Original articleDirect identification of Streptococcus agalactiae and capsular type by real-time PCR in vaginal swabs from pregnant women
Introduction
Streptococcus agalactiae, or Lancefield group B streptococcus (GBS), is a leading cause of neonatal infections with high mortality and morbidity rates [1], [2]. Colonization of GBS, reported to occur in 10%–30% of pregnant women [3], [4], is a risk factor for birth canal infection associated with severe infections in newborns [4], [5], [6].
GBS infections are classified as early-onset disease (EOD), occurring within the first 6 days after birth; and late-onset disease (LOD), occurring 7–89 days after birth [7]. In EOD, sepsis was the most frequent infection, followed by meningitis [8], [9], [10]; vertical transmission from mothers to neonates has been implicated. In contrast, meningitis predominates in LOD, which involves other routes of transmission from the mother or from environmental sources to neonates [11], [12], [13].
To prevent neonatal GBS infection, CDC guidelines issued in 2002 recommend universal screening of pregnant women for GBS colonization at 35–37 weeks of gestation, followed by intrapartum use of an antimicrobial agent for prophylaxis in any carriers identified [14]. These preventive measures were followed by a significant decline in incidence of EOD over the past decade in the US, while incidence of LOD has remained unchanged [15], [16]. Guidelines were revised again in 2010, advocating use of rapid testing methods for GBS identification and changing the antimicrobials suggested for intrapartum prophylaxis [17].
In Japan, similar guidelines including universal screening of pregnant women at 33–37 weeks of gestation and administration of intrapartum antibiotic prophylaxis in GBS-positive cases, was recommended by the Japan Society of Obstetrics and Gynecology in 2008 [18]. Although incidence of GBS infections per 1000 births is low in our country [19] compared to others [4], [17], [20], GBS remains a leading cause of newborn morbidity and mortality.
Recently, rapid screening of GBS by direct PCR and real-time PCR methods that show high sensitivity and specificity has attracted attention as an improvement over conventional culture-based methods [21], [22], [23], [24], [25], [26], [27], [28], [29].
Although capsular polysaccharide serotypes in GBS include 10 types [30], [31], only 3-- Ia, Ib, and III-- account for most neonatal infections [32], [33], [34]. Among these types, about 70–80% of LOD were caused by type III, which has particularly high virulence [32], [33], [34]. With the aim of preventing GBS infection in neonates, a GBS conjugate vaccine targeted to capsular types Ia, Ib, and III has been developed, and clinical trials in pregnant women are underway [35]. Therefore, rapid identification of serotypes Ia, Ib, and III together with species identification in samples is increasingly important in GBS testing.
In the present study, we constructed a system to directly identify serotypes Ia, Ib, and III, as well as the GBS species, with high sensitivity and specificity by real-time PCR methods using a cycling probe. We describe results of this real-time PCR assay in clinical specimens from pregnant women, as compared with findings based on conventional cultures.
Section snippets
Clinical samples
Between September 2008 and August 2012, our laboratory received 1226 vaginal samples from the Department of Obstetrics and Gynecology at Juntendo University Shizuoka Hospital. After informed consent was obtained, vaginal samples were collected by obstetrician-gynecologists from pregnant women 14–45 years old (mean, 31 years) at 36–39 weeks of gestation, and sent immediately to our laboratory at Kitasato Institute for Life Sciences.
Application form of GBS examination in every case-patient was
Sensitivity and specificity of real-time PCR
In identifications of GBS and capsule type by real-time PCR, the Ct value for a positive result was defined as the point at which the horizontal threshold line crossed the primary fluorescence curve. Sensitivities for 4 genes, dltS, cps Ia, cps Ib, and cps III, were 10 copies per reaction tube corresponding to 33 to 35 Ct, at the same level. As shown in Supplement 1, if GBS was included among the test samples and had a capsular type of Ia, Ib, or III, capsular type could be established at the
Discussion
To prevent neonatal GBS infections, US CDC guidelines recommend universal GBS screening for pregnant women at 35–37 weeks of gestation as well as intrapartum prophylaxis with an antimicrobial agent in GBS-positive cases [7]. Epidemiologic studies later determined that these recommendations had contributed to a decrease in EOD, but not in LOD [15], [16]. Presumably, prophylactic antibiotic treatment decreased numbers of GBS colonizing in the vagina and rectum, but could not completely eliminate
Conflict of interest
None.
Acknowledgments
Our research was supported by a grant under the category “Research on Emerging and Re-emerging Infectious Diseases (number H22-013)” from the Japanese Ministry of Health, Labour and Welfare (to Dr. K. Ubukata).
References (50)
- et al.
Group B streptococcal colonization and serotype-specific immunity in pregnant women at delivery
Obstet Gynecol
(2000) - et al.
Genetic analysis of Streptococcus agalactiae strains isolated from neonates and their mothers
FEMS Immunol Med Microbiol
(2003) - et al.
Group B streptococcus colonization of Greek pregnant women and neonates: prevalence, risk factors and serotypes
Clin Microbiol Infect
(2003) - et al.
Early-onset and late-onset group B streptococcal disease in Japan: a nationwide surveillance study, 2004–2010
Int J Infect Dis
(2013) - et al.
Evaluation of the BD MAX GBS assay to detect Streptococcus group B in LIM broth-enriched antepartum vaginal-rectal specimens
Diagn Microbiol Infect Dis
(2012) - et al.
Intrapartum antibiotic prophylaxis for the prevention of perinatal group B streptococcal disease: Experience in the United States and implications for a potential group B streptococcal vaccine
Vaccine
(2013) - et al.
Comprehensive detection of causative pathogens using real-time PCR to diagnose pediatric community-acquired pneumonia
J Infect Chemother
(2008) - et al.
A multiplex PCR assay for the direct identification of the capsular type (Ia to IX) of Streptococcus agalactiae
J Microbiol Methods
(2010) - et al.
Preventing the broad spectrum of perinatal morbidity and mortality through group B streptococcal vaccination
Vaccine
(2013) - et al.
Neonatal meningitis in England and Wales: 10 years on
Arch Dis Child Fetal Neonatal Ed
(2001)
Epidemiology of group B streptococcal disease in the United States: shifting paradigms
Clin Microbiol Rev
Intrapartum antibiotics for known maternal Group B streptococcal colonization
Cochrane Database Syst Rev
Risk factors for early-onset group B streptococcal sepsis: estimation of odds ratios by critical literature review
Pediatrics
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
Prevention of perinatal group B streptococcal disease: a public health perspective
MMWR Recomm Rep
Dynamics of Streptococcus agalactiae colonization in women during and after pregnancy and in their infants
J Clin Microbiol
Group B streptococcus late-onset disease: 2003–2010
Pediatrics
To feed or not to feed?: case presentation and best practice guidance for human milk feeding and group B Streptococcus in developed countries
J Hum Lact
Horizontal transmission of group B streptococcus in a neonatal intensive care unit
Paediatr Child Health
Prevention of perinatal group B streptococcal disease. Revised guidelines from CDC
MMWR Recomm Rep
Trends in perinatal group B streptococcal disease – United States, 2000–2006
MMWR Morb Mortal Wkly Rep
Group B streptococcal disease in the era of intrapartum antibiotic prophylaxis
N Engl J Med
Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention (CDC). Prevention of perinatal group B streptococcal disease–revised guidelines from CDC
MMWR Recomm Rep
Guidelines for obstetrical practice in Japan
Emerging trends in the Epidemiology of invasive group B streptococcal disease in England and Wales, 1991–2010
Clin Infect Dis
Cited by (28)
Immunologic biomarkers for bacterial meningitis
2023, Clinica Chimica ActaRelationship between intrapartum antibiotic prophylaxis and group B streptococcal colonization dynamics in Japanese mother–neonate pairs
2021, Journal of Infection and ChemotherapyCitation Excerpt :After the supernatant was discarded, 55 μL of the pellet was stirred and 50 μL of this used for DNA extraction using SimplePrep reagent for DNA (Takara Bio, Shiga, Japan) and mutanolysin (Sigma Aldrich, St. Louis, Missouri) for 10 min at 37 °C. Subsequently, real-time PCR testing for identifying the dltS gene encoding histidine kinase and genes encoding the capsular polysaccharide of types Ia, Ib, and III was performed according to the methods described previously [18]. The remaining 5 μL of each sample was inoculated onto sheep blood agar (Nippon Becton, Dickinson, Tokyo, Japan) and incubated at 37 °C for 20 h in a chamber containing 5% CO2.
Systematic review of Group B Streptococcal capsular types, sequence types and surface proteins as potential vaccine candidates
2020, VaccineCitation Excerpt :Although our results suggest that serotypes (VI to IX) currently not part of a vaccine represent around 4% of maternal colonisation isolates worldwide, in south-eastern Asia, eastern Asia, and southern Asia, they represent 20%, 12% and 7%, respectively. In specific countries such as Japan, a higher prevalence of less common serotypes VI-IX has been reported [62,71–75]. We calculated a pooled 39% (95%CI:25–53) of maternal colonisation isolates as serotypes VI-IX (from 5 studies, n = 728 isolates) [62,71–74].
Effects of Intrapartum Antibiotic Prophylaxis on Neonatal Acquisition of Group B Streptococci
2017, Journal of PediatricsCitation Excerpt :Relative capsular serotype prevalence is similar to that previously reported worldwide.15 Among GBS isolates from Japanese maternal carriers, the predominant capsular serotype was type Ib, followed by types V and III5; other countries have differed in maternal carrier serotype prevalence.8,9 In Japan, serotype V has shown little involvement in invasive GBS disease during early infancy, whereas in the US serotype V is increasing in prevalence both in pregnancy and in invasive disease during early infancy.16