Group B streptococcal colonization: Prevalence and impact of smoking in women delivering term or near term neonates in a large tertiary care hospital in the southern United States.

BACKGROUND AND HYPOTHESIS: The role of smoking as a risk factor for group B streptococcal (GBS) colonization in women during pregnancy has not been previously adequately explored. We hypothesized that women of term or near term neonates who smoked during pregnancy were more likely to have GBS colonization than their non-smoking counterparts.
METHODS: The electronic health records (EHRs) of a convenience sample of women delivering in an inner-city university tertiary care center were reviewed. The outcome variable of interest was maternal GBS colonization during pregnancy. The primary independent variable of interest was tobacco smoking during pregnancy, determined from the EHRs by the number of cigarettes smoked during gestation. Descriptive statistics were conducted and categorical data were compared by the Fischer's exact test. Multiple logistic regression analysis was further conducted to determine the independent impact of tobacco smoke exposure on GBS colonization.
RESULTS: The prevalence of maternal GBS colonization was 35% among the study population. In the univariate analyses, factors associated with maternal GBS colonization were tobacco smoking during pregnancy (P of trend <0.001), Race (P<0.001), maternal age <20 years (P = 0.006), low birthweight <2500 gm (P = 0.020), maternal drug use (P = 004), and gestational age <37 (P = 0.041). In a multiple logistic regression analysis, tobacco smoking during pregnancy remained the most significant predictor of GBS colonization. Women who smoked during pregnancy were more than twice more likely to be colonized than their non-smoking counterparts (OR = 2.6; 95% CI = 1.5-4.6; p<0.001). Maternal age was the only other significant predictor with younger mothers more than one and a half time more likely to be colonized than their older counterparts (OR = 1.65; 95% CI = 1.02-2.68; P = 0.04).
CONCLUSION: The prevalence of GBS colonization in this institution was consistent with recent national rates. Smoking and maternal age were identified as two independent risk factors for GBS colonization during pregnancy. Further studies are needed to confirm these findings.

Introduction

Maternal group B streptococcal (GBS) colonization is one of the most important risk factors for GBS disease in neonates [1]. In 2002, the Centers for Disease Control (CDC) recommended universal culture-based screening of all pregnant women between 35–37 weeks gestation in order to treat all those positive for GBS with intrapartum antibiotics prior to delivery to prevent against early onset neonatal GBS sepsis [2]. Ever since the institution of this policy, the incidence of early onset GBS sepsis has continued to fall dramatically from a rate of 1.5/1,000 to a rate of 0.24/1,000 live births [3].

The GBS colonization rates vary from country to country, the geographic location, and the method used in determining the colonization. In the USA, since the early 80’s colonization rates ranged from 20% to 35% [4-6]. In Africa, rates have ranged from 20% to 30% [7-9]. In Eastern Europe rates of 30+% have also been determined [10]. In Western Europe rates have ranged from a low of 14% to a high of 36% [6, 11]. In India one study found a rate of 15% [12]. while in Korea, the rate was as low as 8% [13]. In Australia, rates as high as 35% have been demonstrated [14]. In summary, rates of GBS colonization have remained remarkably stable over the past 40 years worldwide.

Tobacco smoke exposure during pregnancy is one risk factor for GBS colonization that has not yet been adequately explored. Tobacco smoke has been shown to be associated with increased colonization of the respiratory, gastrointestinal, and even genital tracts with various bacterial pathogens [15-21]. However, the impact of tobacco smoke exposure on GBS colonization has not been adequately explored. Because over 10% of women in the United States smoke during pregnancy [22], it is important to clearly determine its impact on GBS colonization. We therefore hypothesized that tobacco smoking during pregnancy is independently associated with increased GBS colonization among women delivering term or near term neonates in this large tertiary care hospital in Virginia.

Methods

Ethics statement

The Institutional Review Board (IRB) of the Virginia Commonwealth University School of Medicine approved the study as expedited since the data were purely retrospectively collected from the EHRs. There was no interaction between the subjects and the researchers in any way, hence the need for consent was waived.

Design and setting

This was a retrospective cross-sectional study design. The study was carried out in a large inner-city tertiary care university teaching hospital in Virginia. Almost 2,000 babies are born each year in this hospital of which about 30% are Whites, 40% African Americans and 30% Hispanics or Latinos.

Participants/subjects

Mother-newborn dyad singletons were retrospectively recruited in the study using electronic health records (EHRs). The study was initiated in January 1, 2011 and ended in December 31, 2019. Each year subjects were randomly selected by 2 of the study authors (PK and RW). The authors, at their convenience, were allowed by the IRB to fully access the EHRs of mothers and their neonates anytime during the above study period. Inclusion criteria consisted of only women delivering term or near-term neonates 35+ weeks gestational age. Women of preterm neonates < 35 weeks were excluded from the study.

Variables and definitions of terms

The outcome variable of interest was GBS colonization of pregnant mothers during gestation. GBS colonization status was determined by recto-vaginal swabs at 35–37 weeks gestation and consisted of placement of the genital swab approximately 2 cm into the vagina followed by placement through the anal sphincter. The result of the test was defined as either positive or negative by the PCR using standard techniques as described [23]. Tobacco smoking was defined by the number of cigarettes smoked per day during gestation. This variable was accurately recorded by the attending obstetrician during prenatal visits. In the rare instances when this information was missing in the EHR of the obstetrician, e.g. because of lack of prenatal care, the pediatrician usually recorded the smoking status after delivery by direct questioning of the new mother during her Nursery stay. If the mother smoked during pregnancy, the number of cigarettes was also recorded in the mother’s EHR by the obstetrician or the pediatrician. Information on tobacco smoking status of all subjects was thus available. Maternal variables abstracted were: age in years, parity, gestational diabetes, race, and substance use. Infant factors abstracted were: birthweight in grams, gestational age in weeks, and gender of the newborn baby (male or female). Maternal substance use was defined as any use during pregnancy of various drugs such as cocaine, various opiates (e.g., methadone, OxyContin, buprenorphine (Subutex), cannabinoids, or other illegal substances. These potential risk factors were all gleaned from the literature.

Data analyses

A total sample of 736 mother newborn dyad was recruited in the study. The outcome variable of interest was GBS colonization among pregnant women delivering term or near term infants 35+ weeks gestational age. The primary independent variable of interest was active tobacco smoking throughout pregnancy as determined by the number of cigarettes smoked per day. In the univariate analysis, tobacco smoking was categorized as 0 cigarette/day, 1 cigarette/day, 2 cigarettes/day, and 3 + cigarettes/day respectively for trend analysis. Test of trend was also explored for maternal age. Initially maternal age was empirically categorized into 2 groups (< 20 year of years versus 20+ years). A trend analysis for this variable was then conducted to determine whether GBS rates varied by the various maternal age groups. When a clear trend emerged, maternal age was subsequently empirically expanded to 5 groups (<20, 20–24, 25–29, 30–34, and 35+) to explore whether the trend continued. Tests of trends were also explored for the other independent variables of interest (birthweight, gestational age) but were not significant so these variables were left dichotomized in the univariate analysis. In the multiple logistic regression analyses, all the independent variables of interest were left dichotomized for simplicity of analyses.

Descriptive statistics were conducted. Univariate analysis was conducted to determine if each of the selected variables was associated with maternal GBS colonization using Fischer’s exact test for comparisons of proportions. Next, a multiple logistic regression analysis was conducted using only the statistically significant variables in the univariate analysis to determine if each of these factors was still predictive of maternal GBS colonization after controlling for the other potential confounders. The SPSS statistical software (IBM, Armonk, NY 2018) was used in the analyses. A p value of <0.05 was used as test of significance in all cases.

Results

Characteristics of the study population

In this study almost 35% of the mothers were colonized with GBS (see Table 1). Overall 11% of the mothers smoked during pregnancy, 2.6% smoked 1 cigarette per day while almost 9% smoke two or more cigarettes daily. The rest of the Table is self-explanatory.

Table 1

Sociodemographic characteristics of the study population.

Continuous Variables N = 736)	Mean (SD)	Range
Maternal Age (years)	28.1 (5.6)	15–44
Gestational Age (weeks)	39.1 (1.4)	35–42
Birthweight (grams)	3335.2 (485.7)	2030–4805
Parity	2.2 (1.3)	0–9
Number of cigarettes/day	0.6 (2.5)	0–30
Categorical Variables	Number (%)
Group B Strep Colonized?
Yes	257 (34.9.)
No	479 (65.1)
Mother Smoked?
Yes	84(11.4.0)
No	652 (88.6)
Maternal Age (years)
< 20	84 (11.4)
20+	652 (88.6)
Birthweight (grams)
<2500	27 (3.7)
2500 +	709(96.3)
Gestational Age (weeks)
< 37	32 (4.3)
37+	704 (96.7)
No of Cigarettes per day
0	652 (88.6)
1	19 (2.6)
2	19 (2.6)
3+	46 (6.2)
Gender
Males	365 (49.6)
Females	371 (50.4)
Race/Ethnicity
White	242 (32.9)
African American	249 (33.9)
Hispanic/Latino	174 (23.6)
Asia/Pacific Islander	71 (9.6)
Maternal Substance Use
Yes	46(6.3)
No	690 (93.7)
Infant of Diabetic Mother
No	669 (91.8)
Yes	60 (8.2)
Missing	7 (1.0)
Parity
<2	269 (36.5)
2+	467 (63.5)

Prevalence of GBS colonization by sociodemographic characteristics

GBS colonization was least prevalent among the none smokers (32%) but rose sharply to 50% if the subjects smoked 1 cigarette/day and the rate almost doubled to 61% if the subjects smoked 2+cigarettes/day (p of trend = 0.001). When maternal age was categorized into 2 groups (teens vs older), GBS was higher among the teen mothers (<20 years) as compared to their older counterparts (20+ years, P = 0.004; also see Table 2). Furthermore, when maternal age was categorized into 5 groups, the prevalence of GBS was highest among women <20 years (49%), dropped to 40% in women 20–24 years, and 30% in women 25 to 29years. However, there was a small spike to 36% in women 30–35 years but the rate dropped again to 28% in women 35 + years. In general, therefore, there was a decreasing trend of GBS colonization with increasing maternal age (P of Trend = 0.023; also see Fig 1). Interestingly, this trend was only true among non-smokers. Among the smokers, however, there was no downward trend of maternal in GBS colonization by maternal age (P of Trend = 0.935; also see Fig 2).

Table 2

Prevalence of GBS colonization by sociodemographic characteristics.

Variable (N = 736)	Number (% with MGBS)	P Value
Maternal Variables
Mother Smoked?
Yes	49 (58.3)
No	208 (31.9)	< 0.001
Tobacco Smoking (No of cigs/day)
0	210 (32.0)
1	8 (50.0)
2+	39 (60.9)	<0.001
Race
White	74 (30.6)
African American	122 (49.0)
Hispanic	45 (25.9)
Asian /Pacific Islanders	16 (22.5)	<0.001
Maternal Age (years)
< 20	39 (48.1)
20+	218 (33.3)	0.006
Maternal Substance Use
No	232 (33.6)
Yes	25 (54.3)	0.004
Gestational Diabetes*
No	230 (34.4)
Yes	25 (41.7)	0.257
Parity
<2	85 (31.6)
2+	172 (36.8)	0.088
Infant Variables
Gestational Age (Weeks)
<37	241 (34.2)
37+Birthweight (gm)	16 (50.0)	0.051
<2500 (LBW)	15 (55.6)
2500+ (NBW)	242 (34.2)	0.020
Gender of Newborn
Male	134 (36.7)
Female	123 (33.2)	0.311

* Numbers may not add up due to missing information.

Fig 1

Prevalence of maternal GBS colonization by age group.

The Figure demonstrates that overall the youngest mothers (<20 years) had the highest rates of GBS colonization (49%) while the oldest mothers (35+ years) had the lowest rates (28%) although there was a small spike in mothers 30 to 34 years (P of Trend = 0.023).

Fig 2

Prevalence of maternal GBS colonization by maternal age group and smoking status.

This Figure shows that, for smoking mothers, there was no relationship between maternal age and GBS colonization (P of Trend = 0.935) whereas for the non-smoking mothers there was a clear inverse-relationship between maternal age and GBS colonization (P of Trend <0.05).

GBS colonization varied widely among the racial groups and was lowest among Asian /Pacific Island Americans (22.5%) as compared to the Latinos (26%), White Americans (30.6%) or African American (49%, P <0.001). Furthermore, the proportion of colonization was more prevalent among women with any history of substance use (54%) as compared to those without any use (35%; P = 0.004). Thus, of the nine variables explored in this study, only 3 viz. gestational diabetes mellitus, parity, and gender were not predictive of GBS carriage (also see Table 2).

Multiple logistic regression of factors associated with GBS colonization

All the 6 variables significantly associated with GBS colonization in the univariate analyses (smoking, maternal age, birthweight, gestational age, race, and use of substance) were now selected for inclusion in the multiple logistic regression analysis. Tobacco smoking was by far the most significant predictor of GBS colonization even after controlling for the other confounders (adjusted OR = 2.6, 95% CI: 1.53–4.34; P <0.001). The only other risk factor independently associated with GBS colonization was maternal age < 20 years (adjusted OR = 1.65, 95% CI: 1.02–2.68; P = 0.041, also see Table 3).

Table 3

Multiple logistic regression of factors associated with GBS colonization among pregnant mothers showing unadjusted and adjusted Odds Ratios (OR) and their 95% Confidence Intervals (CI).

Variables	Unadjusted OR (95% CI)	P Value	Adjusted OR (95% CI)	P Value
Mother Smoked?
No (Referent)	1	-	1	-
Yes	2.99 (1.88–4.75)	<0.001	2.55 (1.54–4.34)	<0.001
Maternal Age (years)
20+(Referent)	1	-	1	-
<20	1.86 (1.17–2.96)	0.009	1.65 (1.02–2.68)	0.04
Birthweight (grams)
2500+ (Referent)	1	-	1	-
< 2500	2.41 (1.11–5.23)	0.026	1.89 (0.83–4.25)	0.132
Race
White (Referent)	1	-	1	-
Non-White	1.34 (1.0–1.86)	0.05	1.22 (0.87–1.72)	0.246
Gestational Age (weeks)
37 + (Referent)	1	-	1	-
<37	1.92 (0.94–3.91)	0.07	1.29 (0.83–1.99)	0.258
Maternal Substance Use
No (Referent)	1	-	1	-
Yes	2.35 (1.29–4.29)	0.005	1.27 (0.64–2.52)	0.487

Discussion

There were three important findings in this study: 1) Almost a third of all women delivering term or near neonates were colonized with GBS in this hospital; 2) even after controlling for various confounders, smoking during pregnancy was more than twice more likely to result in GBS colonization among pregnant women of term or near term neonates.; 3) teen mothers less than 20 years had the highest rates of GBS colonization and the rates steadily decreased with age with the lowest rates occurring among the oldest mothers 35 year+. Interestingly this inverse relationship was only true for women who were nonsmokers.

This high prevalence of GBS colonization found in our study is consistent with previous studies in the USA and elsewhere [4-12]. The lowest rates were observed mainly in some East Asian nations [6, 12, 13]. The across-country differences may be due to differences in techniques [23-35]. PCR testing after broth enrichment has been has been shown to be a reliable and is now the standard method for GBS testing [24]. However, testing conducted intrapartum may show somewhat lower prevalence rates as compared to antepartum testing [25]. If this is so, then there may be excessive use of intrapartum prophylactic antibiotics to prevent GBS sepsis in neonates. Unfortunately, intrapartum testing is not yet practical since it can take up to 2 to 3 days for the results of tests to be made available whereas knowing the results prior to delivery is very important to make a decision to treat prophylactically or not. Hopefully, new rapid PCR tests are being developed for such a purpose [25]. In spite of the dramatic drop of early onset neonatal GBS sepsis in the United States, the reasons for the persisting high prevalence rates of GBS colonization during pregnancy throughout the past 4 decades need to be further explored.

This study further confirms that, potentially, more than a third of all women were eligible to receive intrapartum prophylactic antibiotics prior to delivery. This is indeed a high rate of pre-delivery antibiotics exposure of term or near term neonates. Several authors have recently suggested that at least 40% of children may be exposed to intrapartum antibiotics [26, 27]. Although intrapartum antibiotic prophylaxis have successfully reduced the incidence of GBS sepsis in neonates, the dangers of such prophylaxis are concerning [28]. For instance, intrapartum antibiotic prophylaxis have been associated with complications such as increased prevalence of atopic dermatitis [29], antimicrobial resistance [30], and changes in the neonatal gut microbiota [21, 31, 32].

The prevalence of active tobacco smoking among pregnant women in this study was 11%, and this is consistent with recent national trends [22]. However, in our study, the intensity of smoking during pregnancy was rather low and most women who smoked admitted to smoking no more than 3 cigarettes each day although this may have been largely an underestimate. Since this was a retrospective study the assumption is that women who admitted to smoking actually smoked throughout pregnancy. The study may, therefore, not take account of those who stopped smoking sometime during pregnancy or those who only initiated smoking sometime during pregnancy.

Few studies in the literature have explored the impact of smoking during pregnancy on GBS colonization. Our study was also able to demonstrate a significant dose-response relationship suggesting that this was not just a spurious finding. In the United States, Terry et al. [33] were the first to show that smoking during pregnancy was predictive of GBS colonization but the authors did not perform a multivariable analysis to control for other confounders to determine if smoking was independently predictive. Edwards and colleagues [34], in a large retrospective study, recently demonstrated that smoking was also predictive of GBS colonization in the univariate analysis but not in the multiple regression analysis. However, smoking was not the primary independent variable of their study. Two studies respectively from Korea and China [13, 35], failed to demonstrate an association of smoking during pregnancy with GBS colonization. However smoking was also not the primary independent variable of interest of these studies and there were was the additional problem of smallness of numbers in some cells. Our finding that smoking during pregnancy was predictive of GBS colonization during pregnancy is consistent with a smaller study from Iran [36]. However, their study population was different as it also included preterm neonates that were excluded in our current study. Surprisingly, Regan and colleagues demonstrated in 1991 that smoking was rather protective of GBS colonization [37]. The authors offered no biological plausibility of their finding. However, only women delivering preterm neonates (<36 weeks) were enrolled in the latter study whereas our study focused only on term or near term neonates. We speculate that the population of women delivering term babies may be quite different from the population delivering only preterm babies.

The finding that tobacco smoke exposure during pregnancy is an independent risk factor for increased GBS colonization has significant public health implications. Because smoking exposure is a modifiable risk factor, women can be counseled to stop smoking during pregnancy in order to reduce colonization with this organism which can result in GBS sepsis in their newborn baby. The association between tobacco smoking and increased GBS colonization during pregnancy is biologically plausible. We speculate that tobacco smoke exposure during pregnancy may actually enhance the colonization of GBS in the gastrointestinal and the genital tracts. Indeed, previous studies have shown that tobacco smoke exposure is associated with increased colonization of the respiratory and genital tract with pathogenic bacteria [38, 39], possibly through alteration of the microbiome [18, 21, 40]. Tobacco smoke contains more than 4,500 chemical intoxicants [41] many of which can result in increased suppression or modulation of both active and passive immune response [42]. For instance, nicotine, an important component of tobacco smoke, has been shown to enhance the adherence of bacteria in mucous membranes of the respiratory tract leading to easy penetration of bacteria into the tissues to cause infections [43]. This could also be true of the genital tract of pregnant women where nicotine could actually result in the persistence of GBS in the mucous membranes of the gastrointestinal and genital tracts. Indeed some studies have actually demonstrated higher nicotine levels in the cervical mucous membranes of smokers as compared to non-smokers [44, 45]. In another study, nicotine of the cervical mucus of female smokers resulted in DNA damage of epithelial cells of these women resulting in easy penetration of bacteria into the adjacent tissues [46]. It can be speculated that the overall effect of tobacco smoke is the alteration of the microbiome of the female genital tract resulting in increased prevalence of pathogenic microorganisms such as GBS in the present study.

To our knowledge only a few studies in the United States have been conducted in recent years to determine potential sociodemographic risk factors associated with this rather high colonization rates among pregnant women. In the ‘70’s Anthony et al showed that Mexican Americans had the lowest rates as compared to Whites or Blacks [47]. In our study, black mothers had the highest rates as compared to White or Hispanic/Latino mothers. These findings are consistent with those of Regan and colleagues whose study was conducted almost 30 years ago [37]. The finding that younger mothers had significantly higher rates of GBS colonization was consistent with one previous study conducted more than 40 years ago by Anthony and colleagues [47]. They demonstrated that younger mothers had higher rates of GBS colonization than their older counterparts. Indeed, GBS sepsis in neonates has also been shown to be more common among young mothers < 20 years, as demonstrated by Schuchat et al. [48, 49]. This may be explained by the fact that young mothers also have higher rates of GBS colonization than their older counterparts as shown in our study. However, 10 years after the study by Anthony et al [47], Regan et al [37], showed that GBS colonization was less common among women less than 20 years of age even after controlling for the other sociodemographic confounders. The difference in findings between the two studies may be due to the fact that cultures for GBS were obtained very early in gestation (23–26 weeks gestation) in the latter study, whereas the current recommendation is to obtain cultures at 35–37 weeks of gestation [2]. Again as stated above, our study clearly demonstrates that maternal age was only predictive of GBS colonization among the non-smoking women. Non-smokers < 20 years had the highest GBS colonization rate while those >35 years of age had the lowest rate.

This study has several limitations. First the retrospective nature of the data implies that there is no causality attributed to the findings. Second, because the study only involved subjects recruited from one local hospital, there may therefore be lack of both internal and external validity of the findings as this sample was not necessarily representative of the population of the state or of the nation. Third, the information of tobacco smoking was retrospectively obtained so there is likelihood of misclassification bias. The association between tobacco smoking and GBS colonization would have been even stronger if the smoking status was determined objectively by the use of a biomarker such as serum or urine cotinine levels. This may have reduced the likelihood of misclassification bias of the smoking status of the subjects. However, some of the findings are consistent with previous works. Fourthly, we were not able to control for all the confounders such as obesity and the frequency of sexual relationships which have been shown in some studies to be predictive of GBS colonization [49]. The significance of this study lies in the fact that this is the first robust study with the main focus of determining the impact of tobacco smoke on GBS colonization on women of term or near neonates. In other studies GBS was not the main focus and a test of trend was not explored.

Summary and conclusion

This is one of the few studies to clearly determine that smoking during pregnancy is an independent predictor of GBS colonization specifically in women of term or near term neonates. GBS colonization was highest among the youngest mothers and tended to decrease with age especially among non-smoking mothers. This is another reason why women should be counseled to stop smoking if they are pregnant in order to avoid risking their newborn from developing GBS disease.

(SAV)

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12 Jun 2020

PONE-D-20-15744

GBS colonization: prevalence and impact of smoking in women delivering term or near term neonates in a large tertiary care hospital in the southern United States

PLOS ONE

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Reviewer #1: PONE-D-20-15744

GBS colonization: prevalence and impact of smoking in women delivering term or near term neonates in a large tertiary care hospital in the southern United States

This manuscript describes the relationship between smoking during pregnancy with group B streptococcal (GBS) colonization among women of term and near term neonates. While the manuscript is of some interest, I do not think the manuscript in its current form is of sufficient interest and quality to warrant publication in PLOS ONE.

I suggest the authors to look for STROBE check list for cross-sectional studies to ensure reporting is complete and transparent.

https://www.strobe-statement.org/fileadmin/Strobe/uploads/checklists/STROBE_checklist_v4_cross-sectional.pdf

My sense is that the manuscript could be strengthened by several modest changes as outlined below. Inserting line numbers may facilitate to give comments and feedbacks.

Title

- Suggest to expand GBS long form. Otherwise, the readers may confuse with Genotyping-by-sequencing or Guillian-Barre Syndrome or etc.

INTRODUCTION

- Introduction section is not well organized and not well-written.

- Introduction section needs to include - What is the problem and its supporting details? What is the added scientific value from the study and why are you conducting it? - Both globally and locally.

- Paragraph 2 – the information is overloaded and recommended a concise statement. Some detailed information can be shown by references.

- Need to add reference for the sentence “Maternal group B streptococcal (GBS) colonization is one of the most important risk factors for GBS disease in neonates.”

- The authors described “Potential sociodemographic risk factors for GBS colonization among pregnant women have not been recently explored. Perusal of the literature within the last 2 decades demonstrates a paucity of information on other potential sociodemographic risk factors associated with maternal GBS colonization during pregnancy.” in the introduction but I find myself many papers in the references as well as online. Moreover, this does not reflect the justification of the stated hypothesis.

METHODS

- The authors mentioned “the data ….. divided almost equally yearly (i.e. almost 90 patients selected each year)”. Is this selected by the authors or by the data set?

- Abbreviate first before using it (E.g. IRB, x2)

- Check grammar and spelling mistake throughout the manuscript

- The authors mentioned only the significant variables were put for a multiple logistic regression analysis. What do authors mean by significant? Statistically significant? What is the cut-off point, <0.05 or <0.2?

- Many items of the STROBE check list are written; however, most of them are jumbled up under different sub heading and some are missing (inclusion criteria, exclusion criteria, etc.)

RESULTS

- The authors tend to cover all information in the text and again in the table. You should only include the most important details in the text when you also have a table.

- Any reason behind for including 67 women who outcome status were unknown as study population?

- Any reason behind for categorizing maternal age in two ways?

- Trend analysis, Figure 1 and 2 may not be necessary for testing the study hypothesis.

- Foot note is missing for * in table 2

- Tables should be structured properly (check the published PLOS ONE papers).

- To me, this is a cohort study which the authors can calculate and present relative risk (strong statistic).

- All things considered, re-analysis and rewriting of results section is necessary.

DISCUSSION

- The discussion is the best part of the manuscript.

- Repeating the finding statements and analysis result (E.g. P of trend = 0.02) should be avoided here.

CONCLUSION

- The authors concluded “GBS colonization as rather high but consistent with recent national trend” which is not clear.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Kyaw Lwin Show

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Reviewer comments.docx

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16 Jul 2020

RESPONSES TO REVIEWER of PLoS ONE: Group B Streptococcal colonization: prevalence and impact of smoking in women delivering term or near term neonates in a large tertiary care hospital in the southern United States

The Editor PLoS ONE,

Thank you for allowing us to rewrite our manuscript titled above following your recommendations and those of the Reviewer.

RESPONSES TO SPECIFIC ACADEMIC EDITOR COMMENTS

1) The PLoS ONE’s style requirements were followed

2) Consent Issues: As stated in out manuscript (page line ) the manuscript was reviewed as expedited and no consent was required since this was purely retrospective data from the HER. There was no interaction between the subjects and the researchers in any way, hence the need for consent was waived by the IRB of the Virginia Commonwealth University.

3) EHRs were assessed variously on throughout May 1 to July 31 of 2011, and subsequently same dates through 2019

4) A caption of Fig 1 and Fig 2 included

5) We have included the Tables as part of the main manuscript

6) Table 3 has been referenced in the main manuscript. Thanks for spotting that error

7) Captions for the Supporting Information files at the end of the manuscript have now been included.

RESPONSES TO REVIEWER’s COMMENTS

We have endeavored to follow the STROBE checklist while rewriting this manuscript as suggested by the Reviewer # 1. We furthermore inserted line numbers to facilitate comments and feedback

TITLE

We have changed the abbreviation “GBS” to its expanded form “Group B Streptococcal”. We thank the Reviewer for pointing out this error.

INRODUCTION

- We have reorganized the Introduction: In paragraphs 1 and 2 we have restated the importance of GBS colonization in causing GBS disease worldwide. In paragraph 3 we have stated that one of the risk factors for GBS colonization not adequately explored is tobacco smoke exposure during pregnancy. If this is found to be an independent risk factor, then this may be one additional reason why women may benefit from refraining from smoking during pregnancy

- Paragraph 1. We have added a very important reference to the opening statement “Maternal group B streptococcal (GBS) colonization is one of the most important risk factors for GBS disease in neonates” (Verani et al). We thank the Reviewer for pointing this out.

- Paragraph 2. We have deleted the first sentence in this paragraph but we have left the rest of the 2nd paragraph 2 intact even though there may some information overload as stated by this Reviewer.

- As suggested by the Reviewer we have remove a controversial statement about lack of studies on sociodemographic risk factors associated with GBS colonization. We have rewritten this 3rd paragraph to emphasize ONLY the lack of studies on tobacco smoking during pregnancy as a risk factor for GBS colonization. We thank the Reviewer for pointing this out.

METHODS

- Yes this was a convenience sample selected by the authors form EHRs between May and July of each year.

- Abbreviation issues corrected

- We have endeavored to the best of our ability to check the grammar and spellings as suggested by the Reviewer.

- We have added the word “statistically” in the sentence since only statistically significant variables in the univariate analysis (p<0.05) were included in the multivariate analysis (This is stated see last sentence last paragraph of Data Analysis).

- STROBE checklist: We have endeavored to rewrite and include all items of the checklist although some of the items may not necessarily be in the same order as in the STROBE checklist. We have clearly stated that only women of term or near term neonates were included (35+ week gestational age). All preterm neonates <35 weeks gestational age were excluded.

RESULTS

- We agree with the Reviewer that the Results section may be overloaded with information already in the Tables. We have therefore rewritten the Results section summarizing only the most important findings.

- The Reviewer suggested that we remove the 67 subjects with unknown GBS status in the analyses. This has been done and the sample size is now 736 subjects. The analysis has been redone but mainly affects Table 1 as can be seen. Table 2 and 3 remain unchanged since these tables only took account of the 736 subjects with known GBS status.

- The Reviewer wanted to know why we categorized maternal age (and tobacco smoke) in two different ways. As stated in our Methods section we did a trend analysis only of all the statistically significant independent predictors of GBS colonization in the univariate analysis. In the multiple logistic regression analysis, all variables were dichotomized for simplicity.

- The Reviewer felt trend analyses and Fig 1 and 2 may not be needed. However we feel that a trend analysis gives the average reader additional insight on how smoking and maternal age affect GBS colonization. The Figures clearly show that maternal age is only predictive of GBS among the non-smoking women regardless of age group.

- Footnote of Table 2 has been inserted

- The Reviewer suggests that this study should be labeled as a cohort. But because all the data were retrospectively collected from EHRs without any interactions with the subjects this is still a retrospective study and we still think Odds Ratios rather than relative risks should be calculated.

DISCUSSION

- We are very thankful for this Reviewer’s appreciation of our discussion in this study.

- We have followed the Reviewer’s suggestion to avoid any finding statements and analysis result have been deleted these (e.g P of Trend=0.02)

Submitted filename: GBS. RESPONSES TO REVIEWER of PLoS ONE.docx

Click here for additional data file.

23 Jul 2020

PONE-D-20-15744R1

Group B streptococcal colonization: prevalence and impact of smoking in women delivering term or near term neonates in a large tertiary care hospital in the southern United States

PLOS ONE

Dear Dr. Kum-Nji,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

SPECIFIC ACADEMIC EDITOR COMMENTS: There are some additional revisions that are requested and must be addressed.

Please submit your revised manuscript by Sep 06 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Frank T. Spradley

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: PONE-D-20-15744-R1

Group B Streptococcal colonization: prevalence and impact of smoking in women delivering term or near term neonates in a large tertiary care hospital in the southern United States

My sense is that the manuscript could be strengthened by minor modifications as outlined below.

- Check the spelling mistakes (E.g. LINE 89)

- Table 2 – what do you mean *? In the responses, the authors stated that footnote was inserted but I could not find it.

- Check number in LINE 152, 153 – first 736, out for 67 , then 736 again

- Discrepancies of proportion in the text and table (check carefully)

- The above errors mean that the authors should have checked every single word in the manuscript before submission. There may be more that I couldn’t find.

- The authors stated “Using x2 analysis of comparisons of proportion with Fisher’s exact test” which is not clear. Used chi-square or Fisher’s exact?

- Table 1 - Maternal smoked during pregnancy? � 803 participants?

- Table 3 should be revised (check Table 4 as example from https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176875)

- The authors concluded “GBS colonization as rather high but consistent with recent national data” which is not clear. Why do you say high here? Is nationally already high? High compared to what?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Reviewer comments.docx

Click here for additional data file.

28 Jul 2020

RESPONSES TO THE ACADEMIC EDITOR AND REVIEWER

We have checked the spelling mistakes and corrected lines 88 and 89: “out” is inserted between “carried” and “in” to read “carried out in” Also in a large “hospital.in Virginia” now becomes “hospital in Virginia”. Thus the period between “hospital” and “in” is deleted

Table 2. We have deleted the asterisk “*” from this Table (see “Tobacco Smoking (No of cigs/day)” and “Race”. Since we have reanalyzed the data there is no longer a need for a footnote for these variables. We thank the editor for consistently spotting this error.

Lines 152-153: We have deleted this sentence completely since it was indeed rather confusing and did not make sense. We thank the reviewer for spotting this error!

Discrepancies of proportions in the text and Tables. We again thank the reviewer for spotting these errors! After reanalyzes of the data as suggested by the previous reviewer we completely forgot to go back and to very carefully edit the numbers in the texts. All these have now been carefully corrected as listed in the lines of the new “Track Changes” as noted below:

Table 1:

- Line 150 : “2%” becomes “2.6”

- Line 151: becomes “10%” becomes “9%”

Table 2:

- Line 155: 35% becomes “32%” and “53%” becomes “50%”.

- Line 156: “63%” becomes “61%” .

- Line 157: “P of Trend= 0.003” becomes “P of Trend=0.001” .

- Line 159: “P=0.006” becomes “P=0.004”.

- Line 169: Pacific Island Americans (22%)” becomes “22.5%”.

- Lines 169-170: “White Americans (30.1%)” now becomes “(30.6)”

Data Analysis : Lines 140-141: We have deleted the portion on X2 analyses and left it with Fischer’s exact test since this is what was used in the analyses throughout

Table 1. “Mother Smoke during Pregnancy? “ the numbers have now been corrected and they add up to 736 not 803

Table 3. This Table has been revised as suggested by the academic reviewer: Unadjusted ORs have therefore been added to the adjusted ORs in the previous analysis and heading of the Table has been updated.

Conclusion: Because there is confusion about the use of the word “high”, the authors have decided to simply delete the use of this word. The conclusion has therefore been slightly reworded.

We very much appreciate the academic reviewer for their thorough editing of our manuscript and their suggestions for improvement. We hope you can publish it in its present form.

Please, do not hesitate to let us know if we can further improve our manuscript fit for publication in your esteemed journal.

Philip Kum-Nji, MD, MPH

(On behalf of the authors)

Submitted filename: RESPONSES TO GBS PLoS One ACADEMIC EDITOR AND REVIEWER.docx

Click here for additional data file.

3 Aug 2020

PONE-D-20-15744R2

Group B streptococcal colonization: prevalence and impact of smoking in women delivering term or near term neonates in a large tertiary care hospital in the southern United States

PLOS ONE

Dear Dr. Kum-Nji,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

SPECIFIC ACADEMIC EDITOR COMMENTS: There are still comments that must be addressed. Also, please have your manuscript proofed by a professional to assess English grammar and syntax.

Please submit your revised manuscript by Sep 17 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Frank T. Spradley

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: PONE-D-20-15744-R2

Group B Streptococcal colonization: prevalence and impact of smoking in women delivering term or near term neonates in a large tertiary care hospital in the southern United States

Comments

- I agree about adding unadjusted OR however, Table 3 in current form is UNACCEPTABLE. The readers may get confused regarding the reference category.

- There are typo errors still remained especially in the tables. The authors stated they have corrected it but not really corrected. The authors have all the RESPONSIBILITIES for checking each and every words in the manuscript correct rather than submit the revised version fast.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Reviewer comments.docx

Click here for additional data file.

23 Aug 2020

RESPONSES TO ACADEMIC REVIEWER

We are grateful that the editors have given us another opportunity to correct and improve our manuscript for publication.

Table 3. We have rewritten Table 3. We have specified the referent category for each variable for clarity.

Typos in the Tables still noted by the academic reviewer:

- * for race was actually for gestational diabetes. An explanatory footnote has been therefore inserted under the Table 2.

- Typos have been corrected under Table 1:

o Birthweight range corrected

- Typos under Table 2:

o Maternal Age

o Gestational diabetes

o Gestational Age

NB: We also noted on rereading the manuscript that one reference was duplicated (reference 21 and 31). We therefore deleted reference 31 and the text references were therefore also updated carefully.

We hope that we have answered your queries to your satisfaction but please do not hesitate to let us know if further improvements are warranted.

Philip Kum-Nji, MD, MPH

Submitted filename: Responses to the Academic Editor PLoS One.docx

Click here for additional data file.

3 Sep 2020

Group B streptococcal colonization: prevalence and impact of smoking in women delivering term or near term neonates in a large tertiary care hospital in the southern United States

PONE-D-20-15744R3

Dear Dr. Kum-Nji,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Frank T. Spradley

Academic Editor

PLOS ONE

7 Sep 2020

PONE-D-20-15744R3

Group B streptococcal colonization: prevalence and impact of smoking in women delivering term or near term neonates in a large tertiary care hospital in the southern United States

Dear Dr. Kum-Nji:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Frank T. Spradley

Academic Editor

PLOS ONE