A Retrospective Study Comparing of Group B Streptococcus Invasiveness in Pregnant Women and Infants

Background : Group B streptococcus (GBS) is commonly recognized as an opportunistic pathogen, which can cause infections in pregnant women and their newborns. The aim of this study was to explore the invasiveness of GBS by comparing various indices of pregnant mothers and newborns. Methods : This retrospective study involved 6892 consecutive GBS screened pregnant women, and 48 GBS-positive newborns. The data of pregnant women and newborns was compared by Chi-square test and Kruskal-Wallis test. A p -value ≤ 0.05 was considered statistically significant. Results : After excluding the other risk factors which can cause adverse pregnancy outcomes, there were no differences between pregnant women in GBS-positive and GBS-negative groups, except the age group. In the GBS-negative and positive groups the incidence of prematurity, premature rupture of membranes (PROM), and chorioamnionitis were 1.06% and 0.74%, 7.72% and 8.14%, 0.63% and 0.74%, respectively. The corresponding p -values were 0.619, 0.263, and 0.626. The GBS-positive rate was 6.83% (201/2943) in the 19–30 years (y) group, 6.89% in the (262/3802) in the 31–40 y group, and 1.36% (2/147) in the 41–52 y group ( p = 0.031). The indices in the different newborn groups exhibited significant differences. Analysis of the data revealed significant differences in delivery mode, gestational age, neonatal birth weight, and Apgar scores among the GBS-colonization, GBS-infection, and death groups ( p = 0.010, 0.004, 0.022, and 0.000 < 0.05, respectively). Conclusions : After excluding related factors, the evidence showing that GBS-colonization independently induced adverse pregnancy outcomes in pregnant women was insufficient. GBS was more likely to attack premature newborns with low weight and poor health status.


Introduction
Group B streptococcus (GBS or Streptococcus agalactiae) is both a normal commensal and an opportunistic pathogen that colonizes the gastrointestinal tracts of women, men, and children of all ages, and is the source of vaginal and urethral colonization [1][2][3].GBS is a predominant microbe of perinatal infections that can cause puerperium complications, vertical transmission from mothersto-newborns at the time of delivery, and can lead to severe neonatal sepsis, pneumonia, and meningitis [4,5].So, administering intravenous antibiotics during labor to GBSpositive women could prevent invasive disease of their newborns.However, intrapartum overtreatment with antibiotics increases the risk of maternal and neonatal untoward effects, such as antibiotic resistance, anaphylactic shock, and intestinal complaints [6].Especially, the mucosal immune system of newborns is closely related to the initial bacterial colonization of the intestines, while overuse of antibiotics can alter intestinal flora, then lead to childhood asthma, diabetes, allergy, obesity, and autism [7].To reduce the incidence of GBS disease in newborns [8] and the overuse of antibiotics, universal screening for GBS at 35-38 weeks gestation and intrapartum antibiotic prophylaxis (IAP) is the standard of care [9].
It has been demonstrated by other studies that conventional bacterial culture methods have high requirements for transportation conditions and identification level.Timeconsuming culture methods are another problem in labor which gets results within 24-72 hours after sampling and gets the antibiotic susceptibility testing results after 72 hours [10].On the other hand, the GBS gene targeting realtime polymerase chain reaction (PCR) method is more accurate, more sensitive, and faster due to the results are available within 1-2 hours after sampling [11].Thus, we collected the PCR results of pregnant women and newborns to identify the epidemiological characteristics of GBS from our hospital.

Samples and Data Collection
This retrospective study was conducted involving clinical data of 946 consecutive cases from 6892 pregnant women with late antenatal screening of GBS tests from January to December 2020, and the files of GBS-positive neonates from January 2019 to December 2020.Two swabs were collected from the anus and vagina of each pregnant woman, respectively, at 35-38 weeks gestation.The swabs were collected from the nasopharynx or ears of the new- borns with clinical symptoms of infection after birth from GBS-positive mothers.All information was obtained from the laboratory information system of YuHuangding Hospital (Yantai, Shandong, China).

The Data of Pregnant Women
Inclusion and exclusion criteria, the 946 consecutive cases were during the period from all 6892 pregnant women with GBS tests and delivered in our hospital later.At the same time excluded the cases with other risk factors which can cause adverse pregnancy: myoma uteri, uterine malformations, multiple pregnancies, preeclampsia, hypertension, diabetes mellitus, fetal malformations, fetal growth restriction, intra-uterine fetal demise, and other genital tract pathogen infections.And then, to devided the 946 cases into three groups, in which 500 GBS-negative cases and 446 GBS-positive cases, shown in Tables 1,2,3.The first group was the premature delivery group at <37 weeks gestation.The second group was the premature rupture of membranes (PROM) group and included pregnant women of all gestational ages with premature rupture of membranes, not limited to preterm women <37 weeks.The third group was the chorioamnionitis group.Inclusion and exclusion criteria of pregnant women are shown in Fig. 1.

The Data of Newborns
There were 15,967 newborns whom were born in our hospital from January 2019 to December 2020, of which 38 cases were GBS-positive.The other 10 GBS-positive cases were transferred from other hospitals after the onset of GBS infection.The inclusion criteria for the newborns are shown in Fig. 2. All data from the 38 GBS-positive cases and the 10 GBS-positive cases transferred from other hospitals were compared in Table 4.According to the clinical symptoms of GBS-positive children, they were divided into three groups: GBS-colonization group; GBS-infection group; and death group.The deaths in the mode of delivery groups were merged into the corresponding infection groups in Table 4.With respect to the GBS-colonization site in the mother group, the death group was also merged into the corresponding infection groups.The result of pairwise comparison between groups by the Kruskal Wallis H-test is shown in Table 5.

GBS Tests
The GBS-specific CAMP gene was detected by polymerase chain amplification and fluorescent labeling of the GBS reagent (Taipu Biological Co. LTD, Fuzhou, Fujian, China), and the results were analyzed using an ABI

Statistical Analysis
Data are presented as a percentage, median and interquartile range for different variables.A Chi-square test was utilized to assess the categorical data and the Kruskal-Wallis test was used for measurement data with SPSS 24.0 (IBM Corp., Armonk, NY, USA).p-value ≤ 0.05 was considered statistically significant.
In Table 3, there were not any differences in the three common complications among the two groups.In the GBSnegative and -positive groups, the incidence of prematurity, PROM, and chorioamnionitis were 1.06% and 0.74%, 7.72% and 8.14%, 0.63% and 0.74%, respectively.The corresponding p-values were 0.619, 0.263, and 0.626.Accordingly, there were no differences in the gestational weeks group (p = 0.262).
In the 15,967 newborns group, the neonatal general GBS-positive rate was 0.24% (38/15,967).A total of 48 GBS-positive newborns were identified, among which 38 were born in our hospital.There was a 0.24% (38/15,967) GBS-positive rate among 15,967 births, the infection rate was 0.94 per 1000 live births (15/15,967), and the mortality rate was 0.06 per 1000 live births (1/15,967) (in Fig. 2).Analysis of the data revealed significant differences in delivery mode, gestational age, neonatal birth weight, and Apgar scores among the GBS-colonization, GBS-infection, and death groups (p = 0.010, 0.004, 0.022, and 0.000 < 0.05, respectively; Table 4), as well as the pairwise comparison between groups by the Kruskal Wallis H-test (Table 5).
In the neonatal GBS-infection group, 19 neonates had symptoms of infection within 24 hours after birth.In the ≤7 days group, 2 newborns had symptoms of infection within 2 days, while in the >7 days group, symptoms of infection occurred after 15, 21, and 22 days.Symptoms of infection occurred in 2 neonatal deaths within ≤24 hours after birth.Most of the neonates had pneumonia and some progressed to pyemia or bacteremia.A neonate with pneumonia complicated by bacteremia and meningitis had GBS-positive blood and cerebrospinal fluid cultures (Table 4).

Basic Distribution of GBS in Pregnant Women
The GBS-colonization rate of pregnant women in our study was 6.75%, which was close to the average level of the GBS-positive rate in mainland China (3.7-14.52%)[8].The GBS-positive rates in the vagina, anus, and rectovagi-  5. # Some groups were merged due to the low frequency.Both in the mode of delivery group and in the GBS-colonization site of the mother group, the death group was merged into the corresponding infection groups.
nal were 0.51% (35/6892), 3.38% (233/6892), and 2.86% (197/6892), respectively; thus, the GBS-positive rate in the anus was higher than the vagina because the GBS primary reservoir is the gastrointestinal tract [3].The positive rate of GBS was different among the different age groups.It showed that the prevalence of GBS in younger women was higher than in older women.These results are similar to previous studies [12][13][14].

Relationship of GBS-Positive and Adverse Pregnancy
Between GBS-positive group and GBS-negative group, there was no difference in gestational weeks, PROM, preterm delivery, and chorioamnionitis groups.Contrary to previous studies [11,15,16], in our current research there was no significant difference between the two groups regarding adverse pregnancy outcomes, which is consistent with the findings by Goel et al. [13] and Ngonzi et al. [17].Accordingly, in the Tano et al. [18] study, there was no pathologic evidence to support a connection between GBSinfection and chorioamnionitis, which is consistent with our study.
We excluded all cases with diabetes, hypertension, and preeclampsia, which may cause premature labor or PROM [19][20][21].The cases with Ureaplasma urealyticum, Chlamydia trachomatis, Candida, Neisseria gonorrhoeae, and Gardnerella vaginalis, which have a pathological role in PROM and chorioamnionitis [12,22] were also eliminated.Rocchetti et al. [12] confirmed that candidiasis and cytolytic vaginosis also increase GBS-colonization.Therefore, the seco-infection cases were deleted because it was difficult to determine the actual pathogenic factor leading to the maternal infection.
Additionally, either diabetes or hypertension are risk factors for premature labor and PROM [23], and also increase the likelihood of GBS-colonization [10,15].These confounding factors not only increase the rate of GBScolonization, but also induce adverse pregnancy outcomes.Therefore, it is necessary to exclude the confounding factors and to compare a single factor for GBS to confirm the role of GBS in adverse pregnancy outcomes.Moreover, these confounding factors may be the reason for the differences in results between studies.

GBS with Strong Invasiveness among Frail Children
Most of the GBS-infected neonates had pneumonia, followed by pyemia, bacteriaemia, meningitis, and even death.The emergence of symptoms of infection and death were mostly concentrated within the first 24 hours.There were significant differences in the three indices (gestational age, neonatal birth weight, and Apgar scores), reflecting the health status of newborns among the three groups.Infants with poor basic conditions are concentrated in the infection and death groups.
In the weight group, there was only a difference between the colonization and death groups.The two dead cases in our study were premature babies with low weight, and similar cases were also reported by Todorova-Christova et al. [24], that low weight at birth or prematurity was confirmed as a substantial risk factor of GBS-infection.In the Apgar score group, there were differences among all three groups.Therefore, we believe that the occurrence and progression of GBS-infections were related to the basic physical health status of newborns, which is consistent with Mousavi et al. [10], which reported that GBS can give rise to life-threatening infections in some vulnerable hosts, especially infants with chronic diseases.

Analysis of GBS Maternal-to-Child Transmission
A difference also existed in the mode of delivery so that the colonization and GBS-infection rates in newborns that were delivered spontaneously were higher than newborns delivered by cesarean section, which is consistent with the results of Verani et al. [3] and Joachim et al. [25] In the GBS-colonization site group, most of the rectovaginal-positive mothers caused vertical transmission (37.5%, 18.75%, and 2.08%).Three GBS-positive newborns were born from GBS-negative mothers, which confirmed the intermittent and transient nature of GBScolonization [11,26].Of the 48 GBS-positive newborns, the mothers of 15 did not have GBS screening or had falsenegative results, thus accounting for 57.7% (15/26) of in-fected children and the infection rate was higher than the newborns from the maternal screening group, which confirmed that prenatal screening of GBS is beneficial to reduce GBS-infections in neonates.
Among the 30 mothers who had GBS screening, there were 10 newborn infections, 1 newborn dead, and 19 newborns colonized.1 dead newborn was a premature infant at 33 weeks + 5 days gestation with a birth weight of 1700 g, and an Apgar score of 8 at 1 min.Although the mother received IAP, the prophylactic antibiotics failed to effectively prevent fetal infection and death.The mother of another dead newborn was not screened for GBS; the newborn was born at 25 weeks + 2 days gestational age with a birth weight of 800 g and Apgar score of 3 at 1 min.The two deceased children had poor basic conditions and were difficult to survive in the case of combined GBS-infection.All of the above data are consistent with most studies that concluded that premature infants in poor health with GBS-infections usually have a poor prognosis [10,24].

Conclusions
In our study we found that GBS may not be a singlefactor pathogenic microorganism.After excluding other related factors, the pathogenicity of GBS in pregnant women was not as significant as described in some other studies [10,18].The colonization rate and invasiveness of GBS were increased when one or more pathogenic factors or clinical complications exist at the same time [12,15].In fact, GBS is more likely to attack premature newborns with poor health status, and poise life-threat to vulnerable individuals.
To reduce the pathogenic effects caused by GBSinfections, IAP has significantly altered the adverse outcomes of neonatal infection.Moreover, to reduce maternal adverse pregnancy related factors and improving the basic conditions of newborns can also prevent GBS-infection of newborns.
thors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.Both authors read and approved the final manuscript.

Fig. 1 .
Fig. 1.Flow chart of the inclusion criteria of the pregnant women.

Fig. 2 .
Fig. 2. Analysis of infants.*When calculating the infection rate and mortality rate of newborns, the 10 cases born in the other hospitals were not included in the 15,967 cases born in our hospital.

Table 1 . GBS-positive results of different positions.
Each pregnant woman collected two swabs at the same time, so a total of 13,784 swabs were collected from 6892 pregnant women.The positive rate was calculated by the number of positive women.GBS, Group B streptococcus.

Table 2 . Characteristics of GBS distribution in pregnant women.
a * (93.11%) 262 a * (6.89%) 3802 41-52 y 145 a * (98.64%) 2 b (1.36%) 147 *Each subscript letter denotes a subset of GBS group categories (such as 'a' and 'b'), the column proportions of which do not differ significantly from each other at the 0.05 level.The difference between different age groups was mainly in the positive rate of 45-50 group.y, years.

Table 4 . Clinical characteristics of 48 neonates with GBS-positive.
*The results were compared by the Kruskal Wallis H-test and shown in Table