Ia to 12/72 for genotype V (p = 0.028); the resistance to erythromycin from 3/67 (Ia) to 13/72 (V) (p = 0.016), and the resistance to tetracycline from 59/67 (Ia) to 24/42 (Ib) (p b 0.001).
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In this large unselected group of pregnant women colonized with GBS, we found that 13.8% of isolates could not be satisfactorily typed by latex agglutination, an unacceptably high failure rate. In contrast, using a combination of capsular genotyping methods, only 0.7% of isolates were not identified. Previous studies applying capsular latex
231 232
Please cite this article as: Brigtsen, A.K., et al., Comparison of PCR and serotyping of Group B Streptococcus in pregnant women: The Oslo GBSstudy, J. Microbiol. Methods (2014), http://dx.doi.org/10.1016/j.mimet.2014.11.001
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Percentage of isolates
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0 Ib
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IV
V Genotype
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Fig. 2. Distribution of GBS capsular genotypes among 426 isolates using a combination of 4 PCR assays. a NT, non-typeable.
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246 247
Genotype
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n
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Ia Ib II III IV V VI VII VIII IX NT Sum genotyped Not genotyped Total (%)
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Table 3 Distribution of resistance among GBS genotypes.
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distribution in pregnant women, identifying serotypes Ia, II and III as predominant in a subset of 26 culture-positive women (Hordnes et al., 1996). In the present study, serotypes III, V, Ia, IV and Ib were the most common isolates using the molecular gene typing results. The predominance of serotype III is in accordance with results from other authors (Davies et al., 2001; Hakansson et al., 2008; Lu et al., 2013; Savoia et al., 2008). A trivalent GBS capsular polysaccharide conjugate vaccine prepared with capsular polysaccharides to serotypes Ia, Ib and III is undergoing phase-II evaluation among pregnant women (Heath, 2011; Madhi et al., 2013). Based on the current observations, this vaccine may not cover more than half of the colonizing strains in the general population of pregnant women. We observed resistance rates of 10.2% to erythromycin and 9.6% to clindamycin. This is relatively low compared to rates as high as 66% and 54% for erythromycin, and 56% and 33% for clindamycin reported for colonizing strains in China and USA, respectively (DiPersio and DiPersio, 2006; Lu et al., 2013). A restrictive policy for administering antibiotics in Norway may partially explain this. A risk-based rather than a screening-based approach for identifying women who should receive intrapartum antibiotic prophylaxis to prevent invasive neonatal GBS disease keeps the use of antibiotics low in women giving birth (Verani et al., 2010). Erythromycin is the treatment of choice in
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agglutination methods have in most cases been able to serotype more than 90% of the isolates (Savoia et al., 2008; Turner et al., 2012). An exception to this is the study by Yao et al. in which 27% of 281 human isolates could not be typed by latex agglutination (Yao et al., 2013). Some of their isolates were selected because they were problematic when serotyped by latex agglutination. Our results demonstrate that difficulties with serotyping are common in the clinical setting of an unselected population of pregnant women. The GBS colonization rate of 26.0% was lower than the 34.7% reported in a Norwegian study of perinatal samples from 251 women (Bergseng et al., 2007). However, it is in the range of previous reports from North America and several European countries (Campbell et al., 2000; Davies et al., 2001; Hakansson et al., 2008; Jones et al., 2006; Kunze et al., 2011), but higher than recently reported rates from Asia (7%–12%) and Denmark (10%) (Lu et al., 2013; Stokholm et al., 2013; Turner et al., 2012). The proportions of different serotypes may vary considerably between geographical areas, between subgroups of the population, and over time. Knowing the serotype distribution is essential for finding the most suitable capsular polysaccharide based vaccine for a given country. Limited data are available on the distribution of GBS serotypes in Europe. In Norway, only one study has examined the serotype
E
236 237
67 42 59 106 60 72 2 0 3 12 3 426 26 452
a
Resistance
n
Clindamycin No. (%)
Erythromycin No. (%)
Both clindamycin and erythromycin No. (%)
67 42 57 105 60 72 2 0 3 12 3 423 26 449
3 (4.5) 4 (9.5) 8 (13.6) 11 (10.4) 3 (5.0) 12 (16.7)
3 (4.5) 5 (11.9) 9 (15.3) 10 (9.4) 4 (6.7) 13 (18.1)
2 (3.0) 4 (9.5) 8 (13.6) 9 (8.5) 3 (5.0) 12 (16.7)
Tetracycline No. (%) 59 (88.1) 24 (57.1) 46 (78.0) 92 (86.8) 50 (83.3) 49 (68.1) 1 (50.0)
T-sb No. (%)
Penicillin No. (%)
2 2
3 (100.0) 2 (7.7) 43 (9.6)
2 (7.7) 46 (10.2)
2 (7.7) 40 (8.9)
20 (76.9) 344 (76.6)
4 (0.9)
0 (0)
Three samples were genotyped (2 genotype II and one genotype III), but were not available for susceptibility testing. T-s: Trimethoprim-sulfametoxazole.
Please cite this article as: Brigtsen, A.K., et al., Comparison of PCR and serotyping of Group B Streptococcus in pregnant women: The Oslo GBSstudy, J. Microbiol. Methods (2014), http://dx.doi.org/10.1016/j.mimet.2014.11.001
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We wish to thank everyone involved in enrolment, sampling and culturing the GBS strains at the Department of Obstetrics and Gynaecology and the Department of Microbiology at Oslo University Hospital Ullevaal. We are grateful to Cathrine Nygaard for expert technical assistance. This work was partially supported by funds from Renée and Bredo Grimsgaard's Foundation and Eckbo's Foundation. The sponsors were not involved in the design and execution of the study.
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pregnant women allergic to penicillin in Norway, and the choice of antibiotics to this group should be guided by contemporary information concerning antimicrobial resistance. We further observed an association between erythromycin and clindamycin resistance and capsular serotype V. Serotype V displayed the highest resistance to erythromycin (18.1%) and clindamycin (16.7%), which is in accordance with other studies (Borchardt et al., 2006; DiPersio and DiPersio, 2006; Lin et al., 2000). Strengths of our study include the prospective inclusion of patients in an unselected population as well as the size of the study, which to the best of our knowledge is the largest study investigating GBS colonization in pregnant women in Scandinavia (Hakansson et al., 2008; Hansen et al., 2004). Limitations of our study include the selection of consenting participants, who may not be fully representative of the whole population of pregnant women, and the fact that the midwives were not able to obtain vaginal–rectal samples from all the women participating in the study. This was mainly due to a high level of activity in the delivery ward during the study period and is thus unlikely to have introduced a systematic bias. In conclusion, GBS colonization rate and serotype distribution in the Oslo GBS-study are compatible with other Western countries. GBS capsular serotyping, in particular, but also GBS capsular genotyping has shortcomings. For epidemiological studies of GBS to be accurate, identification should include a combination of capsular gene typing methods in addition to traditional serotyping by latex agglutination.
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Please cite this article as: Brigtsen, A.K., et al., Comparison of PCR and serotyping of Group B Streptococcus in pregnant women: The Oslo GBSstudy, J. Microbiol. Methods (2014), http://dx.doi.org/10.1016/j.mimet.2014.11.001
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