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Comparison of three real-time PCR assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in young pregnant women
Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Comparison of three real-time PCR assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in young pregnant women Olivia Peuchant a,b,c,⁎, Sabrina de Diego a,b, Chloé Le Roy a,b, Sandrine Frantz-Blancpain d, Claude Hocké d, Cécile Bébéar a,b,c, Bertille de Barbeyrac a,b,c a
Univ. Bordeaux, USC EA 3671 Infections Humaines à Mycoplasmes et à Chlamydiae, Bordeaux, France INRA, USC EA 3671 Infections Humaines à Mycoplasmes et à Chlamydiae, Bordeaux, France Centre Hospitalier Universitaire de Bordeaux, Laboratoire de Bactériologie, Bordeaux, France d Centre Hospitalier Universitaire de Bordeaux, Service de Gynécologie Obstétrique, Bordeaux, France b c
a r t i c l e
i n f o
Article history: Received 12 May 2015 Received in revised form 1 September 2015 Accepted 3 September 2015 Available online xxxx Keywords: Chlamydia trachomatis Neisseria gonorrhoeae Real-time PCRs Vaginal swabs Pregnant women
a b s t r a c t We compared 3 commercial real-time PCR assays, the Abbott RealTime CT/NG, the cobas® 4800 CT/NG, and the Cepheid Xpert® CT/NG, for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in vaginal swabs collected prospectively from pregnant women aged b25 years. The overall agreement among 2 assays ranged from 98.9% to 99.5% with a kappa score between 0.94 and 0.97 for C. trachomatis. For N. gonorrhoeae, the overall agreement was 100%. All kits allowed prompt and specific results for C. trachomatis and N. gonorrhoeae in young pregnant women. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Chlamydia trachomatis is the most common pathogen causing sexually transmitted infections especially in young people. Neisseria gonorrhoeae is much less prevalent, but coinfections with C. trachomatis are common. The majority of these infections are asymptomatic in women. Today, nucleic acid amplification tests (NAATs) are widely used for highthroughput screening of C. trachomatis and N. gonorrhoeae. However, the detection of N. gonorrhoeae using NAATs has been compromised historically by unacceptably high rates of nonspecific reactivity with nongonococcal Neisseria species, and international guidelines recommended that N. gonorrhoeae–positive NAAT results should be confirmed by using supplementary assays with different target if the positive predictive value is b90% (Bignell et al., 2012). At the Bordeaux University Hospital, all pregnant women requesting abortion and aged b25 years are screened for C. trachomatis and N. gonorrhoeae infections on vaginal swabs, using the Abbott RealTime CT/NG test (Abbott, Rungis, France). Indeed, the prevalence of C. trachomatis and N. gonorrhoeae was evaluated at 12.5% and 2.4%, respectively, in this population in 2012 (de Barbeyrac, personal data). In a previous study realized among pregnant women attending for routine antenatal care and aged b25 years, we reported a prevalence of 7.9% for C. trachomatis, whereas no gonorrhea was detected (Peuchant et al., 2015). These results were obtained using vaginal swabs and the cobas® 4800 CT/NG test (Roche diagnostics, Meylan, France). The gap in ⁎ Corresponding author. Tel.: +33-5-57-57-16-25; fax: +33-5-56-93-29-40. E-mail address: [email protected] (O. Peuchant).
N. gonorrhoeae prevalence in these 2 populations was surprising and could be due to differences in the performances of tests used or differences link to the studied population. The aim of this study was to compare the performances of the Abbott RealTime CT/NG, the cobas® 4800 CT/NG, and the Cepheid Xpert® CT/NG (Cepheid, Maurens-Scopont, France) assays, tested simultaneously, for C. trachomatis and N. gonorrhoeae detection in vaginal swabs collected from pregnant women aged b25 years. 2. Materials and methods 2.1. Samples analyzed This study was conducted prospectively from September 2012 to February 2013 on pregnant women aged b25 years and presenting for the first visit prior to abortion or attending for routine antenatal care at the Bordeaux University Hospital, France. Vaginal swabs were collected before abortion or during pregnancy follow-up. 2.2. Sample process All dry vaginal swabs were first discharged in 2 mL of 2SP (saccharose-phosphate) media. According to the manufacturer's package insert instructions, the volume of specimens tested was 400 μL for the Abbott RealTime CT/NG and the cobas® 4800 CT/NG tests and 1 mL for the Cepheid Xpert® CT/NG assay. Each NAAT contains an internal control, as well as positive and negative controls. Results were interpreted
http://dx.doi.org/10.1016/j.diagmicrobio.2015.09.002 0732-8893/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Peuchant O, et al, Comparison of 3 real-time PCR assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in young pregnant women, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.09.002
2
O. Peuchant et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
according to the manufacturer's instructions, including recommended procedures for resolution of indeterminate results. For each vaginal swab, the 3 NAATs were performed at the same time. 2.3. Data analysis Concordant results for 1 specimen were defined as positive if all three NAATs gave positive results and negative if all 3 NAATs gave negative results. For discordant results, a true-positive result was assigned if at least 2 out of the 3 NAATs gave a positive result and a true-negative was assigned if at least 2 out of the 3 NAATs gave a negative result. Patient infected status was defined as being infected with C. trachomatis or N. gonorrhoeae if all 3 NAATs or 2 out of 3 NAATs gave positive results and as being not infected if all 3 NAATs or 2 out of 3 NAATs gave negative results for the collected vaginal swab, as previously described (Martin et al., 2004). Overall percent agreement was calculated based on initial results using head-to-head comparison of the 3 assays (Feinstein and Cicchetti, 1990; Simel et al., 1991). The unweighted kappa statistic was calculated and interpreted as follows: ≤0.20, poor agreement; 0.21–0.40, fair agreement; 0.41–0.60, moderate agreement; 0.61–0.80, good agreement; ≥0.80, excellent agreement. 3. Results A total of 377 pregnant women were included in the study, 265 from women requesting abortion (median age = 21 years [15–24]) and 112 from routine antenatal care (median age = 21 years [14–24]). Overall, the prevalence of C. trachomatis infection was 10.1% (38/377), ranging from 7.1% (8/112) for women with routine antenatal care to 11.3% (30/265) for those requesting abortion. For N. gonorrhoeae, the prevalence was similar among both population studied (1.8% (2/112) and 1.9% (5/265), respectively). 3.1. C. trachomatis results The first analysis on C. trachomatis results obtained with the 3 NAATs showed concordant positive results for 36 vaginal swabs and concordant negative results for 336 specimens (Table 1). Only 5 vaginal swabs provided discordant results between the 3 assays. Three discrepant specimens were Cepheid Xpert® CT/NG and cobas® 4800 CT/NG negative and assigned as true-negative. Two specimens were Abbott RealTime CT/NG and Cepheid Xpert® CT/NG positive and Abbott RealTime CT/NG and cobas® 4800 CT/NG positive, respectively, and were assigned as true-positive. Using head-to-head comparison, the overall agreement between the Abbott RealTime CT/NG assay and the cobas® 4800 CT/NG or the Cepheid Xpert® CT/NG test was of 98.9%, respectively. The value of kappa coefficient was 0.94. Head-to-head comparison of cobas® 4800 CT/NG assay and Cepheid Xpert® CT/NG test yielded an overall agreement of 99.5%. The value of kappa coefficient was 0.97. Table 1 Comparison of C. trachomatis results using the 3 real-time PCR assays. Test result Abbott RealTime CT/NG
cobas® 4800 CT/NG
Cepheid Xpert® CT/NG
+ − + + +
+ − + − −
+ − − − +
TP = true-positive; TN = true-negative.
Number of specimens
Specimen status
Patient
36 336 1 3 1
TP TN TP TN TP
Infected Not infected Infected Not infected Infected
Infected status
3.2. N. gonorrhoeae results A total of 370 specimens were negative, and 7 were positive using the 3 kits. All results were concordant, leading to 100% overall agreement and a value of kappa coefficient of 1. There were 3 C. trachomatis/ N. gonorrhoeae coinfections, 1 case in a woman requesting abortion and 2 cases in pregnant women attending for routine antenatal care. 4. Discussion In this study, the 3 real-time PCR tests presented excellent performances on vaginal swabs. All kappa scores were ≥0.94 for the detection of C. trachomatis, indicating excellent agreement between the assays. A 100% overall agreement was found using head-to-head comparison for N. gonorrhoeae detection. Thus, the 3 systems performed equivalent for the detection of C. trachomatis and N. gonorrhoeae using vaginal swabs from asymptomatic subjects, without need of confirmatory testing for N. gonorrhoeae–positive results. These results are in agreement with those previously described (Gaydos et al., 2013; Geelen et al., 2013; Le Roy et al., 2012; Van Der Pol et al., 2013). One of the strengths of this study is the inclusion of commercially available assays that are frequently used for C. trachomatis and N. gonorrhoeae detection. Only 1 previous report compared the performances of 2 assays tested in our study, the cobas® 4800 CT/NG and the Abbott RealTime CT/NG assays, and high concordance rates (≥96%) were observed between the 2 NAATs for C. trachomatis/N. gonorrhoeae detection in both rectal and self-collected vaginal swabs (Geelen et al., 2013). Since few laboratories have access to multiple platforms, head-to-head comparisons data may be difficult to obtain. Thus, reports of these findings are particularly relevant to today's laboratory management needs. Head-to-head analysis of the results provided an unbiased observation of the performance of all 3 assays without assigning true- and false-positive status. All assays used are fully automated systems but are designed to detected different targets of each bacterium. The Abbott RealTime CT/NG assay targets 2 regions of the cryptic plasmid of C. trachomatis and a conserved region of the opacity gene of N. gonorrhoeae (Geelen et al., 2013). The cobas® 4800 CT/NG assay targets the cryptic plasmid and the major outer membrane protein gene of C. trachomatis and a highly conserved direct repeat region DR-9 and a conserved sequence variant from this region of N. gonorrhoeae (Gaydos et al., 2013). The Cepheid Xpert® CT/NG test was designed to target a conserved chromosomal sequence of C. trachomatis and 2 highly conserved, noncontiguous chromosomal sequences of N. gonorrhoeae (Gaydos et al., 2013). Targeting at least 2 different genetic sequences reduce the potential for false-positive results and enhance specificity. Among discrepant results for C. trachomatis, there were 3 Abbott RealTime CT/NG positive/cobas® 4800 CT/NG and Cepheid Xpert® CT/NG negative samples. With the Abbott RealTime CT/NG assay, these specimens were initially tested equivocal because of a cycle number beyond the cutoff but were scored as C. trachomatis positive based on a positive repeat testing using the same specimen as recommended by the manufacturer. According to the specimen status, these vaginal swabs were considered as Abbott false-positive result. However, for discordant results, one limitation of our study is that we could not sequence amplicons obtained as sequence of primers used in each assay was not available. These data may challenge the proposed algorithm for the Abbott RealTime CT/NG assay and raises the question of interpretation of equivocal result. We reported a prevalence of 7.1% for C. trachomatis infections among pregnant women aged b25 years and attending for routine antenatal care. These results confirmed those obtained in the same population in a previous study performed in 2011 in the same hospital, using the cobas® 4800 CT/NG test (Peuchant et al., 2015), and are in agreement with those reported in the literature (Borges-Costa et al., 2011). Among women requesting abortion, the prevalence of C. trachomatis was 10.4% in our study. Similar findings were reported in 2 French
Please cite this article as: Peuchant O, et al, Comparison of 3 real-time PCR assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in young pregnant women, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.09.002
O. Peuchant et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
recent studies (Lavoué et al., 2012; Toyer et al., 2012) and in other worldwide works (Baczynska et al., 2008; Garland et al., 2000; Patel et al., 2008), the highest C. trachomatis infection rates being described in women b25 years. For N. gonorrhoeae, we found a prevalence of 1.8% among pregnant women aged b25 years and attending for routine antenatal care, while no gonorrhea infection was found in our previous study in the same population (Peuchant et al., 2015). This difference, not statistically significant (P = 0.16, Fischer's exact test), could be likely due to the low prevalence of this infection in pregnant women. Among subjects consulting for induced abortion, our prevalence of N. gonorrhoeae infection was 1.9%, slightly higher than that reported by Toyer et al. (2012) using the Abbott Real-Time CT/NG test. A weakness of our study is the limited number of patients infected with N. gonorrhoeae. In conclusion, this study demonstrated that the cobas® 4800 CT/NG, the Abbott RealTime CT/NG, and the Cepheid Xpert® CT/NG assays had excellent performances for C. trachomatis and N. gonorrhoeae detection in vaginal samples, without need of confirmatory testing for N. gonorrhoeae. In view of the high prevalence of C. trachomatis infection and the current PCR techniques which simultaneously detect C. trachomatis and N. gonorrhoeae, systematic screening for pregnant women aged b25 years could be benefit. Competing interest The authors declare that they have competing interests with Roche Diagnostics and Cepheid, which provided the cobas® 4800 CT/NG and the Cepheid Xpert® CT/NG kits for this study, respectively. Contributors BB contributed to the design of the study. SD and CLR collected samples and data and performed the real-time PCRs. OP, SD, CLR, CB, and BB performed the data analysis. OP, SFB, CH, CB, and BB wrote the paper and contributed to the interpretation of the results.
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References Baczynska A, Hvid H, Lamy P, Birkelund S, Christiansen G, Fedder J. Prevalence of Mycoplasma genitalium, Mycoplasma hominis and Chlamydia trachomatis among Danish patients requesting abortion. Syst Biol Reprod Med 2008;54:127–34. Bignell C, Ison C, Fitzgerald M. BASHH. United Kingdom National Guideline for Gonorrhoea Testing 2012. Clinical Effectiveness Group; 2012. Borges-Costa J, Matos C, Pereira F. Sexually transmitted infections in pregnant adolescents: prevalence and association with maternal and foetal morbidity. J Eur Acad Dermatol Venereol 2011;26:972–5. Feinstein AR, Cicchetti DV. High agreement but low kappa: I. The problems of two paradoxes. J Clin Epidemiol 1990;43:543–9. Garland SM, Tabrizi S, Hallo J, Chen S. Assessment of Chlamydia trachomatis prevalence by PCR and LCR in women presenting for termination of pregnancy. Sex Transm Infect 2000;76:173–6. Gaydos CA, Van Der Pol B, Jett-Goheen M, Barnes M, Quinn N, Clark C, et al. Performance of the Cepheid CT/NG Xpert Rapid PCR test for detection of Chlamydia trachomatis and Neisseria gonorrhoeae. J Clin Microbiol 2013;51:1666–72. Geelen TH, Rossen JW, Beerens AM, Poort L, Morré SA, Ritmeester WS, et al. Performance of cobas® 4800 and m2000 real-time assays for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in rectal and self-collected vaginal specimen. Diagn Microbiol Infect Dis 2013;77:101–5. Lavoué V, Vandenbroucke L, Lorand S, Pincemin P, Bauville E, Boyer L, et al. Screening for Chlamydia trachomatis using self-collected vaginal swabs at a public pregnancy termination clinic in France: results of a screen-and-treat policy. Sex Transm Dis 2012;39:622–7. Le Roy C, Papaxanthos A, Liesenfeld O, Mehats V, Clerc M, Bébéar C, et al. Swabs (dry or collected in universal transport medium) and semen can be used for the detection of Chlamydia trachomatis using the cobas 4800 system. J Med Microbiol 2012;62:217–22. Martin DH, Nsuami M, Schachter J, Hook III EW, Ferrero D, Quinn TC, et al. Use of multiple nucleic acid amplification tests to define the infected-patient “gold standard” in clinical trials of new diagnostic tests for Chlamydia trachomatis infections. J Clin Microbiol 2004;42:4749–58. Patel A, Rashid S, Godfrey EM, Panchal H, et al. Prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae genital infections in a publicly funded pregnancy termination clinic: empiric vs. indicated treatment? Contraception 2008;78:328–31. Peuchant O, Le Roy C, Desveaux C, Paris A, Asselineau J, Maldonado C, et al. Screening for Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium should it be integrated into routine pregnancy care in French young pregnant women? Diagn Microbiol Infect Dis 2015;82:14–9. Simel DL, Samsa GP, Matchar DB. Likelihood ratios with confidence: sample size estimation for diagnostic test studies. J Clin Epidemiol 1991;44:763–70. Toyer AL, Trignol-Viguier N, Mereghetti L, Joly B, Blin E, Body G, et al. Interest of simultaneous Chlamydia trachomatis and Neisseria gonorrhoeae screening at the time of preabortion consultation. Contraception 2012;86(5):572–6. Van Der Pol B, Taylor SN, Liesenfeld O, Williams JA, Hook III EW. Vaginal swabs are the optimal specimen for detection of genital Chlamydia trachomatis or Neisseria gonorrhoeae using the Cobas 4800 CT/NG test. Sex Transm Dis 2013;40:247–50.
Please cite this article as: Peuchant O, et al, Comparison of 3 real-time PCR assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in young pregnant women, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.09.002
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Comparison of three real-time PCR assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in young pregnant women Olivia Peuchant a,b,c,⁎, Sabrina de Diego a,b, Chloé Le Roy a,b, Sandrine Frantz-Blancpain d, Claude Hocké d, Cécile Bébéar a,b,c, Bertille de Barbeyrac a,b,c a
Univ. Bordeaux, USC EA 3671 Infections Humaines à Mycoplasmes et à Chlamydiae, Bordeaux, France INRA, USC EA 3671 Infections Humaines à Mycoplasmes et à Chlamydiae, Bordeaux, France Centre Hospitalier Universitaire de Bordeaux, Laboratoire de Bactériologie, Bordeaux, France d Centre Hospitalier Universitaire de Bordeaux, Service de Gynécologie Obstétrique, Bordeaux, France b c
a r t i c l e
i n f o
Article history: Received 12 May 2015 Received in revised form 1 September 2015 Accepted 3 September 2015 Available online xxxx Keywords: Chlamydia trachomatis Neisseria gonorrhoeae Real-time PCRs Vaginal swabs Pregnant women
a b s t r a c t We compared 3 commercial real-time PCR assays, the Abbott RealTime CT/NG, the cobas® 4800 CT/NG, and the Cepheid Xpert® CT/NG, for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in vaginal swabs collected prospectively from pregnant women aged b25 years. The overall agreement among 2 assays ranged from 98.9% to 99.5% with a kappa score between 0.94 and 0.97 for C. trachomatis. For N. gonorrhoeae, the overall agreement was 100%. All kits allowed prompt and specific results for C. trachomatis and N. gonorrhoeae in young pregnant women. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Chlamydia trachomatis is the most common pathogen causing sexually transmitted infections especially in young people. Neisseria gonorrhoeae is much less prevalent, but coinfections with C. trachomatis are common. The majority of these infections are asymptomatic in women. Today, nucleic acid amplification tests (NAATs) are widely used for highthroughput screening of C. trachomatis and N. gonorrhoeae. However, the detection of N. gonorrhoeae using NAATs has been compromised historically by unacceptably high rates of nonspecific reactivity with nongonococcal Neisseria species, and international guidelines recommended that N. gonorrhoeae–positive NAAT results should be confirmed by using supplementary assays with different target if the positive predictive value is b90% (Bignell et al., 2012). At the Bordeaux University Hospital, all pregnant women requesting abortion and aged b25 years are screened for C. trachomatis and N. gonorrhoeae infections on vaginal swabs, using the Abbott RealTime CT/NG test (Abbott, Rungis, France). Indeed, the prevalence of C. trachomatis and N. gonorrhoeae was evaluated at 12.5% and 2.4%, respectively, in this population in 2012 (de Barbeyrac, personal data). In a previous study realized among pregnant women attending for routine antenatal care and aged b25 years, we reported a prevalence of 7.9% for C. trachomatis, whereas no gonorrhea was detected (Peuchant et al., 2015). These results were obtained using vaginal swabs and the cobas® 4800 CT/NG test (Roche diagnostics, Meylan, France). The gap in ⁎ Corresponding author. Tel.: +33-5-57-57-16-25; fax: +33-5-56-93-29-40. E-mail address: [email protected] (O. Peuchant).
N. gonorrhoeae prevalence in these 2 populations was surprising and could be due to differences in the performances of tests used or differences link to the studied population. The aim of this study was to compare the performances of the Abbott RealTime CT/NG, the cobas® 4800 CT/NG, and the Cepheid Xpert® CT/NG (Cepheid, Maurens-Scopont, France) assays, tested simultaneously, for C. trachomatis and N. gonorrhoeae detection in vaginal swabs collected from pregnant women aged b25 years. 2. Materials and methods 2.1. Samples analyzed This study was conducted prospectively from September 2012 to February 2013 on pregnant women aged b25 years and presenting for the first visit prior to abortion or attending for routine antenatal care at the Bordeaux University Hospital, France. Vaginal swabs were collected before abortion or during pregnancy follow-up. 2.2. Sample process All dry vaginal swabs were first discharged in 2 mL of 2SP (saccharose-phosphate) media. According to the manufacturer's package insert instructions, the volume of specimens tested was 400 μL for the Abbott RealTime CT/NG and the cobas® 4800 CT/NG tests and 1 mL for the Cepheid Xpert® CT/NG assay. Each NAAT contains an internal control, as well as positive and negative controls. Results were interpreted
http://dx.doi.org/10.1016/j.diagmicrobio.2015.09.002 0732-8893/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Peuchant O, et al, Comparison of 3 real-time PCR assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in young pregnant women, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.09.002
2
O. Peuchant et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
according to the manufacturer's instructions, including recommended procedures for resolution of indeterminate results. For each vaginal swab, the 3 NAATs were performed at the same time. 2.3. Data analysis Concordant results for 1 specimen were defined as positive if all three NAATs gave positive results and negative if all 3 NAATs gave negative results. For discordant results, a true-positive result was assigned if at least 2 out of the 3 NAATs gave a positive result and a true-negative was assigned if at least 2 out of the 3 NAATs gave a negative result. Patient infected status was defined as being infected with C. trachomatis or N. gonorrhoeae if all 3 NAATs or 2 out of 3 NAATs gave positive results and as being not infected if all 3 NAATs or 2 out of 3 NAATs gave negative results for the collected vaginal swab, as previously described (Martin et al., 2004). Overall percent agreement was calculated based on initial results using head-to-head comparison of the 3 assays (Feinstein and Cicchetti, 1990; Simel et al., 1991). The unweighted kappa statistic was calculated and interpreted as follows: ≤0.20, poor agreement; 0.21–0.40, fair agreement; 0.41–0.60, moderate agreement; 0.61–0.80, good agreement; ≥0.80, excellent agreement. 3. Results A total of 377 pregnant women were included in the study, 265 from women requesting abortion (median age = 21 years [15–24]) and 112 from routine antenatal care (median age = 21 years [14–24]). Overall, the prevalence of C. trachomatis infection was 10.1% (38/377), ranging from 7.1% (8/112) for women with routine antenatal care to 11.3% (30/265) for those requesting abortion. For N. gonorrhoeae, the prevalence was similar among both population studied (1.8% (2/112) and 1.9% (5/265), respectively). 3.1. C. trachomatis results The first analysis on C. trachomatis results obtained with the 3 NAATs showed concordant positive results for 36 vaginal swabs and concordant negative results for 336 specimens (Table 1). Only 5 vaginal swabs provided discordant results between the 3 assays. Three discrepant specimens were Cepheid Xpert® CT/NG and cobas® 4800 CT/NG negative and assigned as true-negative. Two specimens were Abbott RealTime CT/NG and Cepheid Xpert® CT/NG positive and Abbott RealTime CT/NG and cobas® 4800 CT/NG positive, respectively, and were assigned as true-positive. Using head-to-head comparison, the overall agreement between the Abbott RealTime CT/NG assay and the cobas® 4800 CT/NG or the Cepheid Xpert® CT/NG test was of 98.9%, respectively. The value of kappa coefficient was 0.94. Head-to-head comparison of cobas® 4800 CT/NG assay and Cepheid Xpert® CT/NG test yielded an overall agreement of 99.5%. The value of kappa coefficient was 0.97. Table 1 Comparison of C. trachomatis results using the 3 real-time PCR assays. Test result Abbott RealTime CT/NG
cobas® 4800 CT/NG
Cepheid Xpert® CT/NG
+ − + + +
+ − + − −
+ − − − +
TP = true-positive; TN = true-negative.
Number of specimens
Specimen status
Patient
36 336 1 3 1
TP TN TP TN TP
Infected Not infected Infected Not infected Infected
Infected status
3.2. N. gonorrhoeae results A total of 370 specimens were negative, and 7 were positive using the 3 kits. All results were concordant, leading to 100% overall agreement and a value of kappa coefficient of 1. There were 3 C. trachomatis/ N. gonorrhoeae coinfections, 1 case in a woman requesting abortion and 2 cases in pregnant women attending for routine antenatal care. 4. Discussion In this study, the 3 real-time PCR tests presented excellent performances on vaginal swabs. All kappa scores were ≥0.94 for the detection of C. trachomatis, indicating excellent agreement between the assays. A 100% overall agreement was found using head-to-head comparison for N. gonorrhoeae detection. Thus, the 3 systems performed equivalent for the detection of C. trachomatis and N. gonorrhoeae using vaginal swabs from asymptomatic subjects, without need of confirmatory testing for N. gonorrhoeae–positive results. These results are in agreement with those previously described (Gaydos et al., 2013; Geelen et al., 2013; Le Roy et al., 2012; Van Der Pol et al., 2013). One of the strengths of this study is the inclusion of commercially available assays that are frequently used for C. trachomatis and N. gonorrhoeae detection. Only 1 previous report compared the performances of 2 assays tested in our study, the cobas® 4800 CT/NG and the Abbott RealTime CT/NG assays, and high concordance rates (≥96%) were observed between the 2 NAATs for C. trachomatis/N. gonorrhoeae detection in both rectal and self-collected vaginal swabs (Geelen et al., 2013). Since few laboratories have access to multiple platforms, head-to-head comparisons data may be difficult to obtain. Thus, reports of these findings are particularly relevant to today's laboratory management needs. Head-to-head analysis of the results provided an unbiased observation of the performance of all 3 assays without assigning true- and false-positive status. All assays used are fully automated systems but are designed to detected different targets of each bacterium. The Abbott RealTime CT/NG assay targets 2 regions of the cryptic plasmid of C. trachomatis and a conserved region of the opacity gene of N. gonorrhoeae (Geelen et al., 2013). The cobas® 4800 CT/NG assay targets the cryptic plasmid and the major outer membrane protein gene of C. trachomatis and a highly conserved direct repeat region DR-9 and a conserved sequence variant from this region of N. gonorrhoeae (Gaydos et al., 2013). The Cepheid Xpert® CT/NG test was designed to target a conserved chromosomal sequence of C. trachomatis and 2 highly conserved, noncontiguous chromosomal sequences of N. gonorrhoeae (Gaydos et al., 2013). Targeting at least 2 different genetic sequences reduce the potential for false-positive results and enhance specificity. Among discrepant results for C. trachomatis, there were 3 Abbott RealTime CT/NG positive/cobas® 4800 CT/NG and Cepheid Xpert® CT/NG negative samples. With the Abbott RealTime CT/NG assay, these specimens were initially tested equivocal because of a cycle number beyond the cutoff but were scored as C. trachomatis positive based on a positive repeat testing using the same specimen as recommended by the manufacturer. According to the specimen status, these vaginal swabs were considered as Abbott false-positive result. However, for discordant results, one limitation of our study is that we could not sequence amplicons obtained as sequence of primers used in each assay was not available. These data may challenge the proposed algorithm for the Abbott RealTime CT/NG assay and raises the question of interpretation of equivocal result. We reported a prevalence of 7.1% for C. trachomatis infections among pregnant women aged b25 years and attending for routine antenatal care. These results confirmed those obtained in the same population in a previous study performed in 2011 in the same hospital, using the cobas® 4800 CT/NG test (Peuchant et al., 2015), and are in agreement with those reported in the literature (Borges-Costa et al., 2011). Among women requesting abortion, the prevalence of C. trachomatis was 10.4% in our study. Similar findings were reported in 2 French
Please cite this article as: Peuchant O, et al, Comparison of 3 real-time PCR assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in young pregnant women, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.09.002
O. Peuchant et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
recent studies (Lavoué et al., 2012; Toyer et al., 2012) and in other worldwide works (Baczynska et al., 2008; Garland et al., 2000; Patel et al., 2008), the highest C. trachomatis infection rates being described in women b25 years. For N. gonorrhoeae, we found a prevalence of 1.8% among pregnant women aged b25 years and attending for routine antenatal care, while no gonorrhea infection was found in our previous study in the same population (Peuchant et al., 2015). This difference, not statistically significant (P = 0.16, Fischer's exact test), could be likely due to the low prevalence of this infection in pregnant women. Among subjects consulting for induced abortion, our prevalence of N. gonorrhoeae infection was 1.9%, slightly higher than that reported by Toyer et al. (2012) using the Abbott Real-Time CT/NG test. A weakness of our study is the limited number of patients infected with N. gonorrhoeae. In conclusion, this study demonstrated that the cobas® 4800 CT/NG, the Abbott RealTime CT/NG, and the Cepheid Xpert® CT/NG assays had excellent performances for C. trachomatis and N. gonorrhoeae detection in vaginal samples, without need of confirmatory testing for N. gonorrhoeae. In view of the high prevalence of C. trachomatis infection and the current PCR techniques which simultaneously detect C. trachomatis and N. gonorrhoeae, systematic screening for pregnant women aged b25 years could be benefit. Competing interest The authors declare that they have competing interests with Roche Diagnostics and Cepheid, which provided the cobas® 4800 CT/NG and the Cepheid Xpert® CT/NG kits for this study, respectively. Contributors BB contributed to the design of the study. SD and CLR collected samples and data and performed the real-time PCRs. OP, SD, CLR, CB, and BB performed the data analysis. OP, SFB, CH, CB, and BB wrote the paper and contributed to the interpretation of the results.
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References Baczynska A, Hvid H, Lamy P, Birkelund S, Christiansen G, Fedder J. Prevalence of Mycoplasma genitalium, Mycoplasma hominis and Chlamydia trachomatis among Danish patients requesting abortion. Syst Biol Reprod Med 2008;54:127–34. Bignell C, Ison C, Fitzgerald M. BASHH. United Kingdom National Guideline for Gonorrhoea Testing 2012. Clinical Effectiveness Group; 2012. Borges-Costa J, Matos C, Pereira F. Sexually transmitted infections in pregnant adolescents: prevalence and association with maternal and foetal morbidity. J Eur Acad Dermatol Venereol 2011;26:972–5. Feinstein AR, Cicchetti DV. High agreement but low kappa: I. The problems of two paradoxes. J Clin Epidemiol 1990;43:543–9. Garland SM, Tabrizi S, Hallo J, Chen S. Assessment of Chlamydia trachomatis prevalence by PCR and LCR in women presenting for termination of pregnancy. Sex Transm Infect 2000;76:173–6. Gaydos CA, Van Der Pol B, Jett-Goheen M, Barnes M, Quinn N, Clark C, et al. Performance of the Cepheid CT/NG Xpert Rapid PCR test for detection of Chlamydia trachomatis and Neisseria gonorrhoeae. J Clin Microbiol 2013;51:1666–72. Geelen TH, Rossen JW, Beerens AM, Poort L, Morré SA, Ritmeester WS, et al. Performance of cobas® 4800 and m2000 real-time assays for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in rectal and self-collected vaginal specimen. Diagn Microbiol Infect Dis 2013;77:101–5. Lavoué V, Vandenbroucke L, Lorand S, Pincemin P, Bauville E, Boyer L, et al. Screening for Chlamydia trachomatis using self-collected vaginal swabs at a public pregnancy termination clinic in France: results of a screen-and-treat policy. Sex Transm Dis 2012;39:622–7. Le Roy C, Papaxanthos A, Liesenfeld O, Mehats V, Clerc M, Bébéar C, et al. Swabs (dry or collected in universal transport medium) and semen can be used for the detection of Chlamydia trachomatis using the cobas 4800 system. J Med Microbiol 2012;62:217–22. Martin DH, Nsuami M, Schachter J, Hook III EW, Ferrero D, Quinn TC, et al. Use of multiple nucleic acid amplification tests to define the infected-patient “gold standard” in clinical trials of new diagnostic tests for Chlamydia trachomatis infections. J Clin Microbiol 2004;42:4749–58. Patel A, Rashid S, Godfrey EM, Panchal H, et al. Prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae genital infections in a publicly funded pregnancy termination clinic: empiric vs. indicated treatment? Contraception 2008;78:328–31. Peuchant O, Le Roy C, Desveaux C, Paris A, Asselineau J, Maldonado C, et al. Screening for Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium should it be integrated into routine pregnancy care in French young pregnant women? Diagn Microbiol Infect Dis 2015;82:14–9. Simel DL, Samsa GP, Matchar DB. Likelihood ratios with confidence: sample size estimation for diagnostic test studies. J Clin Epidemiol 1991;44:763–70. Toyer AL, Trignol-Viguier N, Mereghetti L, Joly B, Blin E, Body G, et al. Interest of simultaneous Chlamydia trachomatis and Neisseria gonorrhoeae screening at the time of preabortion consultation. Contraception 2012;86(5):572–6. Van Der Pol B, Taylor SN, Liesenfeld O, Williams JA, Hook III EW. Vaginal swabs are the optimal specimen for detection of genital Chlamydia trachomatis or Neisseria gonorrhoeae using the Cobas 4800 CT/NG test. Sex Transm Dis 2013;40:247–50.
Please cite this article as: Peuchant O, et al, Comparison of 3 real-time PCR assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in young pregnant women, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.09.002