A rapid intrapartum test for group B Streptococcus to reduce antibiotic usage in mothers with risk factors: the GBS2 cluster RCT

Maternal colonisation

The gastrointestinal tract is the natural reservoir of GBS in humans and is the likely source of vaginal colonisation. Asymptomatic maternal colonisation with GBS has been reported at a global level of 15%,6 although this figure can vary with race, region and the method of laboratory culture used for its detection. 6,7 A previous review of UK studies indicated an overall colonisation rate of 18% from vaginal/rectal swabs [95% confidence interval (CI) 16% to 21%], higher than from vaginal swabs alone. 8

Transmission

Neonates with early-onset GBS infection show initial colonisation mainly in the mucous membranes of their respiratory tract, and the major route of vertical transmission at the time of birth is thought to be through aspiration of vaginal, rectal and amniotic aerosols during birth. Vertical transmission in utero is thought to occur as a consequence of prolonged rupture of membranes (although GBS can cross intact membranes9) and is regarded as one of the causes of stillbirth. 10 Colonisation of the mother is less predictive for late-onset GBS infection, with prematurity being the major risk factor. 11

The association between the rates of maternal colonisation, transmission and infection has been established (Figure 1). A meta-analysis of six studies of the maternal and baby colonisation rates in an untreated general population showed a transmission rate between the colonised mother and her baby of 36.4% (95% CI 28.1% to 45.0%). A further analysis in the same report gave an average incidence of 3.0% (95% CI 1.6% to 4.7%) of babies born to colonised mothers who went on to develop early-onset GBS disease. 8 There is some evidence that maternal colonisation with GBS is associated with preterm birth, particularly ascending infection due to GBS bacteriuria. 12

FIGURE 1.

Model of colonisation, transmission and early-onset GBS disease.

Burden of early-onset group B Streptococcus infection

Group B Streptococcus remains one of the most important causes of severe early-onset infection in newborn infants. A systematic review estimated that the global incidence of early-onset GBS infection was 0.41 (95% CI 0.36 to 0.47) per 1000 live births, with the highest burden found in the Caribbean and South Africa. 13 Enhanced surveillance in the UK and Ireland from 2014 to 2015 showed an incidence of culture-proven GBS infection in babies aged < 90 days of 0.94 (95% CI 0.88 to 1.00) per 1000 live births. 14 In this study, 60% of cases were early-onset infection, a rate of 0.57 (95% CI 0.52 to 0.62) per 1000 live births, an increase from 0.48 per 1000 live births in 2000. In the USA, the incidence of early-onset GBS infection fell from 1.7 per 1000 live births in the early 1990s to 0.22 per 1000 live births in 2014, without any concurrent rise in Gram-negative sepsis. 15

The global case–fatality ratio is estimated at 8.4%, with a fourfold variation between Africa and high-income countries,13 whereas in the UK it was 6.2% in 2014, an improvement from 9.6% in 2001, possibly because of the improvements in neonatal care. Mortality is much higher in preterm babies. Oddie and Embleton16 found that preterm infants accounted for 38% of all cases and 83% of the deaths from early-onset GBS infection. Information on morbidity among survivors is less clear, but significant long-term morbidity, including impaired psychomotor development, has been reported in up to 30% of survivors. 17

It is highly likely that some cases of serious early-onset sepsis caused by GBS are unrecognised because cultures of blood and cerebrospinal fluid are negative. By taking into account superficial swab culture results from all neonates who underwent a septic screen in the first 72 hours of life, Luck et al. 18 concluded that the true incidence of early-onset GBS disease in the UK may be as high as 3.6 per 1000 live births (i.e. over seven times higher than previously estimated). Epidemiological studies have suggested that various factors present at the time of birth are associated with the neonate having an increased risk of developing GBS disease, presenting as either an early- or late-onset infection. UK surveillance data suggest that only 35% of infants with early-onset GBS infection were born to mothers with one or more risk factors,14 as defined by the RCOG guidelines implemented at that time. 19

Prematurity

Colonised premature babies are at a high risk of developing early-onset GBS infection, as their immune system is immature and they are less likely to have received passive immunity transplacentally. The pooled odds of maternal colonisation in preterm births, compared with births at > 37 weeks’ gestation, was 1.53 (95% CI 1.14 to 2.05). Birth weight is highly correlated with prematurity and inversely related to developing early-onset GBS infection. The UK surveillance study indicated an incidence of 2.24 (95% CI 1.31 to 3.59) early-onset GBS infection cases per 1000 live births in babies weighing < 1500 g at birth, compared with 0.43 (95% CI 0.38 to 0.49) per 1000 live births in those weighing ≥ 2500 g at birth. 14

Prelabour rupture of membranes

Prelabour rupture of membranes (PROM) with a delay in progress to established labour would be expected to lead to an increased likelihood of ascending infection and baby colonisation in utero, although there is debate as to what, if any, role the presence of GBS plays in the induction of PROM. Rupture of the membranes > 18 hours before birth is significantly associated with early-onset GBS infection (odds ratio 25.8, 95% CI 10.2 to 64.8) compared with non-infected infants. 17 Therefore, babies born to mothers who experience preterm labour with PROM of any duration, or preterm labour if there is suspected or confirmed intrapartum rupture of membranes lasting > 18 hours, are especially thought to be at risk of developing early-onset GBS infection. UK NICE guidelines recommend that induction of labour at term is offered at 24 hours after PROM. 3

Maternal fever

Intrapartum fever is also highly associated with the development of early-onset GBS infection (odds ratio 10.0, 95% CI 2.4 to 40.8). 16 In a UK surveillance study, 19% of babies with early-onset GBS infection were born to mothers with intrapartum pyrexia and 31% to mothers with suspected chorioamnionitis. 14

Previous baby with group B Streptococcus disease

The proportion of women giving birth at term who have had a previous baby with early-onset GBS infection was estimated at 0.08%. These women are considered to be at higher risk of another infected baby, than multiparous women whose previous babies were not affected, although the data are insufficient to describe the size of this increased risk. 20

Group B Streptococcus detected in current pregnancy

Group B Streptococcus bacteriuria is associated with higher risk of chorioamnionitis and neonatal disease, although the data are insufficient to quantify the increased risk. 21,22 In the 2014 UK surveillance study, 9% of mothers of GBS-infected babies were known to be colonised with GBS, although a figure of 5% was used in a model of the effectiveness of screening. 23

Bacteriological culture

The sensitivity of testing methods based on the culture identification of maternal colonisation of GBS depends on the timing of specimen collection, the source of the specimen and the culture technique used by the microbiology laboratory. A systematic review of prospective studies showed that a single vaginal/rectal swab has a positive predictive value (i.e. the proportion positive at 35–37 weeks’ gestation who remain positive at term labour) of 70.2% and a negative predictive value of 95.2%, but there were variations in testing methods. 24 Overall, using retrospective and prospective studies and different types of swabs and culture methods, sensitivities of the antenatal tests ranged from 51% to 71% and specificities were consistently around 85%. 24

Molecular tests for group B Streptococcus colonisation

Development of molecular methods that allow rapid bedside detection of microorganisms, such as polymerase chain reaction (PCR) methods, offers the potential to target use of antibiotics more specifically than was previously possible. Previous experience shows that implementation of complex point-of-care (POC) tests for GBS colonisation are technically feasible. 25 However, the practical value of any POC test depends on accurate results being reliably available within the clinically required time frame. To this end, careful consideration is required of a number of factors, including the expected frequency of testing, achievable results turnaround times, the amount of hands-on test time, strategies to deal with test failure and assurance of the ongoing availability of sufficient trained staff able to undertake testing when required.

The majority of the commercially available test systems required multiple preparation and incubation steps. What is required, is a system that can use swabs directly and be operated by midwives on a maternity unit. In the light of these limitations, the technology used in the GBS2 trial was the Cepheid GeneXpert^® system (Cepheid, Maurens-Scopont France), which is feasible as a POC test. The Xpert^® GBS test (Cepheid) for this platform allows detection of GBS within 35 minutes of placing a swab into a cartridge and loading one machine, with a hands-on preparation time of < 2 minutes.

Accuracy of GeneXpert rapid test to detect group B Streptococcus colonisation

The Cepheid GeneXpert GBS system has been available since 2008 and several groups have assessed its accuracy in studies, although with different swabbing strategies and reference standard comparators. The manufacturer cites a sensitivity (1 – false-positive rate) of 91.9% (95% CI 84.7% to 96.5%) and a specificity of 95.6% (95% CI 95.0% to 98.9%) for intrapartum vaginal/rectal samples, compared with selective enrichment culture of a second intrapartum swab. 26 A meta-analysis, undertaken in 2014, of nine studies, which used an enrichment culture method for the reference standard, obtained estimates of the average accuracy of the test of sensitivity 96.4% (95% CI 90.8% to 98.6%) and specificity 98.9% (95% CI 97.5% to 99.5%) (Jane Daniels, University of Nottingham, 2014, personal communication). Overall, the quality of the studies was rated as being adequate, but the potential for biases remained, from the lack of blinding, the use of only vaginal swabs and the inappropriate use of non-enrichment culture as the reference standard, which could underestimate colonisation rates. A subsequent meta-analysis of 15 studies using various test PCR platforms produced a pooled sensitivity of 93.7% (95% CI 92.1% to 95.3%) and specificity of 97.6% (95% CI 97.0 % to 98.1%). 27 These accuracy parameters, together with the turnaround time, suggest that the GeneXpert system meets the proposed criteria set by the US Centers for Disease Control and Prevention for a clinically useful GBS test, namely that it should consist of a simple bedside kit that can be used by delivery suite staff, have a turnaround time of < 30 minutes, and have a sensitivity and specificity of ≥ 90%. 26

Current policy for prevention of early-onset group B Streptococcus infection

The strategy recommended by the UK’s RCOG is to consider and offer IAP to women who have risk factors identified during the pregnancy or in labour for having a baby with early-onset GBS infection. 4 Reviews by the UK National Screening Committee (NSC)24,28 and NICE3 have endorsed this approach.

The risk factors to consider in this approach, and the management options available in the original,29 the 201219 and the 20174 revised guidance are summarised below.

Evidence for group B Streptococcus testing strategies

Introducing universal testing for maternal GBS colonisation into the UK health-care system has been considered alongside other testing and vaccination strategies from the perspectives of both cost-effectiveness and overtreatment. The UK NSC reviewed the evidence for universal and risk factor-based screening in 2012 and again in 2017, and at both times concluded that there was insufficient evidence against its standardised criteria to justify a change from the current risk factor-based screening approach to guide administration of IAP. 24,28 The NSC estimated that the number of women needing to receive IAP on the basis of culture-based testing at 35–37 weeks to prevent one case of early-onset GBS infection missed by the risk factor approach was 1000–1500 in the 2012 analysis and 1675–1854 in the 2017 analysis. In the 2019 hypothetical cohort, an additional 96,260 women would receive IAP under a testing policy; however, with the test having a positive predictive power of 0.2%, the overwhelming majority of infants born to women receiving IAP would never have been at risk. 23 However, the model’s input parameters have been called into question, as the risk factor strategy emerged with an early-onset GBS infection rate of 0.49 per 1000 live births, which is significantly lower than UK surveillance data suggest. 14,23

A French study31 assessed whether or not rapid intrapartum testing for GBS reduced the proportion of women inappropriately given IAP, compared with a hypothetical situation in which the national screening programme standard of antenatal testing prevailed. The French investigators assessed the diagnostic performance of both the GeneXpert test and microbiological screening at 34–38 weeks’ gestation compared with a reference standard of microbiological culture of intrapartum swabs. IAP was directed during the study by the rapid test, and the adequacy of this strategy compared with what hypothetically would have been followed by use of the 35–37 weeks’ gestation culture result. The authors31 concluded that the universal rapid intrapartum test would correspond to an absolute risk reduction of 0.925%, equivalent to 108 more women needing to be tested in labour and provided IAP to prevent a single case of early-onset GBS infection that would be missed by the culture-based testing. 31

Acceptability and implementation of testing for maternal group B Streptococcus colonisation

Even if the trial shows the superiority of one test over the other, the likelihood of successful implementation will depend on the acceptability of the test for childbearing women and for maternity care professionals. The previous UK GBS rapid testing study24 did examine the acceptability of the swabbing process and the information provided, but in a hypothetical situation in which the results were not being used to direct care. Although study participation was less than half of those approached, among the women who did agree to participate, there was a high level of satisfaction with the process, with 80.5% of women satisfied or very satisfied with the information provided, 94.3% of women happy or very happy with the way the swabs were taken and 94.1% of women confident in its use in routine care. The NSC consider evidence that the criterion, namely that any test should be acceptable to the target population, has been met as uncertain. 24

Cost-effectiveness of group B Streptococcus testing for maternal group B Streptococcus colonisation

At the time of the initiation of the GBS2 trial, the most relevant cost-effectiveness evidence was a 2010 decision model, which suggested that a policy of routine culture testing at 35–37 weeks was considered the most cost-effective once routine untargeted IAP was eliminated as a potential strategy. 32 Intrapartum testing using first-generation PCR tests was not cost-effective unless the average cost of testing was reduced from £29.95 to £7.00. The 2017 NSC review did not identify any new cost-effectiveness estimates relevant to testing in a UK setting. 4 Economic models do not yet have the data to incorporate aspects such as the effect of widespread use of antibiotics on the development of antibiotic resistance and the impact this will have, the impact of maternal and neonatal microbiomes, and the effect of very rare but potentially catastrophic anaphylaxis in labour.

Prevalence of antibiotic resistant group B Streptococcus

Penicillin remains the first choice of antibiotic for GBS prophylaxis and treatment. 44–47 However, since 2008, strains with reduced susceptibility have emerged. Studies in Japan have detected alarming rates of resistance, with 14.7% of GBS isolates testing resistant to penicillin, a marked increase from 2.3% in 2005–6, albeit from a range of samples and populations and not vaginal/rectal swabs from pregnant women. 48 Although resistance to penicillin appears to be low outside Japan, resistance to the secondary antibiotics of choice, clindamycin and erythromycin, is high, and so it is possible that these will not forever be available as an alternative for penicillin-allergic women. Clindamycin was removed as a recommended alternative to benzylpenicillin in the 2017 RCOG guidelines, as the resistance rate in the UK is reported as 16%. 14 Other research highlights substantial risks of adverse consequences arising from exposure of the fetus and newborn infant to unnecessary antibiotics, including necrotising enterocolitis, inflammatory bowel disease, fungal infection49 and cerebral palsy. 50

Cluster randomised controlled trial

The GBS2 trial was designed as a cluster RCT that was randomised at the level of the maternity unit. Sites were randomised to the rapid test strategy (i.e. IAP administration based on the rapid test results) or to usual care (i.e. the standard risk-based screening strategy and IAP offered to all women with risk factors).

Nested test accuracy study

To establish the real-time accuracy of the Cepheid GeneXpert system rapid POC test for GBS colonisation in women presenting to a labour ward with risk factors for early-onset GBS infection, the results of the rapid test were compared with the reference standard of selective enrichment culture in a prospective cohort study. The results of the rapid test preceded those of the culture test and interpretation of the reference standard was performed blind to the rapid test.

Process evaluation in rapid test sites

In the units that were allocated to rapid testing in the cluster randomised trial, we undertook an observational study to determine in real time the timings between rapid test results and commencement of IAP, and to identify if there were any failures in producing results once the tests were initiated on the machine.

Economic evaluation

The economic analysis is based on primary clinical data collected in the cluster RCT, adopting the UK NHS perspective. Full methods of the health economics analysis are described in Chapter 7.

Microbiological substudy

A substudy embedded in the GBS2 trial estimated the relatedness of certain bacterial species that are of public health concern that may colonise the mother during the birth of her baby and her child when they reach 6 weeks of age. The substudy also aimed to detect the antibiotic susceptibility of these strains. This substudy took place in participating units in London and the south-east of England (LSE) that were randomised to receive a rapid test system. The methods are described in Chapter 8.

Eligibility of centres

Maternity units were eligible to participate in the GBS2 trial if they were prepared to accept a policy of rapid test-directed IAP administration to all women with risk factors for the duration of the trial period when allocated to the rapid test strategy. Maternity units also had to be prepared to include all preterm labours considered as high risk, irrespective of the implementation date of the RCOG’s guidelines and its current local policy. The trusts hosting the maternity units were also required to have access to microbiology facilities that were able to perform a selective enrichment bacteriological culture to detect GBS.

Randomisation

Randomisation of clusters was performed at the Birmingham Clinical Trials Unit using a minimisation algorithm programed in a Microsoft Excel^® spreadsheet (Microsoft Corporation, Redmond, WA, USA) incorporating the following factors:

• region [the Midlands (MID) or LSE]

• pre-trial IAP rate (above or below the median)

• the number of vaginal or emergency caesarean births (above or below the median).

The pre-trial IAP rates and the birth rates over a period of 12 months were determined from all potential recruiting units prior to randomisation. This estimate provided the trial with the size of the population eligible to be tested, and served as a minimisation variable and as the denominator for the IAP use calculation. Antibiotic data were derived from interrogation of hospital records and pharmacy prescribing databases at the level of the maternity unit, not at the individual level. Using the standard dosing regimen of antibiotics, the size of the eligible population and the assumptions regarding the average duration of antibiotic administration, we estimated the number of women who received IAP prior to the trial. The median value of the eligible population sizes and pre-trial IAP rates were calculated for the first 20 maternity units that were intending to participate in the GBS2 trial, and these were used as thresholds for dichotomising the minimisation variable data.

Blinding

Owing to the different study procedures under the two testing strategies, it was not possible to blind maternity unit staff to their allocation. A summary of the blinding status along the trial pathway is shown in Figure 3.

FIGURE 3.

Cluster randomised trial blinding timeline for the GBS2 trial. EOGBS, early-onset group B Streptococcus (disease/infection). This figure was produced using the Timeline Cluster Tool. 53

Setting

The cluster was defined as the maternity unit. Women were identified and screened for eligibility by clinical midwives and doctors in various locations, including the delivery suite, the maternity triage unit and the induction ward. No research-specific consent was obtained, although women in the rapid test units provided verbal clinical assent to have the vaginal/rectal swab.

Usual-care units (risk factor-based provision of intrapartum antibiotic prophylaxis)

The study procedure for usual-care sites, in which IAP is offered to women with clinical risk factors, is outlined in Figure 5. All women with risk factors for early-onset GBS infection, whether initially apparent or emerging, were considered eligible for the study.

FIGURE 5.

Study procedures for usual practice maternity units.

Rapid test strategy

The units that were randomised to the rapid test received a GeneXpert^® Dx IV GBS rapid testing system (Cepheid), which was installed and commissioned by a field specialist from the manufacturers. Each unit allocated to the rapid test screening policy was required to house the GeneXpert machine and computer centrally in the unit and have it operational at all times. Units were provided with a sufficient supply of Xpert GBS test cartridges (Cepheid) and double-headed swabs in transport tubes containing Stuart transport medium. In units participating in the substudy, additional single-headed swabs were also supplied. The use of the rapid test for screening was restricted to women who presented to the labour ward and who were eligible according to the participant eligibility criteria. A summary of the pathway for rapid test units is shown in Figure 6.

FIGURE 6.

Study procedures for rapid test maternity units. a, Failed tests include, but are not limited to, machine failure and failure to process swab in required time frame; and b, unless otherwise clinically indicated. This was at the discretion of local clinical care.

Obtaining a vaginal/rectal swab

Swabs were taken using a double-headed swab. Depending on the stage of labour, the swabs were obtained by either the woman herself or a suitably trained member of the woman’s care team. This could be on admission to the labour or induction ward before a vaginal examination was performed, or after a risk factor was detected (e.g. when maternal fever was observed). Swabs were first taken by gently rotating the swabs across the mucosa of the lower vagina. The same swab was then used to take a sample from the rectum, inserting the swab through the anal sphincter then gently rotating. The shafts of the double-headed swabs were then separated carefully. One was returned to the transport tube and sent to the local microbiology laboratory for selective enrichment culture to detect GBS (see Enrichment culture method) and the other was immediately used for the rapid test. For sites taking part in the microbiological substudy, and for participants who had consented to take part, an additional single-headed swab was taken and sent to the substudy laboratory (see Chapter 8).

In practice, a number of substances containing antimicrobial compounds administered vaginally may interfere with the results of the rapid test. The use of lubricant that contains antimicrobial substances, such as K-Y Jelly (Reckitt Benckiser, Slough, UK), for vaginal examination or for taking swabs was discouraged. It was suggested that sterile non-bacteriostatic fluid (e.g. sterile water or saline) was used when lubrication was necessary. Other procedures that may have involved antimicrobial substances include placing a pessary to induce labour, or chlorhexidine to cleanse the perineum or as a vaginal douche (although this is not currently indicated in NICE guidelines). 54 Nevertheless, as the GBS2 trial aimed to establish the use of a rapid test in a real-world setting and, indeed, vaginal examinations are usually undertaken to establish labour, women for whom antimicrobial substances were used were not excluded from the trial. Instead, the use of any antimicrobial substances after admission to the maternity and prior to the swab being taken was recorded.

Delayed labour

If more than 48 hours had elapsed since the test result became available and the woman had yet to give birth, the test result was regarded as invalid. In this situation, it was advised that the woman should be re-swabbed and the presence of GBS tested for again using both the rapid test and the microbiological techniques.

Conducting the rapid intrapartum test

After sampling, one swab was used to inoculate a test cartridge. Should the inoculated cartridge not have been loaded into the rapid test system and the test commenced within 15 minutes of the swab being introduced into the cartridge, the test was be deemed to have failed. The NHS or hospital number was entered into the GeneXpert software alongside the woman’s unique trial number. This trial number was identical on each item of study material associated with one woman and her child (or children). The test was conducted as per the operator manual. 55 It takes, on average, 35 minutes to get a result if GBS is present, 55 minutes to confirm if there is no GBS present and an error message is presented if the test has failed. Used test cartridges and swabs were disposed of in accordance with local policies for clinical waste. Instructions provided to sites for conducting the rapid test are available on request from the manufacturer.

Enrichment culture method

The second vaginal/rectal swab was sent to the local microbiology department where it was used to inoculate a selective enrichment medium prior to plating to detect the presence of any GBS, as per the current Public Health England recommendations. 56 Results from the microbiological cultures were returned to the care team using the usual reporting pathways and were recorded in the woman’s notes. Swabs, sent to the microbiology laboratory for the determination of the GBS colonisation status, were inoculated into Todd–Hewitt broth for overnight enrichment at 37 °C. This enriched broth was subcultured on chromogenic GBS agar and incubated aerobically overnight at 37 °C. GBS was identified by the presence of pink/red colonies. Microbiological data were transcribed to the GBS2 trial database by either a member of the microbiology department or a local research nurse. When manual data entry was required, the data entry screen did not allow review of the rapid test results by those outside the study office, therefore reducing the risk of review bias.

Neonatal ear canal swab

For eligible women in rapid test units, a single swab was taken from the baby’s ear canal as soon as convenient after birth. This swab was put into a transport tube, labelled with a numbered sticker and sent to microbiology for culture using the hospital’s usual request system to detect the presence of GBS, as per the mother’s swab (see Enrichment culture method). The neonatal swab was not used in the rapid test machine.

Antibiotic regimen

Subsequent clinical management of mother and baby was the responsibility of the local health-care team and was not directed by the GBS2 trial. Recommendations for appropriate antibiotic care are included in the NICE guidelines57 and RCOG’s Green-top GBS guidelines. 4

Primary outcomes

• The proportion of women who received only IAP for GBS prophylaxis, of all women identified with one or more risk factors for neonatal GBS infection.

• Measures of test accuracy (i.e. the sensitivity and specificity of the GeneXpert GBS rapid test).

The indication for any intrapartum antibiotic administration was ascertained by making available the following options: GBS prophylaxis (in usual-care units only), a positive rapid test and a failed rapid test (both in rapid test units), pyrexia during labour, a caesarean birth, maternal request and other reason (to be specified). Multiple reasons could be recorded.

Cluster randomised trial

Test accuracy secondary outcomes

Maternal and neonatal colonisation rates, and the mother-to-baby transmission rate.

Process outcomes

A number of process outcomes were also evaluated, for the rapid test units only, including the:

• duration between a positive test becoming available on the GeneXpert machine and the time the result is collected by a midwife, and the duration between that point and the start of IAP

• proportion of the cartridges on which the tests were not commenced within 15 minutes of inoculation, which is defined as an invalid test

• proportion of tests initiated on the Cepheid GeneXpert machine that failed to produce a result within 55 minutes, which is defined as a failed test, or were reported as failed by the system.

Serious adverse event outcomes

Serious adverse events are expected to be extremely rare. There may be significant consequences of the failure to offer IAP to a woman with GBS risk factors and to women identified by rapid test as being colonised with GBS in terms of the increased risk of their baby developing early-onset GBS infection. Conversely, there is a risk of overtreatment if IAP is administered to women without risk factors or with a negative rapid test. However, these instances were considered outcomes of interest within the study, and not adverse events. The risk-based approach involves the noting of historical risk factors and the monitoring of women for emerging risk factors, such as chorioamnionitis. This presents no risk to the women other than a failure to identify and act on these risk factors. The rapid test requires a vaginal/rectal swab to be collected during labour, which is benign and presents no foreseeable risk of harm. As with almost any diagnostic test, there was a risk that testers may suffer an inoculation injury (most likely mucous membrane exposure) with clinical material. A proportion of women will receive antibiotics, particularly benzylpenicillin for IAP, which carry a very small risk of anaphylaxis. 58

The outcomes of economic evaluation and for microbiological substudy are provided in Chapters 7 and 8.

Statistical methods

For baseline characteristics, categorical data are summarised by frequencies and percentages. Continuous data are summarised by the number of responses, mean and standard deviation (SD) if deemed to be normally distributed, and number of responses, median and interquartile range (IQR) if data appear skewed. Tests of statistical significance were not undertaken. In accordance with the Consolidated Standards of Reporting Trials (CONSORT) reporting guideline, both absolute and relative measures of treatment effects are reported, and so for the primary and secondary outcomes in the cluster randomised trial component both risk differences (RDs) and relative risks (RDs) are presented where possible. Estimates of differences between groups are presented with 95% two-sided CIs. p-values are reported from two-sided tests at the 5% significance level and no corrections are made for multiple testing.

In the primary analysis for the cluster randomised trial, a mixed-effects binomial regression with a log-link was used to estimate the RR, and a binomial model with identity link was used to estimate the RD. In the case of non-convergence of the binomial model with a log-link, a Poisson model with robust standard errors was fitted. If the binomial model with the identity link did not converge, then only a RR was reported. Both models allowed for clustering by maternity unit as a random effect. To correct the potential inflation of the type I error rate due to small number of clusters, the Kenward and Roger method59 was used.

Comparative estimates of differences between groups were adjusted for the hospital and the minimisation variables of region (i.e. LSE or MID), baseline IAP rate (i.e. equal to or greater than the median or less than the median) and the number of vaginal or emergency caesarean births (i.e. equal to or greater than the median or less than the median). These effects were all be assumed to be fixed in the regression models used. A secondary analysis for the primary outcome additionally adjusted for maternal temperature of ≥ 38 °C observed while in labour, any previous baby with GBS disease, GBS bacterium detected in current pregnancy, and woman in suspected/diagnosed/established preterm labour. When covariate adjustment was not practical, unadjusted estimates were produced and it was made clear in the output why this occurred (e.g. not possible because of low event rate, lack of model convergence or poor recording accuracy of covariates). Multiple imputation analysis, accounting for clustering, was planned if the proportion of observations with missing complete information on these covariates was > 5%.

Prespecified subgroup analyses were limited to the primary outcome. The following subgroup effects were investigated:

• maternal temperature of ≥ 38 °C observed while in labour

• previous baby with GBS disease

• GBS bacterium detected in current pregnancy

• preterm labour (< 37 weeks’ gestation) with intact membranes or rupture of membranes of any duration, whether suspected, diagnosed or established.

Treatment effects were summarised within each subgroup separately, and an interaction test was performed between each subgroup variable and the received allocation.

The diagnostic accuracy of the rapid test was estimated through the standard calculations of sensitivity and specificity. Estimates are presented with 95% CIs calculated using binomial exact methods. 60 We also undertook a binomial proportions test to compare the observed sensitivity with the hypothesised minimal performance value of 90%.

Sample size for the cluster randomised trial

The focus of sample size calculation was on the effectiveness of the rapid test. We aimed to recruit a minimum of eight clusters per randomisation group (i.e. a total of 16 maternity units). This was a realistic recruitment goal and was deemed sufficient to allow estimation of model parameters. We estimated that the sample size per cluster would be approximately 83 (this is informed by routinely collected data on number of eligible women giving birth). This equated to a total sample size of approximately 664 per strategy group to allow recruitment of the required number of individuals for the test accuracy study. As there were two strategy groups (i.e. usual care and rapid test), each with at least 664 participants, it was expected to provide a sample size of 1328 women. This was rounded up to a target sample size of 1340 women. To allow for dropouts at the level of the cluster, anticipating implementation issues in maternity units, the number of clusters was increased from 16 to 20.

The proportion of women receiving antibiotics to prevent vertical GBS transmission (i.e. the primary outcome in the cluster RCT) was expected to be in the region of 50–75% (based on the previous study51 and assuming better compliance with RCOG guidelines since that study). This primary outcome was a process outcome and so the within-cluster correlation of this outcome [the intracluster coefficient (ICC)] was expected to be higher than it would be for a clinical outcome. We therefore considered sensitivity of our calculations to a range of proportions in the usual-care units and a range of ICC values, which we believe to be quite conservative. All of our calculations allowed for 90% power and 5% significance. We did not acknowledge varying cluster sizes in our calculations, as we considered the coefficient of variation of unit birth sizes to be small (approximately 0.21 based on annual birth rates for 2013) and could be altered by varying the study duration at maternity units. However, given our conservative assumptions, and that the impact of the testing strategies on the primary outcome was expected to be large (i.e. a priori we expect the effect size to be large), we expected the impact of any varying cluster sizes to be minimal. For a range of values for the important parameters, as described above, we worked out the detectable differences and equivalent relative RR (Table 1).

This means that this trial would have around 90% power to detect a reduction in proportion of women prescribed antibiotics to prevent GBS transmission from 75% to 63% (i.e. a RR of about 20%) for a low value of the ICC to a reduction from 75% to 38% for a very conservative value of the ICC (0.2), equating to a RR reduction of 50%.

Sample size for the test accuracy study

The sample size of the test accuracy study was dependent on the sensitivity of the rapid test. For the test to be proven useful, we needed to show that it would detect a higher proportion of GBS colonisation than other tests, but not at the cost of low specificity and/or unnecessary administration of IAP. Results from the Group B Streptococcus 1 (GBS1) study51 suggested that the most cost-effective test (if untargeted universal IAP was excluded) was antenatal culture for GBS at 35–37 weeks’ gestation. In the GBS1 study51 the sensitivity of this test was 75.8% (95% CI 47.2% to 91.5%). Therefore, if we could prove that the sensitivity of rapid test was > 90% then the results of the GBS2 study51 would be convincing. This was a stringent test and a lower threshold might also be adequate. We did not compare the rapid test with antenatal GBS culture testing within the study, but compared with this result from external literature. Therefore, we undertook a ‘one sample, sample size’ computation comparing against a fixed value.

We had data from a systematic review on the performance of the GeneXpert GBS test (J Daniels, personal communication). From the meta-analysis of nine studies, the pooled accuracy of the test was estimated, giving a sensitivity of 96.4% (95% CI 90.8% to 98.6%) and specificity of 98.9% (95% CI 97.5% to 99.5%). Sample size calculations are therefore based on showing that a test with sensitivity of 96.4% is greater than a fixed value of 90%. With a power of 90% to demonstrate this sensitivity, 167 cases of maternal GBS colonisation are required (or 136 at 80% power).

A sample size of 676 women would provide 90% chance of us accruing enough GBS-colonised women to have 90% power to show the sensitivity of the rapid intrapartum test to be statistically significantly (with p < 0.05) > 90% should the meta-analytical estimate of its performance (96.4%) be correct, while allowing for 10% loss from failed tests, based on the GBS prevalence observed in the GBS1 study,51 which was 29.8% (89/299, 95% CI 24.6% to 35.2%). Of the 606 participants with data, we would expect 167 to be GBS carriers and 439 to be negative for GBS colonisation. If the prevalence of GBS colonisation was actually at the lower end of the 95% CI from GBS1 study,51 namely 24.6%, then a total of 673 women would give a 90% chance of observing 136 cases of GBS colonisation, including 10% lost tests. The 95% CIs we would observe on sensitivities and specificities of 85%, 90%, 95% and 98% with a sample size of 676 women (606 women with data) are shown in Table 2, and these have adequate precision (sensitivity within 10% and specificity within 6%) for modelling.

Maternity sites

Following randomisation, the maternity units were balanced according to the minimisation characteristics (Table 5).

Participants

Overall, 1687 women were identified and added to the trial database during the recruitment window for the 20 randomised sites. There were 52 women from two usual-care maternity units who were added to the database but who were ineligible for the study, as the database eligibility criteria initially did not reflect the protocol. A further three women in each group who, on closer examination, were added to the database in error, as they were not eligible and one woman’s record was duplicated. These women’s data were excluded from the analysis, leaving 1628 women in the data set.

The overall numbers of women recruited were 722 in rapid test sites and 906 in usual-care sites. There were 67 pairs of twins and three trios of triplets and, therefore, the number of babies available for assessment was 749 in the rapid test group and 951 in the usual-care units (Figure 7). Similar proportions of women in the two study groups were recruited from the two regions, and the women were similar in their parity and type of birth. A higher proportion of women in the rapid test sites entered labour after being induced than in the usual-care units (Table 6).

FIGURE 7.

The CONSORT flow diagram of sites and participants in the GBS2 study.

In 93% of women there was one risk factor for neonatal GBS disease, and two risk factors in 6–7% of women. The most common risk factor was preterm labour. The distribution of risk factors among the study population is shown in Tables 7 and 8.

Among those women with only one risk factor, more women in rapid test units had GBS detected in the current pregnancy (41%, 293/722) than those in the usual-care units (31%, 278/906). In addition, 15% (139/906) of women in usual-care units had a raised maternal temperature compared with 8% (55/722) of women in rapid test units. GBS was detected in the vaginal or rectal swab commonly in both groups, followed by diagnosis in the midstream sample (Table 9).

Neonatal antibiotics exposure

The rates of neonatal exposure to antibiotics were lowered by 29% (RR 0.71, 95% CI 0.54 to 0.95) in the rapid test group compared with the usual-care group (Table 15). This included all babies who started antibiotics within the first 7 days of life, regardless of maternal antibiotic exposure or indication for neonatal antibiotic prescription. The number of babies for whom suspected early-onset sepsis was the indication for antibiotics was lower in the rapid test units (25%) than in the usual-care units (39%) (RR 0.63, 95% CI 0.43 to 0.92; p = 0.02).

The predominant reason stated for administration of neonatal antibiotics was suspected early-onset infection, which was the stated reason in one-third of all babies (Table 16).

In most of the babies with suspected early-onset infection who were commenced on antibiotics, following further tests, such as microbiological culture of blood or cerebrospinal fluid, the decision was made not to administer the full course of antibiotics (Tables 17–19). There were 11 reports of GBS infection among 561 babies who received antibiotics, equivalent to a rate of 6.5 per 1000 live births. Of these 11 babies, all of their mothers (n = 11) had received IAP. Two babies died before discharge, both in the usual-care group, and one death was judged to be because of GBS (the other death was due to E. coli meningitis). Three of these 11 babies were in the rapid test group and the mothers of all three returned a positive rapid test result. All three of these babies returned an ear canal swab, but only one baby was positive for GBS. There were three perinatal deaths in the rapid test units and eight in the usual-care units, which included one and four stillbirths, respectively.

Subgroup and sensitivity analyses

Performing subgroup analyses for each of the four risk factors indicated that there was no evidence of any interaction between the subgroup category and the comparison of the primary outcome, nor any significant difference between the strategy groups for any subgroup (see Table 17).

Economic analysis

All statistical analyses were conducted using Stata^® version 15 (StataCorp LP, College Station, TX, USA). Initial analyses included conducting a cost–consequence analysis, detailing all costs and outcomes incurred in each trial strategy in a disaggregated form. This analysis was based on an intention-to-treat basis, meaning that within the analysis all women were analysed according to their maternity unit’s allocated testing strategy, regardless of whether the individual adhered to the site’s testing strategy or IAP was administered appropriately. 68

The total costs incurred by each woman during labour were calculated by multiplying the resource use volume by the unit costs. Moreover, mean costs and outcomes were generated for each trial testing strategy. The principal outcome on which the economic evaluation was based is the number of instances of IAP avoided. The timeliness of the test, in terms of the rapid test producing a result in time to administer intrapartum antibiotics before labour begins, was not factored in, as the cost of the rapid test is incurred regardless of whether or not the result was presented in time to be acted on. An incremental analysis was conducted if appropriately indicated by the cost–consequence analysis. An incremental analysis is estimated by comparing the differences in mean costs and outcomes between the two strategies using seemingly unrelated regression estimates. By dividing the cost differences by the outcome differences, the incremental cost-effectiveness ratio (ICER) is calculated. To account for the inherent skewness of the cost data, 95% CIs around the mean differences were calculated using the bias-corrected and accelerated bootstrap method. 69

Stochastic cost-effectiveness analysis

A stochastic CEA was conducted to examine the sensitivity of the results. The approach taken in the probabilistic sensitivity analysis was that all important variables relating to costs and clinical outcomes were given a distribution that describes the uncertainty surrounding the mean. The distributions were simulated 10,000 times. Each time, random numbers were drawn from the appropriate distributions. After each simulation, the incremental costs and effects were plotted in a cost-effectiveness plane that comprised four quadrants: (1) north-east, (2) north-west, (3) south-east and (4) south-west. The scatterplot that was produced represented the simulations. If dots from the scatterplot were in the north-east quadrant, then this indicated that the addition of the rapid test was more costly and more effective than usual care, based on risk factors. Dots in the north-west quadrant indicated that the rapid test was more costly and less effective than the usual care. Based on these simulations, the probability that the rapid test strategy would be cost-effective was presented. This was the standard approach for health economics following accepted guidelines (i.e. CHEERS)70 and was a presentation of results of cost-effectiveness studies that would be required by decision-makers, such as NICE.

Results were also presented using cost-effectiveness acceptability curves (CEACs) to reflect sampling variation and uncertainties in the cost-effectiveness value, when appropriate. The CEACs show the probability of the rapid test being cost-effective, compared with usual care, based on risk factors and observed data for a range of maximum monetary values (thresholds) that decision-makers might be willing to pay for a particular unit change in outcome. 71 We examine cost-effectiveness across a range of monetary willingness-to-pay (WTP) thresholds.

Additional sensitivity analyses were carried out:

• Sensitivity analysis 3 – the proportion of midwife time to conduct the rapid test was reduced from 40 minutes in the base case to 10 minutes.

• Sensitivity analysis 4 – only the women for whom IAP was given specifically for GBS and no other reason were analysed.

Rapid intrapartum test

A total of 721 women gave birth in units randomised to the rapid test (one woman delivered elsewhere) and 906 women gave birth in usual-care units. Almost all women in the rapid test arm had at least one swab taken and reported (for three women it is unknown whether or not the swab was taken and no test results are available). However, more tests were issued within the rapid test strategy, depending on the length of labour and delivery stage. The data identified 26 women who had a repeat test performed because either the test was not started within 15 minutes of taking the swab or labour had not progressed within 48 hours of initial admission.

Risk factors

Of the total population, the most common risk factor was preterm labour, identified in 757 (46%) women. This was followed by a history of temperatures of ≥ 38 °C during labour, identified in 662 (41%) women (see Table 8). It should be noted that, for this analysis, any costs associated in assessing risk factors were assumed to be included within the ‘usual’ birth costs.

Vaginal deliveries

Vaginal deliveries were grouped into four categories: (1) spontaneous vaginal delivery following spontaneous onset, (2) vaginal delivery following induced labour, (3) instrumental delivery following spontaneous onset of labour and (4) instrumental delivery following induction of labour. These categories are based on the trial data set and the available cost data for these types of birth in the NHS reference costs. 64 Across both strategy groups, 584 (48.1%) women had spontaneous deliveries, 397 (32.7%) had a vaginal birth with induced labour, 124 (10.2%) had an assisted vaginal birth and 109 (9.0%) had an assisted and induced birth.

Caesarean deliveries

Elective caesareans were not included in this analysis, as they were part of the exclusion criteria. Women who gave birth by emergency caesarean section were included. In total, 406 (25.0%) women underwent an emergency caesarean section.

Resource use

The following resource use and costs were reported by category. The estimation of resource use and associated cost was carried out on complete-case data.

Average costs

To calculate the mean costs for each testing strategy, mean resource use was multiplied by the relevant unit cost. The mean cost varied between strategies for individual antibiotics (see Appendix 3,Table 34), but the overall the mean of the total antibiotic cost was higher in the rapid test arm.

Cost–consequences analysis

Table 29 presents the costs and outcomes assessed within the GBS2 trial. In the rapid test strategy, the costs included the following: the mean cost of IAP, the mean costs of birth/deliveries and the mean cost of issuing the rapid test. The mean cost of issuing the rapid test included the additional midwifery time per woman, the costs of vaginal/rectal swabs, the costs of a cartridge for the GeneXpert machine and the machine running time. By contrast, the costs of the usual-care strategy comprised the mean cost of IAP and the mean costs of the baby being born. The assumption was made that assessing for risk factors is an integral component of the overall cost of the baby being born. The mean cost per woman in the rapid testing strategy was approximately £4128, whereas under usual care the mean cost was approximately £4003 per woman.

Outcomes

The primary outcome of the trial was the proportion of women for whom IAP was used as prophylaxis against GBS infection in her baby. In the rapid test sites, IAP for GBS prophylaxis was administered to 297 women [proportion 0.41 (297/722)], compared with 328 women in the usual-care units [proportion 0.36 (328/906)]. A higher proportion of women in the rapid test units received IAP to prevent neonatal GBS disease, but there was no statistically significant difference from those in the usual-care units (see Table 12).

Base-case analysis

The results in Table 29 show that the mean cost per woman in the rapid test units was approximately £4128 and the mean proportion of women treated with antibiotics was 0.41. This compared with the mean cost per woman in usual-care units being approximately £4003 and a mean proportion of women treated with antibiotics of 0.36. Therefore, to achieve the outcome of averting antibiotic use, the rapid test was more costly and less effective than usual care, as it cost more and antibiotics use to prevent GBS infection was greater. The use of the rapid test in this analysis was said to be dominated by usual care. Therefore, producing an ICER is not applicable for the base case, as the cost–consequence analyses revealed the situation of dominance.

The cost-effectiveness plane for the base-case analysis is presented in Figure 9. The majority of the scatterplot dots (depicting paired incremental costs and outcomes) are in the north-west and south-west quadrants. Scatterplot dots falling in the north-west quadrant represent higher costs and worse outcome than the comparator, whereas scatterplot dots in the south-west quadrant represent worse outcome and lower costs. Therefore, Figure 9 suggests that the rapid test is less effective at reducing the use of IAP. However, it is uncertain whether the rapid test is likely to be more costly (north-west quadrant) or less costly (south-west quadrant) compared with usual care (represented by the origin). The CEAC (Figure 10) shows the probability of the rapid test being cost-effective at various values of decision-makers’ WTP per case of antibiotic avoided. The CEAC (see Figure 10) suggests that the probability of the rapid test being cost-effective is > 40% for all WTP values, tending to 0% as the WTP increases. Therefore, the probability of cost-effectiveness decreases as the WTP increases.

FIGURE 9.

Cost-effectiveness plane: the base case.

FIGURE 10.

Cost-effectiveness acceptability curve: the base case.

Sensitivity analyses were carried out:

• Sensitivity analysis 1 – the length of stay for the mother was based on trial data, and the mode of birth (which includes costs of an indicative length of stay that was used in the base case) was removed from the analyses for both trial testing strategy groups.

• Sensitivity analysis 2 – as for sensitivity analysis 1, but the costs of inpatient stay for newborns were added to the costs for both trial testing strategy groups based on the trial data.

The descriptive statistics for inpatient days for the mother and the babies on which these two sensitivity analyses are based are presented in the Appendix 3, Tables 35 and 36, respectively.

Sensitivity analysis 1

The mean length of stay and mean costs per strategy are presented in Appendix 3, Table 35. The cost-effectiveness plane for sensitivity analysis 1 is presented in Figure 11. The majority of the scatterplot dots now resided in the north-west quadrant and there were fewer in the south-west quadrant. This suggested that the true length of stay, based on the length of stay recorded in the trial, was actually more costly than the reference cost data implied. The test remained ineffective, but some uncertainty around the cost data was reduced, as the rapid test strategy was shown to be more costly than usual care. Figure 11 broadly supported the base-case analysis and showed that the rapid test strategy was more costly and less effective than usual care. The CEAC (Figure 12) was similar to that of the base case.

FIGURE 11.

Cost-effectiveness plane: mothers’ length of stay (sensitivity analysis 1).

FIGURE 12.

Cost-effectiveness acceptability curve: mothers’ length of stay (sensitivity analysis 1).

Sensitivity analysis 2

This was similar to sensitivity analysis 1, but the costs of inpatient stay for newborns by their level of care were added to the costs in both strategy groups of the trial, based on the trial data. When needed, assumptions were required as indicated in the Appendix 3, Table 36.

The cost-effectiveness plane for sensitivity analysis 2 is presented in Figure 13. The majority of the scatterplot dots have now shifted to the south-west quadrant, showing that although, overall, more antibiotics were prescribed in the rapid test units than in the usual-care units, the overall costs were now less for the rapid test strategy. This meant that the length of stay of babies and their corresponding costs were higher in the usual-care units. The implication was that these babies required more care than the babies in the rapid tests units, whose mothers had received IAP. Therefore, Figure 13 differed from the base-case analysis and suggested that the addition of the babies’ length of stay changes the costs of the rapid test strategy to be less costly than usual care overall. The CEAC (Figure 14) showed that the probability of being cost-effective declined as the WTP increased, primarily because more antibiotics were prescribed in the rapid test group and so the rapid test had failed to meet the objective of reducing the prescribing of antibiotics, despite being slightly cheaper overall.

FIGURE 13.

Cost-effectiveness plane: mothers’ and babies’ length of stay (sensitivity analysis 2).

FIGURE 14.

Cost-effectiveness acceptability curve: mothers’ and babies’ length of stay (sensitivity analysis 2).

Additional probability sensitivity analyses were also conducted. Sensitivity analysis 3 reduced the length of midwife time in the rapid test strategy from 40 minutes to 10 minutes and showed no substantial change on the base case. Sensitivity analysis 4 was the same as the base case, but woman who were prescribed GBS prophylaxis only and not antibiotics for any other reason were included. No substantial change in the base case was observed, with the cost-effectiveness plane focusing on the origin.

Additional microbiological assessment of the third vaginal/rectal swab

The third swab was sent to the microbiology laboratory at Barts Health NHS Trust and stored in a Brain Heart Infusion Broth (Oxoid Ltd, Basingstoke, UK) with 10% glycerol cryopreservative broth at –80 °C. This swab was processed further only if notification of written consent for substudy participation had been obtained from the mother. The third swab was held for a period of 96 hours in the microbiology department of Barts Health NHS Trust to allow the woman to receive an invitation to participate in research and give informed consent. Should a woman have declined to provide consent, or if 96 hours had passed since the receipt of the third swab, then this third swab was not processed but instead was disposed of in a suitable manner compliant with local policies.

Obtaining a faecal sample from the infant

Once a period of 5 weeks had elapsed and following notification from the trial office that consent had been given, the substudy research assistant supplied the maternity unit with a faecal sample pot labelled with the baby’s unique study number. The research assistant then prompted the dispatch of a follow-up sample collection pack to the consenting mother. The research assistant recorded the number on the pot alongside the mother’s trial number. The maternity unit staff or research midwife/practitioner then checked with the community midwifery team that nothing untoward had happened to the child, then the research midwife/practitioner sent out a follow-up sample collection pack. This pack consisted of a faecal sample pot, a covering letter, instructions, a protective transport container and a prepaid addressed envelope to the woman’s home address. The letter requested that a sample of the child’s faecal material be collected from the child’s nappy. The instructions provided to the mother are shown in Report Supplementary Material 1.

When a sample of the baby’s faecal material was not forthcoming after the first request, then another request was sent to the mother at around 9 weeks after her baby was born. No further attempts to obtain a faecal sample were made.

Microbiological testing of infant faecal samples

Methods for the isolation of GBS are described elsewhere in this monograph. Following receipt of the infant faecal sample, the microbiology laboratory at Barts Health NHS trust informed the research assistant, who then recorded this against the woman’s unique trial number. The faecal sample was diluted 1 in 10 into a cryopreservation broth and then mixed by vortexing, before being stored at –80 °C.

Antibiotic resistance testing

The frozen sample was allowed to defrost at room temperature and then plated on to a range of culture media, including chromogenic MRSA agar (Oxoid Ltd), Slanetz–Bartley agar with vancomycin (Oxoid Ltd) and MacConkey agar (Oxoid Ltd). Presumptive isolates were identified using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Antibiotic resistance was tested using European Committee on Antimicrobial Susceptibility Testing methods and break points. 73 Enterobacterales were tested for sensitivity to ampicillin, piperacillin plus tazobactam, amoxicillin plus clavulanate, cefpodoxime, gentamicin, cefuroxime, amikacin, co-trimoxazole, temocillin, ceftazidime, ertapenem and ciprofloxacin on Muller–Hinton agar.

Molecular testing for Gram-negative antibiotic resistance genes

The EasyScreen™ Sample Processing Kit (SP006, Genetic Signatures Ltd, Newtown, NSW, Australia) is designed to rapidly isolate nucleic acids [deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)] from clinical samples via an automated purification system. The system was used to look for antibiotic-resistant genes prevalent in Gram-negative bacteria. Reagents are supplied to convert all nucleic acids into 3base™ sequences (Genetic Signatures Ltd) and to perform a subsequent purification on the GS-mini platform (Genetic Signatures Ltd). The original maternal vaginal swab was removed from the –80 °C freezer and vortexed briefly in the Amies transport medium in which it was stored to release the bacteria. A 1-ml aliquot was removed and added to the GSL-combined reagents 1 and 2. Infant samples were removed from the –80 °C freezer and, after defrosting, 100 µl/ml was added to combined GSL reagents 1 and 2 for processing with the EasyScreen™ Sample Processing Kit (SP006).

The EasyScreen™ Sample Processing Kit lyses any microorganisms present and converts all cytosine bases to uracil (detected as thymine after PCR amplification) to create 3base™ DNA and RNA. The 3base™ nucleic acids have an increased homology compared with the genome that comprised the native four bases. This complexity reduction results in genomes that are more similar to each other, enabling the design of primers and probes that contain fewer mismatches, are more homologous, produce better amplification and, importantly, have less cross-reactivity when multiple strains of the same organism are present.

Extracted DNA was analysed using the EasyScreen™ ESBL/CPO Detection Kit (BL001, Genetic Signatures Ltd), which uses panels of multiplexed real-time PCR assays to provide rapid and accurate detection of 16 β-lactam and carbapenem-resistant pathogen targets. These were TEM, GES, OXA-48(like), MCR-1, VIM, CMY, CTX-M, KPC, OXA-23 (like), DHA, IMI, IMP, NDM, SHV, SME and OXA-51 (like). The kit includes an extraction control in the reaction mix to determine the reliability of the extracted nucleic acids and indicate the presence of any inhibitors after extraction from primary samples. PCR was performed on a Bio-Rad CFX96/384 Touch™ real-time PCR instrument. Reaction mixtures were set up in accordance with the manufacturer’s instructions and C_t values read in accordance with its recommendations.

Statistical methods

A binomial regression model with a log-link was used to estimate RRs. All estimates are reported along with 95% CIs. All analyses were conducted in SAS^® version 9.4 (SAS Institute Inc., Cary, NC, USA). Fisher’s exact test was used to compare proportions and a p-value of < 0.05 was considered statistically significant.

Interpretation of the substudy

Seale and Millar77 reviewed the literature on perinatal transmission of antibiotic resistance from mothers to infants and found a paucity of relevant literature. A more recent systematic review78 reports evidence of carriage of indistinguishable antibiotic-resistant Gram-negative bacteria by mothers and infants, and also concludes that there is a paucity of relevant research and that ‘none of the included studies reported on risk factors for multidrug resistant Gram-negative bacteria transmission from colonised mothers to infants’. There remains little information on the contribution that maternal carriage of resistant bacteria or associated genes makes to carriage by their infants. Recent reports have suggested a weak (if any) association between mode of birth, maternal and infant antibiotic use or feeding practices on the carriage of antibiotic-resistant Gram-negative bacteria by infants. 79,80 Despite collecting data on parental carriage of antibiotic resistance, Hetzer et al. 81 did not include information in the analysis of risk factors for acquisition of antibiotic-resistant intestinal E. coli during the first year of life.

The identification of risk factors for perinatal transmission has the potential to allow the development of preventative strategies. A study published in 2010 reported that, among 114,999 women who gave birth between 1992 and 2007, approximately one-third had received an antibiotic during pregnancy. 82 The extent to which antibiotic prescribing in pregnancy is inappropriate is unknown. Antibiotic stewardship is a potential remediable route to reducing the selection and transmission of antibiotic-resistant bacteria during this critical period of life; however, the extent to which antibiotic usage in pregnancy can be constrained without prejudicing maternal or neonatal outcomes must be determined.

Clinical collaborators

• Dr Shad Hussain (Consultant Neonatologist).

• Professor Khaled Ismail (Consultant in Obstetrics and Gynaecology).

• Ms Sal Higgins (Lead Research Midwife).

• Dr Stephen Kempley (Consultant Neonatologist).

Trial delivery

• Mr Kostas Tryposkiadis (Trial Statistician).

• Ms Alicia Jakeman (Trial Co-ordinator).

• Ms Gemma Slinn (Trials Management Team leader).

• Mr Adrian Wilcockson (Database Developer).

Trial Steering Committee

• Professor Paul Heath (chairperson).

• Professor Kerry Hood.

• Professor Stavros Petrou.

• Professor Ben Stenson.

• Dr Sarah McMullen.

• Professor Julia Sanders.

• Ms Alison Stanley.

Data Monitoring Committee

• Professor Stephen Walters (chairperson).

• Professor Patrick Bossuyt.

• Professor Ruth Gilbert.

• Dr Rhona Hughes.

Principal investigators at participating maternity units

See Appendix 1, Table 32.