European Journal of Obstetrics & Gynecology and Reproductive Biology 176 (2014) 137–141
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Intrapartum detection of Group B streptococci colonization by rapid PCR-test on labor ward Martin Mueller a,1,*, Ariane Henle a,1, Sara Droz b, Andre B. Kind c, Susanne Rohner a, Marc Baumann a, Daniel Surbek a a b c
Department of Obstetrics and Gynecology, Inselspital, Bern University Hospital, University of Bern, Switzerland Institute of Infectious Diseases, Inselspital, Bern University Hospital, University of Bern, Switzerland Department of Obstetrics and Gynecology, University Hospital Basel, Switzerland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 February 2013 Received in revised form 25 June 2013 Accepted 24 February 2014
Objective: Group B streptococci (GBS) may lead to early onset neonatal sepsis with severe morbidity and mortality of newborns. Intrapartum detection of GBS is needed. The objective was to compare a PCRbased test performed in the laboratory versus labor ward. Study design: 300 patients were included prospectively. In phase I, swabs were analyzed by selective culture and rapid PCR in the laboratory. In phase II, swabs were analyzed accordingly, but the PCR test was conducted in labor ward. Test performances were analyzed and compared. Results: In phase I the rapid PCR test had a sensitivity of 85.71% and a specificity of 95.9%. The GBS colonization rate was 18.67%. Overall 8.5% of the PCR results were invalid. In phase II the PCR test showed a sensitivity of 85.71% and a specificity of 95.65%. The GBS colonization rate was 23.3%. Overall 23.5% of swabs tested with PCR were invalid. Initiation of specific, short 2-hour training for operating personnel in the labor ward reduced the invalid test rate to 13.4%. Conclusion: The rapid PCR-based test yields adequate results to identify GBS colonization when performed in labor ward. In order to reduce the number of invalid tests a short training period is needed. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Group B streptococci Neonatal sepsis
Introduction Group B streptococci (GBS) became generally known as an important pathogen in the 1960s and are a major cause of neonatal morbidity and mortality [1]. Early onset GBS neonatal disease (GBS-EOD) occurs during the first seven days of life and typically manifests as neonatal sepsis and pneumonia. Late onset GBS disease (GBS-LOD) develops between the first week and 3rd month of life and usually manifests as meningitis [2]. Mortality in GBSEOD is 10–30%, with the highest risk in preterm neonates, whereas late onset has a lower mortality but may lead to long-term neurologic sequelae. The source of colonization for neonates is the GBS-positive mother, with a colonization rate of 10–30% [3]. Transmission in early onset cases occurs vertically during labor and delivery, with a transmission rate of 50–65% [4]. Of those
* Corresponding author at: Department of Obstetrics and Gynecology, University Hospital Insel, Effingerstrasse 102, CH-3010 Bern, Switzerland. Tel.: +41 31 632 11 03; fax: +41 31 632 11 05. E-mail address: [email protected] (M. Mueller). 1 These authors contributed equally to the study. http://dx.doi.org/10.1016/j.ejogrb.2014.02.039 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
neonates 1–2% of term infants and 8% of preterm infants develop GBS-EOD [5]. Intrapartum antibiotics have been shown to reduce significantly mother-to-child transmission as well as GBS-EOD [6]. Clinical guidelines have therefore been developed, aiming to reduce the incidence of neonatal GBS-sepsis. The Center for Disease Control and Prevention (CDC) issued guidelines for prevention of GBS-EOD and revised them in 2002 and 2010 [7]. Currently, intrapartum antibiotic prophylaxis (IAP) is recommended for women with antenatal GBS colonization in late pregnancy or with unknown maternal GBS status and risk factors [7,8]. Implementation of these guidelines resulted in reduction of GBS-EOD incidence by 80% at the price of increased intrapartum antibiotic use [9,10]. There are still several limitations to this approach. A vaginal-rectal smear with subsequent selective GBS culture at 35–37 weeks’ gestation is currently recommended. Although GBS culture remains the reference standard the current method requires 18–72 h and therefore takes too long to be performed at onset of labor. Additionally, the positive predictive value of antepartum cultures performed 3–5 weeks prior delivery is 67–87% and the negative predictive value is 90–95%, leading to overtreatment or undertreatment [11,12]. Moreover, approximately 7–11% of all pregnant
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women are affected by preterm delivery and these newborns are at highest risk of GBS-EOD [13]. Most infants with GBS-sepsis are born to mothers who screened negative for GBS colonization [14]. Rapid intrapartum detection of GBS may be an additional diagnostic tool enabling preventive strategies for decreasing the incidence of GBS-EOD. Several studies have been published investigating the use of different nucleic acid amplification tests (NAAT) to detect GBS [15– 20]. Immunological or hybridization based methods have been assessed but poor sensitivity and specificity were reported [21]. GeneXpert1 (Cepheid, Sunnyvale, CA, USA) is a commercially available PCR assay allowing rapid GBS detection and can be used as a point-of-care diagnostic method by non-laboratory staff. The aim of our study was to compare the accuracy and feasibility of GBS intrapartum detection using a new rapid PCR assay in the central microbiology laboratory and in our maternity ward setting.
StrepB and analyzed after 18–24 h and 48 h. Suspected colonies were identified according to standard laboratory protocol. Statistical analysis Culture and PCR results were used to calculate the GBS carrier rate in each phase. The sensitivity, specificity, predictive values, and likelihood ratios of the PCR-test in phases I and II were calculated by using the selective GBS culture as gold standard reference. Statistical analysis was performed using Wilson score and 95% confidence intervals (CI’s) were used to obtain the sensitivities and specificities (OpenEpi Version2). Positive, negative, and invalid results of both phases and mean time values were analyzed using two-tailed Fisher’s exact test (GraphPad Prism 5). p < 0.05 was considered significant. Results
Material and methods Phase I This prospective cohort study was conducted at the University Hospital of Berne, Switzerland. The study was approved by the ethics committee of the Canton of Berne. All included patients signed a written informed consent before the study began. The following inclusion criteria for potential subjects were used: >24 weeks of gestation, onset of labor at term or preterm, patients with an elective cesarean section, and no contraindications for vaginal examination. Patients were sequentially asked to participate in the study at the time of admission to labor ward. A total of 300 patients were prospectively enrolled between January 2007 and August 2010. Results were neither provided to personnel in charge of the patients nor used for clinical decision making. Specimens were obtained by attending physicians or midwives according to CDC recommendations [7]. Briefly, the lower onethird of the vagina, followed by rectal sampling, was performed using two swabs. The two swabs were brushed together for a more uniform sample distribution. Phase I During the first phase (n = 150) both swabs were processed in the microbiology laboratory (Institute of Infectious Disease, University of Berne) by trained personnel using a molecular GBS test and selective GBS culture (gold standard). The samples were processed during daily routine only. Phase II During the second phase (n = 150) the molecular GBS test was performed in the labor ward by attending obstetricians or midwives, whereas the second swab was sent to the microbiology laboratory for detection of GBS by selective culture. The samples were processed and the PCR test performed as soon as possible. After processing 47 samples a specific, 2-h training for operating personnel in the labor ward was initiated. It involved a 30-min presentation of the correct GeneXpert1 handling. Furthermore obstetric and midwifery personnel were able to process samples under supervision. The real-time PCR system was used in phases I and II following the manual of instructions [22]. The completely automated process is completed in less than 75 min. For GBS culture selective enrichment broths and ChromagarTM StrepB (CHROM agar Microbiology, Paris, France) were used. The swabs were inoculated into Todd-Hewitt broth (BactoTM, Sparks, MD, USA) supplemented with colistin (Fluka, Buchs, Switzerland) (10 mg/ml) and nalidixic acid (Sigma, St. Louis, MO, USA)(15 mg/ml). After overnight incubation at 35 8C they were subcultured onto ChromagarTM
A total of 150 samples were processed successfully during phase I. The average age of pregnant women was 29.7 years and the mean gestational age was 33 weeks. The GBS colonization rate was 18.67% by both specific GBS culture and real-time PCR. Twentyfour specimens tested positive by culture and rapid test (true positives), and 117 samples tested negative in both procedures (true negatives). Four samples were positive in culture but tested negative by GeneXpert1 (presumed false negative). A further five tests were positive with real-time PCR and negative by culture (presumed false positive). Sensitivity of the rapid PCR test was 85.71% (95% CI, 68.5–94.3) and specificity 95.9% (95% CI, 90.8–98.2) compared to culture. The positive predictive value of the rapid test was 82.76% and negative predictive value 96.69%. Positive likelihood ratio was 20.91 and negative likelihood ratio was 0.149. Diagnostic accuracy reached 94%. The mean time period from specimen collection to rapid test conclusion was 685 476 min. In order to successfully obtain 150 samples 164 rapid tests were processed. Overall 8.5% of the rapid PCR tested swabs were invalid. Phase II During phase II we successfully processed 150 samples. The average age of the mother was 29.7 years and the mean gestational age was 36 weeks. The GBS colonization rate was 23.3% by both gold standard and real-time PCR. Thirty specimens tested positive by culture and rapid test (true positives), and 110 samples tested negative in both procedures (true negatives). Five samples were positive in culture but tested negative by GeneXpert1 (presumed false negative) and also five further tests were positive with the real-time PCR process and negative in the gold standard (presumed false positive). The sensitivity of the rapid PCR test was 85.71% (95% CI, 70.6–93.7) and specificity 95.66% (95% CI, 90.2–98.1) compared to culture. The positive predictive value of the rapid test was 85.71% and negative predictive value 95.65%. The positive likelihood ratio was 19.71 and negative likelihood ratio was 0.149. The diagnostic accuracy reached 93%. The mean time period from specimen collection to rapid test conclusion was 165 42 min. In order to successfully obtain 150 samples 196 rapid tests were processed. Overall 23.5% of swabs tested with the rapid method were invalid. In the beginning of phase II, 55.3% (26/47) of our performed tests were invalid. Initiation of specific, 2-h training for operating personnel in the labor ward reduced the invalid test rate to 13.4% (20/149). Analysis of invalid tests revealed that 59% (27/ 46) of errors were due to inadequate handling of the GeneXpert1.
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Table 1 Performance of the PCR-based test in phase I and phase II. Parameter
Performance of the rapid PCR-based test Sensitivity Specificity Positive predictive value Negative predictive value Diagnostic accuracy Likelihood ratio of a positive test Likelihood ratio of a negative test
Phase I
Phase II
Phase I + II
Estimate
Lower–upper 95% CIs
Estimate
Lower–upper 95% CIs
Estimate
Lower–upper 95% CIs
85.71% 95.90% 82.76% 96.69% 94% 20.91 0.149
(68.5–94.3) (90.8–98.2) (65.5–92.4) (91.8–98.7) (89.0–96.8) (13.9–31.4) (0.1–0.2)
85.71% 95.66% 85.71% 95.65% 93.33% 19.71 0.149
(70.6–93.7) (90.2–98.1) (70.6–93.7) (90.2–98.1) (88.2–96.3) (13.2–29.5) (0.1–0.2)
85.71% 95.78% 84.38% 96.19% 93.67% 20.31 0.149
(75.0–92.3) (92.4–97.7) (73.6–91.3) (92.3–98.0) (90.3–95.9) (16.6–24.9) (0.1–0.2)
In phase I selective culture and PCR-based test were processed in laboratory. In phase II selective culture was processed in laboratory and PCR-based test in a labor ward. Performance of the rapid test is summarized and compared to selective culture as ‘‘gold standard’’.
[(Fig._1)TD$IG]
Fig. 1. Results of PCR-based test and selective culture ‘‘gold standard’’. Results of selective culture and PCR-based test in both phases are summarized.
negative by culture (presumed false positive). The sensitivity of the rapid PCR test was 85.71% (95% CI, 75.0–92.3) and specificity 95.78% (95% CI, 92.4–97.7) compared to culture. The positive predictive value of the rapid test was 84.38% and negative predictive value 96.19%. The positive likelihood ratio was 20.31 and negative likelihood ratio was 0.1492. Comparison of phase I and II accuracy with true positive and negative results (P = 0.3673) and false positive and negative results (P > 0.99) revealed no statistical differences. Contrary comparison of invalid tests in phase I and phase II (prior to training: P < 0.001; after training P = 0.2034; overall P < 0.001) showed several significant differences. The mean time period from specimen collection to rapid test conclusion was significantly lower in phase II compared to phase I (165 42 h vs. 685 476; p < 0.0001). Results of both phases are summarized in Table 1 and Figs. 1 and 2.
Phase I + II (overall results)
Comments
The GBS overall colonization rate in both phases was 21% by both gold standard and real-time PCR. Fifty-four specimens tested positive in culture and rapid test (true positives), and 227 samples were negative in both tests (true negatives). Nine samples were positive in culture but tested negative by GeneXpert1 (presumed false negative) and 10 tests were positive in real-time PCR but
Currently the only effective method to prevent GBS-EOD is the use of IAP. A key issue is the accurate identification of GBScolonized pregnant women in order to select the appropriate atrisk women for whom IAP is beneficial. The positive predictive value of GBS screening by culture at 35–37 weeks is limited. New methods are necessary to identify GBS carriers without temporal delay at onset of delivery. This is particularly important in high risk situations, such as prematurity when GBS screening results usually are lacking. Our study demonstrates the feasibility of intrapartum GBS detection using a rapid PCR test during daily routine by attending physicians and midwives in the labor ward. The prevalence of GBS colonization, 21%, in this study corresponds to our previously published data [23]. To our knowledge this is the first study comparing test performance of a PCR-based test by laboratory-trained specialists (performing the test in their laboratory) and in-house obstetricians/midwives (assessing GBS colonization in labor ward). There was no statistically significant difference between the performances of the two phases. We reached a sensitivity of 85.71% by rapid PCR test, which is in line with other studies on intrapartum analysis performed by non-laboratory staff [16,20]. However we also did not fulfill the specified criteria (sensitivity 90%) for nucleic acid amplification tests (NAAT) [7]. There are reports of high sensitivity (90%) PCR GBS-tests but these have included a limited number of patients or the rapid test was not performed at the point of care [15,17]. It needs to be considered that performance of an assay conducted in a microbiologic laboratory is expected to be superior compared to an assay performed in a labor ward affected by emergencies. We expect an increase in sensitivity of the assay when the PCR test is introduced into obstetric routine procedures, since everyday handling of a diagnostic method plays an important role in the test performance
[(Fig._2)TD$IG]
Fig. 2. Invalid tests in phase I and phase II. The number of invalid test results between phase I and II were compared. In phase I 8.5% of the performed PCR-based tests were invalid. In phase II (overall) 23.5% of the tests were invalid and significantly higher than in phase I (P < 0.001). Training event of the operating personnel reduced the number of invalid results in phase II from initially 55.3– 13.4%. The statistically significant difference between phase I and phase II (prior and after training event) was reduced from P < 0.001 to P = 0.2034.
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[20]. Secondly the GBS prevalence in our population is also low compared to the generally accepted prevalence in the US. The positive and negative predictive values should increase with higher GBS prevalence in our population. The mean time period from specimen collection to rapid GBS test conclusion was significantly lower in the labor ward. This is due to the fact that in laboratory specimen processing was performed only during the daily routine. The number of invalid PCR-based results is an important criterion when assessing the feasibility of a test. Recently De Tejada et al. reported 85% sensitivity of GeneXpert1 with 13% of invalid results at the beginning and 2% at the end of the study [20]. Edwards et al. reported in a similar study that 7% of PCR-based results were invalid on first attempt [16]. Several aspects have to be considered. In contrast to the other two studies we did not perform a second attempt to obtain a PCR based result. Additionally De Tajada et al. evaluated the feasibility of 567 patients after a run-in phase of 132 patients, during which training of operating staff was performed. Initially in phase II of our study the percentage of invalid PCR based tests was significantly higher than in phase I (P < 0.001). Training of operating staff reduced the number of invalid PCR based results to 13.4%, which is comparable to the 13% at the beginning of De Tajada et al.’s study, and no statistically significant differences between phase I and phase II (after training) were detected (P = 0.2034). We even speculate that a larger cohort might eventually have further reduced the percentage of invalid test results. The number of invalid rapid samples tests in both phases, especially before and after training, confirms the assumption that processing assays in a laboratory during regular working time is easier than during daily routine in a maternity ward. Secondly training of operating personnel in labor ward is essential to successfully reduce the number of invalid results. It seems that the simplicity and usability of the PCR-based test is one of the major advantages of the assay. Contrary results between culture and PCR assay were detected in phase I (9/150) and in phase II (10/150). False positive results may be due to detecting devitalized or low numbers of bacteria. GBS-EOD occurs in a substantial number of infants with low colonized mothers and a sensitive rapid intrapartum test with proper IAP may even lower the incidence [24]. Although clinical data are missing to show that IAP in culture-negative and PCRpositive women reduces GBS-EOD, this may be an argument for IAP in women with unknown culture result and positive PCR result, as recommended by the CDC [7]. False negative results may be due to inadequate sampling, processing or performance of the rapid GBS assay. Estimation of the cost of intrapartum PCR based screening on GBS-EOD is also an important aspect. Recently a single-institution study showed that PCR-based screening was cost neutral compared to the GBS culture screening. A significant decrease in GBS-EOD was also detected [25]. There are some limitations of our study. The mean gestational week at the time of swab sampling differs between phases I and II. We are unable to explain this difference, and cannot exclude that this difference might have biased the results. In this study the mean gestational week at time of swab sampling is lower than those in similar studies, but still a high sensitivity of the PCR based test was shown [20]. We did not take account of personnel’s consumed time in order to obtain rapid GBS test results. The time for specimen preparation is less than 5 min, however, and results can be obtained in a completely automated matter. Strengths of our study are the use of intrapartum culture as gold standard and the high number of samples processed at point-of-care. Additionally the results of both phases of the study underline the need for adequate training of non-laboratory personnel, especially in a labor ward with high turnover. In certain situations (e.g. after
antibiotic prophylaxis or therapy) rapid PCR tests for GBS detection may even be superior to culture due to the detection of small amounts of GBS genome, while culture remains negative. This requires further investigation. Currently available NAAT cannot fully replace the standard antepartum selective GBS culture screening strategy, at least in high resource settings with high GBS colonialization prevalence. The real-time PCR assay used in this study can be performed by attending personnel and is a robust tool for point-of-care diagnosis to identify GBS carriers at onset of labor in labor ward. This method may enhance the exact identification of candidates requiring IAP, and subsequently reduce the incidence of GBS-EOD. Training of attending residents and midwives in labor ward is crucial in order to minimize the number of invalid rapid tests in a maternity ward. Disclosure None of the authors has a conflict of interest. Acknowledgement This study has been supported by research grants from the University Women’s Hospital. Cepheid Inc. provided the PCR machines and offered the reagents. References [1] Bizzarro MJ, Raskind C, Baltimore RS, Gallagher PG. Seventy-five years of neonatal sepsis at Yale: 1928–2003. Pediatrics 2005;116:595–602. [2] Ohlsson A, Shah VS. Intrapartum antibiotics for known maternal Group B streptococcal colonization. Cochrane Database Syst Rev 2009;D0:CD007467. [3] Barcaite E, Bartusevicius A, Tameliene R, Kliucinskas M, Maleckiene L, Nadisauskiene R. Prevalence of maternal group B streptococcal colonisation in European countries. Acta Obstet Gynecol Scand 2008;87:260–71. [4] Hickman ME, Rench MA, Ferrieri P, Baker CJ. Changing epidemiology of group B streptococcal colonization. Pediatrics 1999;104:203–9. [5] Valkenburg-van den Berg AW, Sprij AJ, Oostvogel PM, et al. Prevalence of colonisation with group B streptococci in pregnant women of a multi-ethnic population in The Netherlands. Eur J Obstet Gynecol Reprod Biol 2006;124:178–83. [6] Boyer KM, Gadzala CA, Kelly PD, Burd LI, Gotoff SP. Selective intrapartum chemoprophylaxis of neonatal group B streptococcal early-onset disease. II. Predictive value of prenatal cultures. J Infect Dis 1983;148:802–9. [7] Verani JR, McGee L, Schrag SJ. Prevention of perinatal group B streptococcal disease – revised guidelines from CDC. MMWR Recomm Rep 2010;59:1–36. [8] Renner RM, Renner A, Schmid S, et al. Efficacy of a strategy to prevent neonatal early-onset group B streptococcal (GBS) sepsis. J Perinat Med 2006;34:32–8. [9] Diminishing racial disparities in early-onset neonatal group B streptococcal disease – United States, 2000–2003. MMWR Morb Mortal Wkly Rep 2004;53:502–5. [10] Schrag SJ, Zell ER, Lynfield R, et al. A population-based comparison of strategies to prevent early-onset group B streptococcal disease in neonates. N Engl J Med 2002;347:233–9. [11] Yancey MK, Schuchat A, Brown LK, Ventura VL, Markenson GR. The accuracy of late antenatal screening cultures in predicting genital group B streptococcal colonization at delivery. Obstet Gynecol 1996;88:811–5. [12] Edwards RK, Clark P, Duff P. Intrapartum antibiotic prophylaxis 2: positive predictive value of antenatal group B streptococci cultures and antibiotic susceptibility of clinical isolates. Obstet Gynecol 2002;100:540–4. [13] Aveyard P, Cheng KK, Manaseki S, Gardosi J. The risk of preterm delivery in women from different ethnic groups. BJOG 2002;109:894–9. [14] Puopolo KM, Madoff LC, Eichenwald EC. Early-onset group B streptococcal disease in the era of maternal screening. Pediatrics 2005;115:1240–6. [15] El Helali N, Nguyen JC, Ly A, Giovangrandi Y, Trinquart L. Diagnostic accuracy of a rapid real-time polymerase chain reaction assay for universal intrapartum group B streptococcus screening. Clin Infect Dis 2009;49:417–23. [16] Edwards RK, Novak-Weekley SM, Koty PP, Davis T, Leeds LJ, Jordan JA. Rapid group B streptococci screening using a real-time polymerase chain reaction assay. Obstet Gynecol 2008;111:1335–41. [17] Gavino M, Wang E. A comparison of a new rapid real-time polymerase chain reaction system to traditional culture in determining group B streptococcus colonization. Am J Obstet Gynecol 2007;197:388 e1–3884e. [18] Daniels JP, Gray J, Pattison HM, Gray R, Hills RK, Khan KS. Intrapartum tests for group B streptococcus: accuracy and acceptability of screening. BJOG 2011;118:257–65. [19] Bergeron MG, Ke D, Menard C, et al. Rapid detection of group B streptococci in pregnant women at delivery. N Engl J Med 2000;343:175–9.
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[23] Rausch AV, Gross A, Droz S, Bodmer T, Surbek DV. Group B streptococcus colonization in pregnancy: prevalence and prevention strategies of neonatal sepsis. J Perinat Med 2009;37:124–9. [24] Baker CJ. Inadequacy of rapid immunoassays for intrapartum detection of group B streptococcal carriers. Obstet Gynecol 1996;88:51–5. [25] El Helali N, Giovangrandi Y, Guyot K, Chevet K, Gutmann L, Durand-Zaleski I. Cost and effectiveness of intrapartum group B streptococcus polymerase chain reaction screening for term deliveries. Obstet Gynecol 2012;119:822–9.
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Intrapartum detection of Group B streptococci colonization by rapid PCR-test on labor ward Martin Mueller a,1,*, Ariane Henle a,1, Sara Droz b, Andre B. Kind c, Susanne Rohner a, Marc Baumann a, Daniel Surbek a a b c
Department of Obstetrics and Gynecology, Inselspital, Bern University Hospital, University of Bern, Switzerland Institute of Infectious Diseases, Inselspital, Bern University Hospital, University of Bern, Switzerland Department of Obstetrics and Gynecology, University Hospital Basel, Switzerland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 February 2013 Received in revised form 25 June 2013 Accepted 24 February 2014
Objective: Group B streptococci (GBS) may lead to early onset neonatal sepsis with severe morbidity and mortality of newborns. Intrapartum detection of GBS is needed. The objective was to compare a PCRbased test performed in the laboratory versus labor ward. Study design: 300 patients were included prospectively. In phase I, swabs were analyzed by selective culture and rapid PCR in the laboratory. In phase II, swabs were analyzed accordingly, but the PCR test was conducted in labor ward. Test performances were analyzed and compared. Results: In phase I the rapid PCR test had a sensitivity of 85.71% and a specificity of 95.9%. The GBS colonization rate was 18.67%. Overall 8.5% of the PCR results were invalid. In phase II the PCR test showed a sensitivity of 85.71% and a specificity of 95.65%. The GBS colonization rate was 23.3%. Overall 23.5% of swabs tested with PCR were invalid. Initiation of specific, short 2-hour training for operating personnel in the labor ward reduced the invalid test rate to 13.4%. Conclusion: The rapid PCR-based test yields adequate results to identify GBS colonization when performed in labor ward. In order to reduce the number of invalid tests a short training period is needed. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Group B streptococci Neonatal sepsis
Introduction Group B streptococci (GBS) became generally known as an important pathogen in the 1960s and are a major cause of neonatal morbidity and mortality [1]. Early onset GBS neonatal disease (GBS-EOD) occurs during the first seven days of life and typically manifests as neonatal sepsis and pneumonia. Late onset GBS disease (GBS-LOD) develops between the first week and 3rd month of life and usually manifests as meningitis [2]. Mortality in GBSEOD is 10–30%, with the highest risk in preterm neonates, whereas late onset has a lower mortality but may lead to long-term neurologic sequelae. The source of colonization for neonates is the GBS-positive mother, with a colonization rate of 10–30% [3]. Transmission in early onset cases occurs vertically during labor and delivery, with a transmission rate of 50–65% [4]. Of those
* Corresponding author at: Department of Obstetrics and Gynecology, University Hospital Insel, Effingerstrasse 102, CH-3010 Bern, Switzerland. Tel.: +41 31 632 11 03; fax: +41 31 632 11 05. E-mail address: [email protected] (M. Mueller). 1 These authors contributed equally to the study. http://dx.doi.org/10.1016/j.ejogrb.2014.02.039 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
neonates 1–2% of term infants and 8% of preterm infants develop GBS-EOD [5]. Intrapartum antibiotics have been shown to reduce significantly mother-to-child transmission as well as GBS-EOD [6]. Clinical guidelines have therefore been developed, aiming to reduce the incidence of neonatal GBS-sepsis. The Center for Disease Control and Prevention (CDC) issued guidelines for prevention of GBS-EOD and revised them in 2002 and 2010 [7]. Currently, intrapartum antibiotic prophylaxis (IAP) is recommended for women with antenatal GBS colonization in late pregnancy or with unknown maternal GBS status and risk factors [7,8]. Implementation of these guidelines resulted in reduction of GBS-EOD incidence by 80% at the price of increased intrapartum antibiotic use [9,10]. There are still several limitations to this approach. A vaginal-rectal smear with subsequent selective GBS culture at 35–37 weeks’ gestation is currently recommended. Although GBS culture remains the reference standard the current method requires 18–72 h and therefore takes too long to be performed at onset of labor. Additionally, the positive predictive value of antepartum cultures performed 3–5 weeks prior delivery is 67–87% and the negative predictive value is 90–95%, leading to overtreatment or undertreatment [11,12]. Moreover, approximately 7–11% of all pregnant
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M. Mueller et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 176 (2014) 137–141
women are affected by preterm delivery and these newborns are at highest risk of GBS-EOD [13]. Most infants with GBS-sepsis are born to mothers who screened negative for GBS colonization [14]. Rapid intrapartum detection of GBS may be an additional diagnostic tool enabling preventive strategies for decreasing the incidence of GBS-EOD. Several studies have been published investigating the use of different nucleic acid amplification tests (NAAT) to detect GBS [15– 20]. Immunological or hybridization based methods have been assessed but poor sensitivity and specificity were reported [21]. GeneXpert1 (Cepheid, Sunnyvale, CA, USA) is a commercially available PCR assay allowing rapid GBS detection and can be used as a point-of-care diagnostic method by non-laboratory staff. The aim of our study was to compare the accuracy and feasibility of GBS intrapartum detection using a new rapid PCR assay in the central microbiology laboratory and in our maternity ward setting.
StrepB and analyzed after 18–24 h and 48 h. Suspected colonies were identified according to standard laboratory protocol. Statistical analysis Culture and PCR results were used to calculate the GBS carrier rate in each phase. The sensitivity, specificity, predictive values, and likelihood ratios of the PCR-test in phases I and II were calculated by using the selective GBS culture as gold standard reference. Statistical analysis was performed using Wilson score and 95% confidence intervals (CI’s) were used to obtain the sensitivities and specificities (OpenEpi Version2). Positive, negative, and invalid results of both phases and mean time values were analyzed using two-tailed Fisher’s exact test (GraphPad Prism 5). p < 0.05 was considered significant. Results
Material and methods Phase I This prospective cohort study was conducted at the University Hospital of Berne, Switzerland. The study was approved by the ethics committee of the Canton of Berne. All included patients signed a written informed consent before the study began. The following inclusion criteria for potential subjects were used: >24 weeks of gestation, onset of labor at term or preterm, patients with an elective cesarean section, and no contraindications for vaginal examination. Patients were sequentially asked to participate in the study at the time of admission to labor ward. A total of 300 patients were prospectively enrolled between January 2007 and August 2010. Results were neither provided to personnel in charge of the patients nor used for clinical decision making. Specimens were obtained by attending physicians or midwives according to CDC recommendations [7]. Briefly, the lower onethird of the vagina, followed by rectal sampling, was performed using two swabs. The two swabs were brushed together for a more uniform sample distribution. Phase I During the first phase (n = 150) both swabs were processed in the microbiology laboratory (Institute of Infectious Disease, University of Berne) by trained personnel using a molecular GBS test and selective GBS culture (gold standard). The samples were processed during daily routine only. Phase II During the second phase (n = 150) the molecular GBS test was performed in the labor ward by attending obstetricians or midwives, whereas the second swab was sent to the microbiology laboratory for detection of GBS by selective culture. The samples were processed and the PCR test performed as soon as possible. After processing 47 samples a specific, 2-h training for operating personnel in the labor ward was initiated. It involved a 30-min presentation of the correct GeneXpert1 handling. Furthermore obstetric and midwifery personnel were able to process samples under supervision. The real-time PCR system was used in phases I and II following the manual of instructions [22]. The completely automated process is completed in less than 75 min. For GBS culture selective enrichment broths and ChromagarTM StrepB (CHROM agar Microbiology, Paris, France) were used. The swabs were inoculated into Todd-Hewitt broth (BactoTM, Sparks, MD, USA) supplemented with colistin (Fluka, Buchs, Switzerland) (10 mg/ml) and nalidixic acid (Sigma, St. Louis, MO, USA)(15 mg/ml). After overnight incubation at 35 8C they were subcultured onto ChromagarTM
A total of 150 samples were processed successfully during phase I. The average age of pregnant women was 29.7 years and the mean gestational age was 33 weeks. The GBS colonization rate was 18.67% by both specific GBS culture and real-time PCR. Twentyfour specimens tested positive by culture and rapid test (true positives), and 117 samples tested negative in both procedures (true negatives). Four samples were positive in culture but tested negative by GeneXpert1 (presumed false negative). A further five tests were positive with real-time PCR and negative by culture (presumed false positive). Sensitivity of the rapid PCR test was 85.71% (95% CI, 68.5–94.3) and specificity 95.9% (95% CI, 90.8–98.2) compared to culture. The positive predictive value of the rapid test was 82.76% and negative predictive value 96.69%. Positive likelihood ratio was 20.91 and negative likelihood ratio was 0.149. Diagnostic accuracy reached 94%. The mean time period from specimen collection to rapid test conclusion was 685 476 min. In order to successfully obtain 150 samples 164 rapid tests were processed. Overall 8.5% of the rapid PCR tested swabs were invalid. Phase II During phase II we successfully processed 150 samples. The average age of the mother was 29.7 years and the mean gestational age was 36 weeks. The GBS colonization rate was 23.3% by both gold standard and real-time PCR. Thirty specimens tested positive by culture and rapid test (true positives), and 110 samples tested negative in both procedures (true negatives). Five samples were positive in culture but tested negative by GeneXpert1 (presumed false negative) and also five further tests were positive with the real-time PCR process and negative in the gold standard (presumed false positive). The sensitivity of the rapid PCR test was 85.71% (95% CI, 70.6–93.7) and specificity 95.66% (95% CI, 90.2–98.1) compared to culture. The positive predictive value of the rapid test was 85.71% and negative predictive value 95.65%. The positive likelihood ratio was 19.71 and negative likelihood ratio was 0.149. The diagnostic accuracy reached 93%. The mean time period from specimen collection to rapid test conclusion was 165 42 min. In order to successfully obtain 150 samples 196 rapid tests were processed. Overall 23.5% of swabs tested with the rapid method were invalid. In the beginning of phase II, 55.3% (26/47) of our performed tests were invalid. Initiation of specific, 2-h training for operating personnel in the labor ward reduced the invalid test rate to 13.4% (20/149). Analysis of invalid tests revealed that 59% (27/ 46) of errors were due to inadequate handling of the GeneXpert1.
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Table 1 Performance of the PCR-based test in phase I and phase II. Parameter
Performance of the rapid PCR-based test Sensitivity Specificity Positive predictive value Negative predictive value Diagnostic accuracy Likelihood ratio of a positive test Likelihood ratio of a negative test
Phase I
Phase II
Phase I + II
Estimate
Lower–upper 95% CIs
Estimate
Lower–upper 95% CIs
Estimate
Lower–upper 95% CIs
85.71% 95.90% 82.76% 96.69% 94% 20.91 0.149
(68.5–94.3) (90.8–98.2) (65.5–92.4) (91.8–98.7) (89.0–96.8) (13.9–31.4) (0.1–0.2)
85.71% 95.66% 85.71% 95.65% 93.33% 19.71 0.149
(70.6–93.7) (90.2–98.1) (70.6–93.7) (90.2–98.1) (88.2–96.3) (13.2–29.5) (0.1–0.2)
85.71% 95.78% 84.38% 96.19% 93.67% 20.31 0.149
(75.0–92.3) (92.4–97.7) (73.6–91.3) (92.3–98.0) (90.3–95.9) (16.6–24.9) (0.1–0.2)
In phase I selective culture and PCR-based test were processed in laboratory. In phase II selective culture was processed in laboratory and PCR-based test in a labor ward. Performance of the rapid test is summarized and compared to selective culture as ‘‘gold standard’’.
[(Fig._1)TD$IG]
Fig. 1. Results of PCR-based test and selective culture ‘‘gold standard’’. Results of selective culture and PCR-based test in both phases are summarized.
negative by culture (presumed false positive). The sensitivity of the rapid PCR test was 85.71% (95% CI, 75.0–92.3) and specificity 95.78% (95% CI, 92.4–97.7) compared to culture. The positive predictive value of the rapid test was 84.38% and negative predictive value 96.19%. The positive likelihood ratio was 20.31 and negative likelihood ratio was 0.1492. Comparison of phase I and II accuracy with true positive and negative results (P = 0.3673) and false positive and negative results (P > 0.99) revealed no statistical differences. Contrary comparison of invalid tests in phase I and phase II (prior to training: P < 0.001; after training P = 0.2034; overall P < 0.001) showed several significant differences. The mean time period from specimen collection to rapid test conclusion was significantly lower in phase II compared to phase I (165 42 h vs. 685 476; p < 0.0001). Results of both phases are summarized in Table 1 and Figs. 1 and 2.
Phase I + II (overall results)
Comments
The GBS overall colonization rate in both phases was 21% by both gold standard and real-time PCR. Fifty-four specimens tested positive in culture and rapid test (true positives), and 227 samples were negative in both tests (true negatives). Nine samples were positive in culture but tested negative by GeneXpert1 (presumed false negative) and 10 tests were positive in real-time PCR but
Currently the only effective method to prevent GBS-EOD is the use of IAP. A key issue is the accurate identification of GBScolonized pregnant women in order to select the appropriate atrisk women for whom IAP is beneficial. The positive predictive value of GBS screening by culture at 35–37 weeks is limited. New methods are necessary to identify GBS carriers without temporal delay at onset of delivery. This is particularly important in high risk situations, such as prematurity when GBS screening results usually are lacking. Our study demonstrates the feasibility of intrapartum GBS detection using a rapid PCR test during daily routine by attending physicians and midwives in the labor ward. The prevalence of GBS colonization, 21%, in this study corresponds to our previously published data [23]. To our knowledge this is the first study comparing test performance of a PCR-based test by laboratory-trained specialists (performing the test in their laboratory) and in-house obstetricians/midwives (assessing GBS colonization in labor ward). There was no statistically significant difference between the performances of the two phases. We reached a sensitivity of 85.71% by rapid PCR test, which is in line with other studies on intrapartum analysis performed by non-laboratory staff [16,20]. However we also did not fulfill the specified criteria (sensitivity 90%) for nucleic acid amplification tests (NAAT) [7]. There are reports of high sensitivity (90%) PCR GBS-tests but these have included a limited number of patients or the rapid test was not performed at the point of care [15,17]. It needs to be considered that performance of an assay conducted in a microbiologic laboratory is expected to be superior compared to an assay performed in a labor ward affected by emergencies. We expect an increase in sensitivity of the assay when the PCR test is introduced into obstetric routine procedures, since everyday handling of a diagnostic method plays an important role in the test performance
[(Fig._2)TD$IG]
Fig. 2. Invalid tests in phase I and phase II. The number of invalid test results between phase I and II were compared. In phase I 8.5% of the performed PCR-based tests were invalid. In phase II (overall) 23.5% of the tests were invalid and significantly higher than in phase I (P < 0.001). Training event of the operating personnel reduced the number of invalid results in phase II from initially 55.3– 13.4%. The statistically significant difference between phase I and phase II (prior and after training event) was reduced from P < 0.001 to P = 0.2034.
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[20]. Secondly the GBS prevalence in our population is also low compared to the generally accepted prevalence in the US. The positive and negative predictive values should increase with higher GBS prevalence in our population. The mean time period from specimen collection to rapid GBS test conclusion was significantly lower in the labor ward. This is due to the fact that in laboratory specimen processing was performed only during the daily routine. The number of invalid PCR-based results is an important criterion when assessing the feasibility of a test. Recently De Tejada et al. reported 85% sensitivity of GeneXpert1 with 13% of invalid results at the beginning and 2% at the end of the study [20]. Edwards et al. reported in a similar study that 7% of PCR-based results were invalid on first attempt [16]. Several aspects have to be considered. In contrast to the other two studies we did not perform a second attempt to obtain a PCR based result. Additionally De Tajada et al. evaluated the feasibility of 567 patients after a run-in phase of 132 patients, during which training of operating staff was performed. Initially in phase II of our study the percentage of invalid PCR based tests was significantly higher than in phase I (P < 0.001). Training of operating staff reduced the number of invalid PCR based results to 13.4%, which is comparable to the 13% at the beginning of De Tajada et al.’s study, and no statistically significant differences between phase I and phase II (after training) were detected (P = 0.2034). We even speculate that a larger cohort might eventually have further reduced the percentage of invalid test results. The number of invalid rapid samples tests in both phases, especially before and after training, confirms the assumption that processing assays in a laboratory during regular working time is easier than during daily routine in a maternity ward. Secondly training of operating personnel in labor ward is essential to successfully reduce the number of invalid results. It seems that the simplicity and usability of the PCR-based test is one of the major advantages of the assay. Contrary results between culture and PCR assay were detected in phase I (9/150) and in phase II (10/150). False positive results may be due to detecting devitalized or low numbers of bacteria. GBS-EOD occurs in a substantial number of infants with low colonized mothers and a sensitive rapid intrapartum test with proper IAP may even lower the incidence [24]. Although clinical data are missing to show that IAP in culture-negative and PCRpositive women reduces GBS-EOD, this may be an argument for IAP in women with unknown culture result and positive PCR result, as recommended by the CDC [7]. False negative results may be due to inadequate sampling, processing or performance of the rapid GBS assay. Estimation of the cost of intrapartum PCR based screening on GBS-EOD is also an important aspect. Recently a single-institution study showed that PCR-based screening was cost neutral compared to the GBS culture screening. A significant decrease in GBS-EOD was also detected [25]. There are some limitations of our study. The mean gestational week at the time of swab sampling differs between phases I and II. We are unable to explain this difference, and cannot exclude that this difference might have biased the results. In this study the mean gestational week at time of swab sampling is lower than those in similar studies, but still a high sensitivity of the PCR based test was shown [20]. We did not take account of personnel’s consumed time in order to obtain rapid GBS test results. The time for specimen preparation is less than 5 min, however, and results can be obtained in a completely automated matter. Strengths of our study are the use of intrapartum culture as gold standard and the high number of samples processed at point-of-care. Additionally the results of both phases of the study underline the need for adequate training of non-laboratory personnel, especially in a labor ward with high turnover. In certain situations (e.g. after
antibiotic prophylaxis or therapy) rapid PCR tests for GBS detection may even be superior to culture due to the detection of small amounts of GBS genome, while culture remains negative. This requires further investigation. Currently available NAAT cannot fully replace the standard antepartum selective GBS culture screening strategy, at least in high resource settings with high GBS colonialization prevalence. The real-time PCR assay used in this study can be performed by attending personnel and is a robust tool for point-of-care diagnosis to identify GBS carriers at onset of labor in labor ward. This method may enhance the exact identification of candidates requiring IAP, and subsequently reduce the incidence of GBS-EOD. Training of attending residents and midwives in labor ward is crucial in order to minimize the number of invalid rapid tests in a maternity ward. Disclosure None of the authors has a conflict of interest. Acknowledgement This study has been supported by research grants from the University Women’s Hospital. Cepheid Inc. provided the PCR machines and offered the reagents. References [1] Bizzarro MJ, Raskind C, Baltimore RS, Gallagher PG. Seventy-five years of neonatal sepsis at Yale: 1928–2003. Pediatrics 2005;116:595–602. [2] Ohlsson A, Shah VS. Intrapartum antibiotics for known maternal Group B streptococcal colonization. Cochrane Database Syst Rev 2009;D0:CD007467. [3] Barcaite E, Bartusevicius A, Tameliene R, Kliucinskas M, Maleckiene L, Nadisauskiene R. Prevalence of maternal group B streptococcal colonisation in European countries. Acta Obstet Gynecol Scand 2008;87:260–71. [4] Hickman ME, Rench MA, Ferrieri P, Baker CJ. Changing epidemiology of group B streptococcal colonization. Pediatrics 1999;104:203–9. [5] Valkenburg-van den Berg AW, Sprij AJ, Oostvogel PM, et al. Prevalence of colonisation with group B streptococci in pregnant women of a multi-ethnic population in The Netherlands. Eur J Obstet Gynecol Reprod Biol 2006;124:178–83. [6] Boyer KM, Gadzala CA, Kelly PD, Burd LI, Gotoff SP. Selective intrapartum chemoprophylaxis of neonatal group B streptococcal early-onset disease. II. Predictive value of prenatal cultures. J Infect Dis 1983;148:802–9. [7] Verani JR, McGee L, Schrag SJ. Prevention of perinatal group B streptococcal disease – revised guidelines from CDC. MMWR Recomm Rep 2010;59:1–36. [8] Renner RM, Renner A, Schmid S, et al. Efficacy of a strategy to prevent neonatal early-onset group B streptococcal (GBS) sepsis. J Perinat Med 2006;34:32–8. [9] Diminishing racial disparities in early-onset neonatal group B streptococcal disease – United States, 2000–2003. MMWR Morb Mortal Wkly Rep 2004;53:502–5. [10] Schrag SJ, Zell ER, Lynfield R, et al. A population-based comparison of strategies to prevent early-onset group B streptococcal disease in neonates. N Engl J Med 2002;347:233–9. [11] Yancey MK, Schuchat A, Brown LK, Ventura VL, Markenson GR. The accuracy of late antenatal screening cultures in predicting genital group B streptococcal colonization at delivery. Obstet Gynecol 1996;88:811–5. [12] Edwards RK, Clark P, Duff P. Intrapartum antibiotic prophylaxis 2: positive predictive value of antenatal group B streptococci cultures and antibiotic susceptibility of clinical isolates. Obstet Gynecol 2002;100:540–4. [13] Aveyard P, Cheng KK, Manaseki S, Gardosi J. The risk of preterm delivery in women from different ethnic groups. BJOG 2002;109:894–9. [14] Puopolo KM, Madoff LC, Eichenwald EC. Early-onset group B streptococcal disease in the era of maternal screening. Pediatrics 2005;115:1240–6. [15] El Helali N, Nguyen JC, Ly A, Giovangrandi Y, Trinquart L. Diagnostic accuracy of a rapid real-time polymerase chain reaction assay for universal intrapartum group B streptococcus screening. Clin Infect Dis 2009;49:417–23. [16] Edwards RK, Novak-Weekley SM, Koty PP, Davis T, Leeds LJ, Jordan JA. Rapid group B streptococci screening using a real-time polymerase chain reaction assay. Obstet Gynecol 2008;111:1335–41. [17] Gavino M, Wang E. A comparison of a new rapid real-time polymerase chain reaction system to traditional culture in determining group B streptococcus colonization. Am J Obstet Gynecol 2007;197:388 e1–3884e. [18] Daniels JP, Gray J, Pattison HM, Gray R, Hills RK, Khan KS. Intrapartum tests for group B streptococcus: accuracy and acceptability of screening. BJOG 2011;118:257–65. [19] Bergeron MG, Ke D, Menard C, et al. Rapid detection of group B streptococci in pregnant women at delivery. N Engl J Med 2000;343:175–9.
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