Neonatal sepsis

Abstract

The invention relates to methods for identifying neonates at an increased risk of neonatal sepsis by screening for the presence of Group B Streptococcus (GBS) in a sample of placenta. The invention also relates to nucleic acid-based detection methods for detecting Streptococcus agalactiae (Group B Streptococcus, GBS) and to compositions and kits for use in said methods.

Description

[0001] NEONATAL SEPSIS

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The invention relates to methods for identifying neonates at an increased risk of neonatal sepsis. The invention also relates to nucleic acid-based detection methods for detecting Streptococcus agalactiae (Group B Streptococcus, GBS) and to compositions and kits for use in said methods.

[0004] BACKGROUND OF THE INVENTION

[0005] Streptococcus agalactiae (Group B Streptococcus, GBS) is the most common cause of sepsis in the first week of life and this is referred to as early onset disease (EOD). GBS is present in the genital tract in about 20% of women and, in the absence of intervention, about 1% of infants born to a colonized mother develop EOD. An analysis of the global burden of disease in 2020 estimated that about 20 million pregnant women were colonised with GBS, 230,000 infants exhibited EOD, and GBS accounted for around 50,000 stillbirths and 50,000 to 100,000 infant deaths. However, EOD remains uncommon, affecting less than 1 in 1,000 births in high income countries. The incidence is higher in low- and middle-income settings but reliable estimates are problematic due to incomplete ascertainment.

[0006] Among women who are colonised by GBS, the risk of EOD is reduced by more than 80% by the use of intrapartum antibiotic prophylaxis (IAP). Many countries, including the USA, routinely screen pregnant women for GBS using a vaginal and rectal swab and administer IAP to those who screen positive. A substantial minority of countries (40%), including the UK, do not perform universal screening and IAP is only given to women who have a positive culture following a clinically indicated swab. The decision not to screen in the UK is based on multiple facets of the problem, including the fact that detection of genital tract colonisation is temporally variable in a given woman, only a small minority of babies exposed to GBS develop a clinical infection, a very large number of women need to be treated to prevent each case of EOD, and there is an absence of Level 1 evidence that universal screening improves outcomes. In addition, the rates of EOD in countries which do not screen (e.g. the UK) are comparable to other countries where screening with existing screening methods takes place.

[0007] There is an urgent and unmet need for sensitive and specific methods to identify neonates having an increased risk of neonatal sepsis.

[0008] SUMMARY OF THE INVENTION

[0009] The inventors have identified for the first time that placental GBS is associated with an increased risk of neonatal morbidity. The inventors discovered that the presence of placental GBS provides a more accurate predictor of neonatal morbidity than the presence of vaginal and / or rectal GBS. Thus, screening for placental GBS allows neonates who are at an increased risk of neonatal sepsis to be identified and monitored and / or treated accordingly.

[0010] The inventors have also developed nucleic acid-based detection methods which are highly sensitive and specific for GBS. These methods are particularly advantageous given the typically very low cell density of GBS in biological samples, e.g. the placenta.

[0011] The invention provides a method of screening for risk of neonatal sepsis in a neonate comprising screening for Streptococcus agalactiae (Group B Streptococcus, GBS) in a sample of placenta, wherein the presence of GBS in the placenta indicates an increased risk of neonatal sepsis in the neonate.

[0012] In some embodiments, if GBS is detected in the placenta, the method further comprises screening the neonate for signs of infection, optionally wherein screening the neonate for signs of infection comprises one or more of: (a) culture from a blood sample and / or swab sample; (b) full blood count and differential; (c) C-reactive protein assay; (d) lumbar puncture; and (e) radiological tests.

[0013] In some embodiments, the method further comprises testing the level(s) of one or more inflammatory cytokines in the umbilical cord blood, plasma or serum, optionally wherein the one or more inflammatory cytokines comprise interleukin (IL)-ip, IL-6 and / or IL-8.

[0014] In some embodiments, the mother of the neonate has previously tested positive for GBS infection by vaginal / rectal swab.

[0015] In some embodiments, the mother of the neonate tested positive for GBS infection during a previous pregnancy and / or has previously given birth to a neonate who suffered neonatal sepsis.

[0016] In some embodiments, the sample of placenta is obtained after delivery of the neonate.

[0017] In some embodiments, the sample of placenta comprises placental terminal villi.

[0018] In some embodiments, the method comprises screening for GBS nucleic acid in the placenta, optionally wherein GBS nucleic acid is detected by polymerase chain reaction (PCR), optionally quantitative PCR (qPCR). In some embodiments, the method comprises screening for GBS RNA in the placenta, optionally wherein GBS RNA is detected by reverse transcription PCR, optionally reverse transcription quantitative PCR (RT-qPCR). In some embodiments, the nucleic acid is 16S rRNA. The invention also provides a method of treating a neonate identified as being at an increased risk of neonatal sepsis by a method of the invention, the method comprising administering antibiotics to the neonate.

[0019] The invention also provides a composition comprising an antibiotic for use in a method of treating a neonate identified as being at an increased risk of neonatal sepsis by a method of the invention.

[0020] In some embodiments, the neonate exhibits one or more signs of infection and / or sepsis.

[0021] In some embodiments, the antibiotic comprises penicillin and / or gentamicin.

[0022] In some embodiments, the antibiotic is administered to the neonate within 6 hours of birth.

[0023] The invention also provides a method for detecting GBS in a sample, the method comprising: (a) contacting nucleic acid extracted from the sample with reagents for amplifying a DNA fragment of GBS 16S rRNA gene under reaction conditions suitable for amplifying said DNA fragment, wherein the reagents comprise a forward primer having a nucleic acid sequence comprising 5'-ACGGAAGTCTGAC- 3' (SEQ ID NO: 1) and a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2); and (b) detecting the presence of the fragment, wherein presence of the fragment is indicative of the presence of GBS in the sample.

[0024] In some embodiments, reagents for amplifying the DNA fragment comprise: (a) a forward primer having a nucleic acid sequence comprising 5'-AATGGACGGAAGTCTGAC-3' (SEQ ID NO: 3); and / or (b) a reverse primer having a nucleic acid sequence comprising 5'-GTTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 4).

[0025] In some embodiments, the method comprises: (a) contacting nucleic acid extracted from the sample with reagents for amplifying a first DNA fragment of the GBS 16S rRNA gene under reaction conditions suitable for amplifying said first DNA fragment, wherein the reagents for amplifying the first fragment comprise a forward primer having a nucleic acid sequence comprising 5'-GGAGCAATTGCTTCACTGTG- 3' (SEQ ID NO: 13) and a reverse primer having a nucleic acid sequence comprising 5'- CAG l I I CCAAAGCGT-3' (SEQ ID NO: 14); then (b) contacting the nucleic acid with reagents for amplifying a second DNA fragment under reaction conditions suitable for amplifying said second DNA fragment, wherein the second DNA fragment is a portion of the first fragment and wherein the reagents for amplifying the second fragment comprise a forward primer having a nucleic acid sequence comprising 5'-ACGGAAGTCTGAC-3' (SEQ ID NO: 1) and a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2); and (c) detecting the presence of the second fragment, wherein presence of the second fragment is indicative of the presence of GBS in the sample.

[0026] In some embodiments, the reagents for amplifying the first DNA fragment comprise: (i) a forward primer having a nucleic acid sequence comprising 5'-TAAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 15); and / or (ii) a reverse primer having a nucleic acid sequence comprising 5'- TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20). In some embodiments, the reagents for amplifying the second DNA fragment comprise: (i) a forward primer having a nucleic acid sequence comprising 5'- AATGGACGGAAGTCTGAC-3' (SEQ ID NO: 3); and / or (ii) a reverse primer having a nucleic acid sequence comprising 5'-GTTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 4).

[0027] In some embodiments, detecting the presence of the fragment comprises contacting the nucleic acid with a probe configured to hybridize to the fragment and to emit a detectable signal when hybridised to the fragment. In some embodiments, the probe has a nucleic acid sequence comprising 5'- CGTTGGTAG-3' (SEQ ID NO: 25); optionally wherein the probe has a nucleic acid sequence comprising: (a) 5'-AGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 26) (b) 5'-AGAGAAGAACGTTGGTAGGAGTGGA-3' (SEQ ID NO: 27); (c) 5'-AGAGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 28); or (d) 5'- AGAAGAACGTTGGTAGGAGTGGAAA-3' (SEQ ID NO: 29).

[0028] The invention also provides a method of detecting GBS in a sample, the method comprising: (a) contacting nucleic acid extracted from the sample with a probe having a nucleic acid sequence comprising 5'-AGAAGAACGTTGGTAGGAG -3' (SEQ ID NO: 26); and (b) detecting binding of the probe to the nucleic acid, wherein binding of the probe to the nucleic acid is indicative of the presence of GBS in the sample.

[0029] In some embodiments, the probe has a nucleic acid sequence comprising: (i) 5'- AGAGAAGAACGTTGGTAGGAGTGGA-3' (SEQ ID NO: 27); (ii) 5'-AGAGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 28); or (ill) 5'-AGAAGAACGTTGGTAGGAGTGGAAA-3' (SEQ ID NO: 29).

[0030] In some embodiments, the method comprises a step of contacting the nucleic acid with reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene (SEQ ID NO: 30) under reaction conditions suitable for amplifying said DNA fragment. In some embodiments, the reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene comprise: (i) a forward primer having a nucleic acid sequence comprising 5'-ACGGAAGTCTGAC-3' (SEQ ID NO: 1), optionally wherein the forward primer has a nucleic acid sequence comprising 5'-AATGGACGGAAGTCTGAC-3' (SEQ ID NO: 3); and / or (ii) a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2); optionally wherein the reverse primer has a nucleic acid sequence comprising 5'-GTTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 4).

[0031] In some embodiments, contacting the nucleic acid with reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene is preceded by a step of contacting nucleic acid extracted from the sample with reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene (SEQ ID NO: 30) under reaction conditions suitable for amplifying said DNA fragment; optionally wherein the reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene comprise:(i) a forward primer having a nucleic acid sequence comprising 5'-GGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 13), optionally wherein the forward primer has a nucleic acid sequence comprising 5'-TAAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 15); and / or (ii) a reverse primer having a nucleic acid sequence comprising 5'-CAG l I I CCAAAGCGT-3' (SEQ ID NO: 14), optionally wherein the reverse primer has a nucleic acid sequence comprising 5'-TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20).

[0032] In some embodiments, the probe comprises a fluorescent tag, optionally wherein the probe further comprises a quencher which is configured to be cleaved from the probe after the probe has hybridised to the fragment.

[0033] In some embodiments, the nucleic acid extracted from the sample is RNA and the method is preceded by a step of reverse transcribing the RNA, optionally wherein reverse transcription is performed using a primer having a nucleic acid sequence comprising: (a) 5'-TCTAGGCCGGTCAGA-3' (SEQ ID NO: 31), optionally a primer having a nucleic acid sequence comprising 5'-TATCTCTAGGCCGGTCAGA-3' (SEQ ID NO: 32); (b) 5'-AGCCTATCTCTAGG-3' (SEQ ID NO: 33), optionally a primer having a nucleic acid sequence comprising 5'-AGAAAGCCTATCTCTAGG-3' (SEQ ID NO: 34); and / or (c) 5'- CAG l I I CCAAAGCGT-3' (SEQ ID NO: 14), optionally a primer having a nucleic acid sequence comprising 5'-TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20).

[0034] In some embodiments, the sample is a sample of placenta.

[0035] In some embodiments, the sample is a biological sample obtained from a patient. In some embodiments, the sample is selected from a sample obtained by vaginal and / or rectal swab and a neonatal blood sample. In some embodiments, the sample is an endometrial biopsy.

[0036] In some embodiments, screening for GBS in the sample of placenta comprises performing the method the invention. The invention also provides a composition comprising a forward primer having a nucleic acid sequence comprising 5'-ACGGAAGTCTGAC-3' (SEQ ID NO: 1) and / or a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2).

[0037] In some embodiments, the forward primer has a nucleic acid sequence comprising 5'-AATGGACGGAAGTCTGAC-3' (SEQ ID NO: 3) and / or the reverse primer has a nucleic acid sequence comprising 5'-GTTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 4).

[0038] The invention also provides a composition comprising a forward primer having a nucleic acid sequence comprising 5'-GGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 13) and / or a reverse primer having a nucleic acid sequence comprising 5'-CAG l I I CCAAAGCGT-3' (SEQ ID NO: 14).

[0039] In some embodiments, the forward primer has a nucleic acid sequence comprising 5'-TAAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 15) and / or the reverse primer has a nucleic acid sequence comprising 5'-TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20).

[0040] In some embodiments, the composition further comprises a probe having a nucleic acid sequence comprising 5'-CGTTGGTAG-3' (SEQ ID NO: 25).

[0041] In some embodiments, the composition further comprises a primer having a nucleic acid sequence comprising: 5'-TCTAGGCCGGTCAGA-3' (SEQ ID NO: 31), optionally a primer having a nucleic acid sequence comprising 5'-TATCTCTAGGCCGGTCAGA-3' (SEQ ID NO: 32). In some embodiments, the composition further comprises a primer having a nucleic acid sequence comprising: 5'- AGCCTATCTCTAGG-3' (SEQ ID NO: 33), optionally a primer having a nucleic acid sequence comprising 5'-AGAAAGCCTATCTCTAGG-3' (SEQ ID NO: 34).

[0042] The invention also provides a composition comprising a probe having a nucleic acid sequence comprising 5'-CGTTGGTAG-3' (SEQ ID NO: 25).

[0043] In some embodiments, the probe has a nucleic acid sequence comprising: (a) 5'-AGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 26); (b) 5'-AGAGAAGAACGTTGGTAGGAGTGGA-3' (SEQ ID NO: 27); (c) 5'-AGAGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 28); or (d) 5'-AGAAGAACGTTGGTAGGAGTGGAAA-3' (SEQ ID NO: 29).

[0044] The invention also provides a kit comprising reagents for amplifying a DNA fragment comprising residues 370-452 of the GBS 16S rRNA gene (SEQ ID NO: 30); and a probe for detecting the fragment, wherein: (a) the reagents for amplifying a DNA fragment comprising residues 370-452 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising 5'-ACGGAAGTCTGAC-3' (SEQ ID NO: 1) and a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2); and / or (b) the probe has a nucleic acid sequence comprising 5'-CGTTGGTAG-3' (SEQ ID NO: 25).

[0045] In some embodiments, the reagents for amplifying a DNA fragment comprising residues 370-452 of the GBS 16S rRNA gene comprise: (a) a forward primer having a nucleic acid sequence comprising 5'-AATGGACGGAAGTCTGAC-3' (SEQ ID NO: 3); and / or (b) a reverse primer having a nucleic acid sequence comprising 5'-GTTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 4).

[0046] In some embodiments, the kit further comprises a primer having a nucleic acid sequence comprising: 5'-TCTAGGCCGGTCAGA-3' (SEQ ID NO: 31), optionally a primer having a nucleic acid sequence comprising 5'-TATCTCTAGGCCGGTCAGA-3' (SEQ ID NO: 32). In some embodiments, the kit further comprises a primer having a nucleic acid sequence comprising: 5'-AGCCTATCTCTAGG-3' (SEQ ID NO: 33), optionally a primer having a nucleic acid sequence comprising 5'-AGAAAGCCTATCTCTAGG-3' (SEQ ID NO: 34).

[0047] The invention also provides a kit comprising reagents for amplifying a cDNA fragment corresponding to residues 370-452 of the GBS 16S rRNA gene (SEQ ID NO: 30); and a probe for detecting the fragment, wherein: (a) the reagents for amplifying a cDNA fragment corresponding to residues 370-452 of the GBS 16S rRNA gene comprise a primer having a nucleic acid sequence comprising: 5'- TCTAGGCCGGTCAGA-3' (SEQ ID NO: 31), optionally a primer having a nucleic acid sequence comprising 5'-TATCTCTAGGCCGGTCAGA-3' (SEQ ID NO: 32), and / or a primer having a nucleic acid sequence comprising: 5'-AGCCTATCTCTAGG-3' (SEQ ID NO: 33), optionally a primer having a nucleic acid sequence comprising 5'-AGAAAGCCTATCTCTAGG-3' (SEQ ID NO: 34); and / or (b) the probe has a nucleic acid sequence comprising 5'-CGTTGGTAG-3' (SEQ ID NO: 25).

[0048] In some embodiments, the kit further comprises reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene (SEQ ID NO: 30); optionally wherein the reagent for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene comprise: (a) a forward primer having a nucleic acid sequence comprising 5'-GGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 13), optionally a forward primer having a nucleic acid sequence comprising 5'-TAAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 15); and / or (b) a reverse primer having a nucleic acid sequence comprising 5'-CAG I I I CCAAAGCGT-3' (SEQ ID NO: 14), optionally a reverse primer having a nucleic acid sequence comprising 5'-TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20). In some embodiments, the probe comprises a fluorescent tag, optionally wherein the probe further comprises a quencher which is configured to be cleaved from the probe after the probe has hybridised to the fragment.

[0049] In some embodiments, the probe has a nucleic acid sequence comprising: (a)

[0050] 5'-AGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 26); (b) 5'-AGAGAAGAACGTTGGTAGGAGTGGA-3' (SEQ ID NO: 27); (c) 5'-AGAGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 28); or (d)

[0051] 5'-AGAAGAACGTTGGTAGGAGTGGAAA-3' (SEQ ID NO: 29).

[0052] In some embodiments, the kit further comprises reverse transcriptase.

[0053] DESCRIPTION OF THE FIGURES

[0054] Figure 1. Selection of participants for studies. Patients in the discovery study have been previously described (de Goffau, M. C. et al. Nature 572, 329-334 (2019)). Cases (n=41) and controls (n=395) are defined as pregnancies with an infant admitted or not admitted, respectively, to neonatal unit within 48 hours of birth and for 48 hours or more. In the validation study, cases are defined as pregnancies with an infant admitted to neonatal unit without time limits for admission or duration of stay. The control group is represented by a cohort of random controls selected within the POP study where the baby was not admitted for neonatal care. The validation study excluded any patient who was included in the discovery study. In total, 1,361 patients were included in the current analyses. PTBs, preterm births.

[0055] Figure 2. Nested PCR-qPCR assay to detect the 16S GBS gene, a, Schematic diagram of the nested PCR-qPCR assay, b, Multiple sequence alignment of the amplicons generated with the nested PCR- qPCR assay and the 16S GBS gene sequence (SnapGene Viewer 6.1). The sequences of the PCR amplicons (n=6 from 4 placentas positive for 16S and sip, and 2 placentas positive for 16S) were determined by Sanger sequencing using the second-round (inner) reverse and forward primers. This analysis confirmed that the sequence of the qPCR products perfectly aligned to the GBS 16S gene sequence, c, Multiple sequence alignment of the 16S gene from various Streptococci: [top to bottom] S. agalactiae (GBS) (NR_040821.1), S. dysgalactiae (MH393517.1), S. salivarius (KM221948.1), S. anginosus (MF578782.1), S. vestibularis (NR_042777.1), S. pyogenes (NR_028598.1), S. suis (NR_115737.1), S. urinalis (NR_115738.1), S. egui (NR_116010.1), S. uberis (U41048.1). The position of the inner primers, outer primers and probe of the nested PCR-qPCR assay are indicated. The frames highlight regions that differ between species amplified using the qPCR inner primers. Figure 3. Sensitivity of the sip qPCR and 16S PCR-qPCR assays for GBS detection. Genomic Streptococcus agalactiae DNA was used at 4000, 400, 40, or 4 copies / reaction in the unnested qPCR and the nested PCR-qPCR assays targeting the sip and 16S GBS genes, respectively. The two curves are described by semi-log equations, which were used to calculate the GBS genome copies at 25 Ct (cycle threshold). Means ± standard deviation are shown; n=4 independent experiments with 2 technical replicates / each. Ct denotes cycle threshold and it is inversely associated with the relative abundance of the target.

[0056] Figure 4. Healthy umbilical cord, healthy membrane roll, funisitis and chorioamnionitis. a, Low power of healthy cord, b, Medium power of healthy umbilical artery, c, High power of healthy umbilical vein, d, Low power of funisitis. e, Medium power of umbilical artery in funisitis (black arrows = extensive infiltration of neutrophils), f, High power of umbilical vein in funisitis (black arrows = neutrophils infiltrating between layers of smooth muscle, white arrow = microabscess), g, Low power of healthy membrane roll, h, Medium power of healthy membrane roll, i, Low power of chorioamnionitis. j, Medium power of chorioamnionitis (black arrows = extensive infiltration of neutrophils).

[0057] Figure 5. Placental GBS is associated with maternal genital tract colonisation and fetal cytokine storm, a, Proportion of pregnancies with GBS positive placentas (with 95% Cl) in relation to maternal genital tract colonisation assessed by high vaginal swab (HVS) (n=57 patients with positive HVS culture, n=410 patients with negative HVS culture and n=458 patients with no HVS culture performed). Chi- square test p value are reported, b, Cytokines were measured in cord serum samples from term pregnancies with GBS positive (n=ll) and negative (n=129) placentas, using the Ella platform (Bio- Techne). Cytokine levels are expressed as the multiple of the median (MoM) of control samples analysed in the same batch. For graphical purposes, samples with MoM=0 for interleukin-1 (n=24) have been plotted using a value of MoM=0.0126 (the lowest MoM in the dataset). The dotted line represents an elevation of MoM >10. Median cytokine levels in the controls were: 0.18pg / ml for interleukin-10 (n=116), 16.6pg / ml for interleukin-6 (n=140), 13.6pg / ml for interleukin-8 (n=140) and 16.6pg / ml for Tumour Necrosis Factor alpha (n=140). IL-10, interleukin-10; IL-6, interleukin-6; IL-8, interleukin-8; TNF-a, Tumour Necrosis Factor alpha; Cl, confidence interval, c, Proportion of pregnancies with fetal cytokine storm (with 95% Cl) in relation to GBS detection in the placenta (n=ll GBS positives and n=129 GBS negatives). Chi-square test p value is reported, d, Cytokines were measured as described herein in 58 term placental samples from term pregnancies with GBS positive (n=26) and negative (n=32) placentas. For graphical purposes, samples with MoM=0 for interleukin- 10 (n=l) and TNF-a (n=9) have been plotted using a value of MoM=0.256 and MoM=0.493, respectively (the lowest MoM in the dataset), e, Cytokines were measured as described herein in 55 maternal serum samples at 36wkGA from term pregnancies with GBS positive (n=25) and negative (n=30) placentas. Samples with MoM=0 for interleukin-1 (n=49) have been plotted using a value of MoM=0.914 (the lowest MoM in the dataset).

[0058] Figure 6. Temporal distribution of deliveries with GBS positive placenta in the validation study (n=36). The graph on the left side of the figure represents the number of weekly deliveries with GBS positive placenta, n numbers of GBS positive and total deliveries per week are displayed in the list on the right side of the figure.

[0059] Figure 7. Spatial distribution of placental samples and controls in the qPCR plates. The qPCR plates include all the placental samples from the validation study, which were run in triplicate. All the plates had a similar layout with the triplicate wells organized in a L-shape, except for plate 5a which had the triplicate wells organized horizontally. GBS positive samples had a detectable 16S signal. Positive controls contained decreasing quantities of GBS genomic DNA (4000, 400, 40, or 4 copies / well) added at the outer PCR or the inner qPCR step. Negative controls contained H2O added at the outer PCR (darker grey) or the inner qPCR (lighter grey) step. DNA extraction blanks (tissue lysis buffer carried through the entire experimental workflow) are indicated in black. This analysis included: 200 and 112 positive control wells for the PCR and qPCR, respectively, and they all had 16S and sip signal; 50 and 28 negative control wells for the PCR and qPCR, respectively, and they were all 16S- and sip-negative; 51 wells containing DNA extraction blanks, which were all 16S- and sip-negative. In total, the inventors ran 129 negative control wells added at different stages of the experimental workflow and none of them had a detectable signal by either assay.

[0060] Figure 8. RT-qPCR assay to detect GBS 16S rRNA. a, Schematic diagram of the RT-qPCR strategy, b, qPCR amplification plot representing 3 positive samples with signal for GBS 16S rRNA, 1 negative sample with no 16S signal and 2 negative controls samples (H2O or without the reverse transcriptase enzyme in the RT reaction in black and grey, respectively). Placental samples were run in duplicate and were considered GBS positive if both wells had signal for 16S rRNA. The relative abundance of the target is expressed using the Ct (cycle threshold) value, which is inversely associated with the signal. Rn (normalized reporter value) represents the fluorescence of the reporter dye normalized to the signal of the passive reference dye for a given reaction. The ARn is the Rn value of an experimental reaction minus the Rn value of the baseline signal generated by the instrument. This parameter indicates the magnitude of the fluorescent signal generated in the qPCR assay. RT denotes reverse transcriptase. Figure 9. Comparison of sip and 16S PCR-qPCR signals for GBS in placental samples. Correlation between the sip and 16S Ct values obtained in the nested PCR-qPCR assays targeting the two GBS genes (n=44) using the data from the validation study. Samples with undetectable sip signal are plotted with Ct = nd (not detected, n=8). Where samples were positive using both methods, the Ct count was higher for sip than for the 16S rRNA PCR-qPCR (mean difference = 2.4, 95% Cl = 1.0 to 3.7, P=0.0009). Pearson's correlation coefficients (r) are reported for 16S Ct <30 (P<0.0001) and Ct >30 (P=0.56). Ct denotes cycle threshold.

[0061] Figure 10. Representative amplification plots of the 16S and sip PCR-qPCR assays in a placental DNA sample. Amplification plots were obtained using the multiplex qPCR assay targeting the sip and 16S GBS genes and the human RNaseP (RPPH1) gene. The relative abundance of the target is expressed using the Ct (cycle threshold) value, which is inversely associated with the signal. Rn (normalized reporter value) represents the fluorescence of the reporter dye normalized to the signal of the passive reference dye for a given reaction. The ARn is the Rn value of an experimental reaction minus the Rn value of the baseline signal generated by the instrument. This parameter indicates the magnitude of the fluorescent signal generated in the qPCR assay.

[0062] Figure 11. Sensitivity analysis of the association between placental GBS and cord cytokine levels. In this analysis, GBS positives were defined as placental samples with one or more of the following: GBS identified by the 16S PCR-qPCR assay, GBS read identified by deep sequencing, or GBS 16S amplicons >0.1% of all reads (as compared to >1% in primary analysis). Cytokine levels were measured in 145 cord serum samples from term pregnancies with GBS positive (n=16) and negative (n=129) placentas, using the Ella platform (Bio-Techne). Cytokine levels are expressed as the multiple of the median (MoM) of control samples analysed in the same batch. For graphical purposes, samples with MoM=0 for interleukin-ip (n=27) have been plotted using a value of MoM=0.0126 (the lowest MoM in the dataset). GBS negative controls are defined as pregnancies with GBS negative placentas on all tests completed and with babies not requiring NNU admission. The dotted line represents an elevation of MoM >10. IL-ip, interleu kin-ip; IL-6, interleukin-6; IL-8, interleukin-8; TNF-a, Tumour Necrosis Factor alpha.

[0063] Figure 12. Power calculations for the secondary outcomes studied in this work. Plots illustrate the power calculations for the cytokine storm (a), probable (b) and proven (c) sepsis, chorioamnionitis (d) and funisitis (e) outcomes and for a range of different odds ratios (OR). Calculations were based on the observed numbers of GBS positive and GBS negative placentas in the validation study, the observed proportion of the given outcome in the GBS negative group of the validation study, and a range of possible odds ratios. Alpha two sided and threshold = 0.05 were applied. Figure 13. PCR reactions using genomic DNA from GBS, S. pneumoniae and S. pyogenes as templates. The same primers developed in Example 1 (ORIG) or a set of modified primers (MOD) were used in the assay, which was carried at two annealing temperatures (Ta).

[0064] Figure 14. PCR reactions using genomic DNA from GBS, S. pneumoniae and S. pyogenes as templates. The same primers developed in Example 1 (ORIG) or a set of primers including a modified inner reverse primer (MOD) were used in the assay, which was carried at two annealing temperatures (Ta).

[0065] FIGURE 15. Design of the RT primer targeting the GBS 16S rRNA. Sequences of the 16S rRNAs from Sterptococcus Agalactiae (GBS), other Streptococci species and bacteria of the vaginal flora. Characteristics of the selected primer candidates are shown.

[0066] DETAILED DESCRIPTION OF THE INVENTION

[0067] The present invention is based on the surprising discovery that placental GBS is associated with an increased risk of neonatal morbidity. The inventors found that infants whose placenta contained GBS were more likely to be admitted to a neonatal unit (NNU). Moreover, the inventors observed that 10-15% of infants who were born with symptoms, signs, or laboratory tests suggestive of sepsis had placental GBS but did not themselves test positive for GBS infection during a septic screen or did not have a septic screen performed. Importantly, the results described herein suggest that GBS causes about 10 times the number of cases of neonatal morbidity following birth at term than is currently recognized (i.e. ~1 in 200 as opposed to ~1 in 2000 births). The results also suggest that neonatal sepsis may be caused by mechanisms other than colonisation of the infant during passage through the birth canal. Indeed, several neonates who were admitted to a NNU with suspected or proven sepsis were delivered by Caesarean section.

[0068] The inventors found that bacterial invasion of the placenta may induce extreme activation of the fetal innate immune system prior to birth leading to neonatal sepsis, regardless of whether the infant is colonised by GBS directly. This may explain the relatively high rates of suspected, but culture negative sepsis identified in infants admitted to the NNU. In addition, these findings provide some explanation as to why routine vaginal / rectal swabs followed by antibiotic treatment of GBS positive women have not been proven to reduce rates of neonatal sepsis.

[0069] Taken together, the findings described herein suggest that screening for placental GBS provides a more accurate diagnostic tool to identify infants who are at an increased risk of neonatal sepsis than current screening methods, e.g. based on vaginal / rectal swabs or screening the neonate. The invention provides a method of screening for risk of neonatal sepsis in a neonate comprising screening for Streptococcus agalactiae (Group B Streptococcus, GBS) in a sample of placenta, wherein the presence of GBS in the placenta indicates an increased risk of neonatal sepsis in the neonate.

[0070] The terms neonate and infant are used interchangeably herein to refer to a newborn child under 28 days of age. As used herein, the term "neonatal sepsis" refers to a systemic infection during the first 28 days of life. A neonate identified as being at an increased risk of neonatal sepsis by a method of the invention is more likely to have or to develop neonatal sepsis than a neonate who has not been exposed to placental GBS.

[0071] The skilled person will appreciate that the "sample of placenta" is from the placenta of neonate(s) for whom the risk of neonatal sepsis is screened. In some embodiments, the method of the invention indicates an increased risk of neonatal sepsis in a single neonate (e.g. single births, or multiple births wherein each baby has a separate placenta). In some embodiments, the method of the invention indicates an increased risk of neonatal sepsis in two or more neonates (e.g. multiple births wherein each baby shares a placenta).

[0072] Infants suffering from neonatal sepsis include those having "proven" GBS sepsis ("proven sepsis") where GBS infection has been confirmed by a postnatal septic screen (e.g. culture from a blood or swab sample, such as a skin swab) and those having probable but culture negative sepsis ("probable sepsis"), i.e. where GBS infection is suspected based on clinical signs and laboratory and / or radiological evidence, but no GBS is cultured. Laboratory evidence for probable sepsis may include leucocytosis (white blood cell [WBC] count > 20 x 109 / l_), leucopenia (WBC count < 5 x 109 / l_), thrombocytopenia (platelet count < 150 x 109 / l_) and / or a blood C-reactive protein (CRP) level of > 10 mg / L. Radiological evidence for probable sepsis may include presence of pulmonary infiltrates, consolidation and / or intestinal dilatation.

[0073] In some embodiments, neonates identified as being at an increased risk of neonatal sepsis are screened for signs of infection and / or sepsis after delivery. In some embodiments, screening the neonate for signs of infection and / or sepsis comprises one or more of: (a) blood culture; (b) full blood count and differential cell count; (c) CRP assay; (d) lumbar puncture; and (e) radiological tests.

[0074] Blood culture typically involves collecting a sample of blood from the neonate under aseptic conditions, adding the blood sample to culture media (e.g. media comprising brain-heart infusion / trypticase soy broth, amino acids, carbohydrate substrates and an anticoagulant) and incubating the mixture at ~35-37 °C for ~36-48 hours. Bacterial growth may be detected visually or by monitoring carbon dioxide production. Samples that are positive for bacterial growth are typically examined further to identify the organism, e.g. by sub-culturing on agar plates (such as sheep blood agar) and performing biological and / or molecular tests. GBS are catalase negative, Gram-positive cocci which typically produce translucent to opaque, whitish grey, smooth colonies with a narrow zone of beta haemolysis on sheep blood agar plates. Colonies suspected of being GBS may be identified by serologic grouping or by molecular methods, e.g. antigen detection, sequencing and / or mass spectrometry, e.g. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS).

[0075] Full blood count and differential cell count typically involves obtaining a blood sample from the neonate and determining the number of red blood cells and platelets in the sample as well as the number of each type of white blood cell. An increased white blood cell count is typically indicative of infection. Laboratory evidence for sepsis based on full blood counts may include leucocytosis (white blood cell [WBC] count > 20 x 109 / l_), leucopenia (WBC count < 5 x 109 / l_), and / or thrombocytopenia (platelet count < 150 x 109 / l_).

[0076] CRP assays typically measure the level of CRP in a blood sample from the neonate. CRP is an inflammatory marker and so levels of CRP that are above a threshold are indicative of inflammation and infection. The CRP threshold may be >5 mg / L or >10 mg / L.

[0077] Lumbar puncture may be used to check for meningitis and typically involves collecting a sample of cerebrospinal fluid (CSF) and checking for the presence of infectious agents and markers of infection, e.g. white blood cells and protein. Infectious agents may be identified by culturing a sample of the CSF and detecting bacterial growth.

[0078] Radiological tests may comprise chest and / or abdominal X-rays. Signs of infection / sepsis may include presence of pulmonary infiltrates, consolidation and / or intestinal dilatation.

[0079] In some embodiments, neonates identified as being at an increased risk of neonatal sepsis are monitored for signs of sepsis after delivery. In some embodiments, monitoring the neonate comprises tracking temperature, food intake and / or breathing rate.

[0080] In some embodiments, the method further comprises testing the level(s) of one or more inflammatory cytokines in a sample of umbilical cord blood or umbilical cord serum. In some embodiments, the one or more inflammatory cytokine(s) comprises one of more of interleukin (IL)-ip, IL-6 and IL-8. Inflammatory cytokine levels may be measured by bioassay, immunoassay, flow cytometry, and / or aptamer-based methods. Immunoassays may include enzyme-linked immunosorbent assay (ELISA), radioimmunoassays (RIA), direct, indirect, or competitive ELISA, enzyme immunoassays (EIA), fluorescence immunoassays (FIA), western blotting, immunoprecipitation or particle-based immunoassay.

[0081] As noted, the inventors discovered that a portion of infants experiencing neonatal sepsis exhibited cytokine storm. As used herein, "cytokine storm" is defined as two or more pro-inflammatory cytokines being elevated >10 times the median level in umbilical cord blood or serum as compared to healthy controls ( / .e. umbilical cord blood / serum from infants who do not develop neonatal sepsis).

[0082] Thus, in some embodiments, the method further comprises testing the levels of two or more inflammatory cytokines in a sample of umbilical cord blood, umbilical cord serum, or umbilical cord plasma. In some embodiments, the two or more inflammatory cytokines comprise two of more of IL-1 , IL-6 and IL-8. The neonate may be identified as experiencing cytokine storm if the level of two or more pro-inflammatory cytokines are elevated >10 times the median level of a healthy control(s).

[0083] In some embodiments, the method is preceded by a step of obtaining the sample of placenta.

[0084] In some embodiments, the sample of placenta is obtained after delivery of the neonate. In some embodiments, the sample of placenta is obtained within an hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours or within 6 hours of delivery.

[0085] It will be readily understood that delivery of the neonate embraces both vaginal delivery and delivery by Caesarean section.

[0086] In some embodiments, the placenta is washed to remove surface contamination prior to the biopsy being obtained.

[0087] In some embodiments, the sample of placenta is a placental biopsy obtained prior to delivery of the neonate. In some embodiments, the placental biopsy is obtained at 35 or more weeks gestational age. In some embodiments, the placental biopsy is obtained at 36 or more, 37 or more, 38 or more, or 39 or more weeks gestational age. Gestational age measures the age of a pregnancy in weeks from the beginning of the woman's last menstrual period.

[0088] In some embodiments, the mother of the neonate has previously tested positive for GBS infection using screening methods known in the art. In some embodiments, the mother of the neonate has previously tested positive for GBS infection based on vaginal and / or rectal swab screening. In some embodiments, the mother of the neonate tested positive for GBS during a previous pregnancy. Vaginal and / or rectal swabs are normally obtained at between 34 and 37 weeks gestational age. GBS may be detected from these swabs by nucleic acid-based screening methods and / or culture-based methods. Nucleic acid-based detection methods may comprise quantitative PCR, e.g. with fluorogenic detection of amplified DNA. Culture based methods typically involve inoculating media (e.g. media adapted for Streptococci growth, such as Todd-Hewitt broth with nalidixic acid and colistin) with the swab and incubating at ~35-37 °C for ~18-48 hours. The media may be subcultured onto agar plates (e.g. sheep blood agar plates) and incubated. Bacterial colonies may then be isolated and identified, e.g. using standardised biochemical and / or molecular GBS identification methods known in the art.

[0089] In some embodiments, the mother of the neonate has previously given birth to a neonate who suffered neonatal sepsis. Subsequent neonates born to such mothers may be at an increased risk of neonatal sepsis.

[0090] In some embodiments, the sample of placenta comprises placental terminal villi. In some embodiments, the sample of placenta is obtained from separate lobules of the placenta, e.g. at least 2, at least 3, or at least 4 separate lobules.

[0091] In some embodiments, the method comprises screening for GBS nucleic acid in the sample of the placenta. In some embodiments, the method comprises polymerase chain reaction (PCR), optionally quantitative PCR (qPCR). In some embodiments, the method comprises screening for GBS RNA in the sample of placenta by reverse transcription PCR.

[0092] The invention provides a method of treating a neonate identified as being at an increased risk of neonatal sepsis by a method of the invention. In some embodiments, the method comprises administering antibiotics to the neonate. The invention also provides a composition comprising an antibiotic for use in a method of treating a neonate identified as being at an increased risk of neonatal sepsis by a method of the invention.

[0093] In some embodiments, the neonate exhibits one or more signs of infection and / or sepsis. In some embodiments, the one or more signs of infection and / or sepsis are identified by: (i) blood culture; (ii) full blood count and differential cell count; (ill) CRP assay; (iv) lumbar puncture; and / or (v) radiological tests.

[0094] In some embodiments, antibiotics are administered to the neonate within 12 hours of delivery. In some embodiments, antibiotics are administered to the neonate within 8, within 6, within 4, within 2, or within 1 hour of delivery. The invention also provides a method of treating the mother of a neonate identified as being at an increased risk of neonatal sepsis by a method of the invention, the method comprising administering an antibiotic to the mother. The invention also provides a composition comprising an antibiotic for use in a method of treating a mother of a neonate identified as being at an increased risk of neonatal sepsis by a method of the invention.

[0095] In some embodiments, the antibiotic comprises intrapartum antibiotic prophylaxis (AIP). In some embodiments, the antibiotic comprises penicillin (e.g. benzylpenicillin), vancomycin, teicoplanin, and / or clindamycin. Beneficially, prioritising women who have placental GBS for IAP treatment may reduce unnecessary or excessive antibiotic exposure. In some embodiments, the antibiotic comprises penicillin (e.g. benzylpenicillin) and / or gentamicin.

[0096] In some embodiments, antibiotics are administered to the mother of the neonate during the intrapartum period.

[0097] The invention also provides nucleic acid-based detection methods for GBS. The 16S ribosomal RNA (16S rRNA) gene encodes the ribosomal RNA which is a key component of the small subunit of the prokaryotic ribosome. Sequencing of the 16S rRNA gene is commonly used in phylogenetic analysis because it is common to all bacteria and contains both highly conserved and highly variable regions. The 16S rRNA gene is not typically used as a target when screening for a particular target organism (e.g. strain) because the high degree of sequence identity between closely related bacterial species can hamper the specificity of the detection method. Instead, nucleic acid-based methods for screening for a particular target organism often rely on gene(s) which are unique to the target organism. For example, a common target for identifying the presence of GBS is the sip gene which encodes surface immunogenic protein. Surprisingly, the inventors have discovered that targeting the 16S rRNA gene of GBS using the methods described herein not only provides high specificity, but also provides higher sensitivity than detection of sip.

[0098] The inventors found that GBS may be present at very low cell density within the placenta. The GBS genome contains seven copies of the 16S rRNA gene, and so by detecting the presence of this gene, the method advantageously achieves highly sensitive detection of GBS as compared to methods which rely on detection of a gene having a lower (e.g. single) copy number. To ensure high specificity, the inventors developed primers and / or probes which amplify and detect a variable region of the 16S rRNA gene. Importantly, the primers and / or probes described herein achieve highly specific and sensitive detection of GBS in complex nucleic acid samples, e.g. nucleic acid samples including human genomic DNA and genomic DNA from closely related Streptococci, such as S. pneumoniae and S. pyogenes. Sensitivity may be further improved by detecting 16S rRNA which is present at very high copy numbers within each cell.

[0099] In some embodiments, the nucleic acid extracted from the sample comprises RNA and the method comprises a step of reverse transcription converting the RNA into cDNA. The inventors determined that methods of detection which are based on RNA in the sample (e.g. which comprise reverse transcription of RNA to cDNA, and detection of the cDNA using methods described herein) can further improve sensitivity of detection. For example, the inventors developed primers having a nucleic acid sequence comprising SEQ ID NO: 32 or SEQ ID NO: 34, which specifically target GBS 16S RNA. Use of such primers to reverse transcribe GBS 16S rRNA in the sample enriches the sample for GBS 16S cDNA, and thereby further increases the specificity (and sensitivity) of DNA-based methods of detection, as described herein.

[0100] The invention provides a method of detecting GBS in a sample, the method comprising: (a) contacting nucleic acid extracted from the sample with reagents for amplifying a fragment of the GBS 16S rRNA gene under reaction conditions suitable for amplifying said DNA fragment, wherein the reagents comprise a forward primer having a nucleic acid sequence comprising 5'-ACGGAAGTCTGAC-3' (SEQ ID NO: 1) and a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2); and (b) detecting the presence of the fragment wherein the presence of the fragment is indicative of the presence of GBS in the sample. In some embodiments, the sample is a biological sample obtained from a patient.

[0101] In some embodiments, the reagents for amplifying a DNA fragment comprise a forward primer having a nucleic acid sequence comprising 5'-AATGGACGGAAGTCTGAC-3' (SEQ ID NO: 3). In some embodiments, the reagents for amplifying a DNA fragment comprise a reverse primer having a nucleic acid sequence comprising 5'-GTTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 4).

[0102] Advantageously, the inventors have found that performing PCR with a forward primer having a nucleic acid sequence comprising SEQ ID NO: 3 and a reverse primer having a nucleic acid sequence comprising SEQ ID NO: 4 results in the amplification of a fragment of GBS 16S DNA only, even in the presence of genomic DNA from closely related Streptococci such as S. pneumoniae and S. pyogenes. The skilled person would readily understand that a primers specificity relies on hybridisation of the 3' end of the primer to the target DNA. Thus, SEQ ID NOs: 1 and 5-8 which comprise the 3' region of SEQ ID NO: 3 and SEQ ID NOs: 2 and 9-12 which comprise the 3' region of SEQ ID NO: 4 would be expected to achieve the same degree of specificity as these primers. Likewise, the skilled person would readily understand that a primer's specificity relies on hybridisation of the 3' end of the primer to the target RNA. SEQ ID NO: 31 comprises the 3' region of SEQ ID NO: 32 and would be expected to achieve the same degree of specificity as SEQ ID NO: 32. SEQ ID NO: 33 comprises the 3' region of SEQ ID NO: 34 and would be expected to achieve the same degree of specificity as SEQ ID NO: 34.

[0103] In some embodiments, the reagents for amplifying a DNA fragment comprise a forward primer having a nucleic acid sequence comprising: (a) 5'-ATGGACGGAAGTCTGAC-3' (SEQ ID NO: 5); (b) 5'-TGGACGGAAGTCTGAC-3' (SEQ ID NO: 6); (c) 5'-GGACGGAAGTCTGAC-3' (SEQ ID NO: 7); or (d) 5'-GACGGAAGTCTGAC-3' (SEQ ID NO: 8).

[0104] In some embodiments, the reagents for amplifying a DNA fragment comprise a reverse primer having a nucleic acid sequence comprising: (a) 5'-TTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 9); (b) 5'-TAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 10); (c) 5'-AGTTACCGTCACTTGGTA-3' (SEQ ID NO: 11); or (d) 5'-GTTACCGTCACTTGGTA-3' (SEQ ID NO: 12).

[0105] Amplification primers are typically employed to amplify a fragment of target / complementary nucleic acid. In the presence of a suitable polymerase and DNA precursors (dATP, dCTP, dGTP and dTTP), forward and reverse primers are extended in a 5' to 3' direction, thereby initiating the synthesis of new nucleic acid strands that are complementary to the individual strands of the target nucleic acid.

[0106] In some embodiments, methods steps (a) and (b) are performed concurrently. In some embodiments, methods steps (a) and (b) are performed sequentially.

[0107] The method may take the form of a nested PCR assay in which a first set of primers are used to amplify a first DNA fragment, and a second set of primers are used to amplify a second, smaller DNA fragment which is within the first DNA fragment. Nested PCR improves the specificity of the method by reducing the likelihood of false positives which may occur because of off-target binding by the primers (e.g. to a closely related Streptococci strain).

[0108] In some embodiments, the method comprises: (a) contacting nucleic acid extracted from the sample with reagents for amplifying a first DNA fragment from the GBS 16S rRNA gene under reaction conditions suitable for amplifying said first DNA fragment, wherein the reagents for amplifying the first fragment comprise a forward primer having a nucleic acid sequence comprising 5'-GGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 13) and a reverse primer having a nucleic acid sequence comprising 5'-CAG l I I CCAAAGCGT-3' (SEQ ID NO: 14); then (b) contacting the nucleic acid with reagents for amplifying a second DNA fragment from the GBS 16S rRNA gene under reaction conditions suitable for amplifying said second DNA fragment, wherein the reagents for amplifying the second fragment comprise a forward primer having a nucleic acid sequence comprising 5'-ACGGAAGTCTGAC-3' (SEQ ID NO: 1) and a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2); and (c) detecting the presence of the second fragment, wherein presence of the second fragment is indicative of the presence of GBS in the sample. In some embodiments, the sample is a biological sample obtained from a patient.

[0109] In some embodiments, the reagents for amplifying the first DNA fragment comprise a forward primer having a nucleic acid sequence comprising 5'-TAAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 15). In some embodiments, the reagents for amplifying the first DNA fragment comprise a forward primer having a nucleic acid sequence comprising: (a) 5'-AAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 16); (b) 5'-AAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 17); (c) 5'-AAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 18) or (d) 5'-AGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 19).

[0110] In some embodiments, the reagents for amplifying the first DNA fragment comprise a reverse primer having a nucleic acid sequence comprising 5'-TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20). In some embodiments, the reagents for amplifying the first DNA fragment comprise a reverse primer having a nucleic acid sequence comprising: (a) 5'-CCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 21); (b) 5'- CTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 22); (c) 5'-TCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 23); or (d) 5'- CCAGTTTCCAAAGCGT-3' (SEQ ID NO: 24).

[0111] In some embodiments, the reagents for amplifying the second DNA fragment comprise a forward primer having a nucleic acid sequence comprising 5'-AATGGACGGAAGTCTGAC-3' (SEQ ID NO: 3). In some embodiments, the reagents for amplifying the second DNA fragment comprise a forward primer having a nucleic acid sequence comprising: (a) 5'-ATGGACGGAAGTCTGAC-3' (SEQ ID NO: 5); (b) 5'-TGGACGGAAGTCTGAC-3' (SEQ ID NO: 6); (c) 5'-GGACGGAAGTCTGAC-3' (SEQ ID NO: 7); or (d) 5'-GACGGAAGTCTGAC-3' (SEQ ID NO: 8).

[0112] In some embodiments, the reagents for amplifying the second DNA fragment comprise a reverse primer having a nucleic acid sequence comprising 5'-GTTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 4). In some embodiments, the reagents for amplifying the second DNA fragment comprise a reverse primer having a nucleic acid sequence comprising: (a) 5'-TTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 9); (b) 5'-TAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 10); (c) 5'-AGTTACCGTCACTTGGTA-3' (SEQ ID NO: 11); or (d) 5'-GTTACCGTCACTTGGTA-3' (SEQ ID NO: 12).

[0113] In some embodiments, methods steps (b) and (c) are performed concurrently. In some embodiments, methods steps (b) and (c) are performed sequentially. The fragment may be detected by any suitable method known in the art, e.g. agarose gel electrophoresis.

[0114] In some embodiments, detecting the presence of the fragment comprises contacting the nucleic acid with a probe that is configured to produce a detectable signal when hybridised to the fragment. In some embodiments, the probe comprises a fluorophore and a quencher. In some embodiments, the fluorophore is selected from 6-FAM (6-carboxyfluorescin), ABY, VIC and JUN. In some embodiments, the quencher is selected from minor groove binder non-fluorescent quencher (MGB-NFQ) or QSY. In some embodiments, the fluorophore is located at the 5' end of the probe and the quencher is located at the 3' end of the probe. In unbound probes comprising a fluorophore and a quencher, the fluorophore is typically quenched by the quencher to prevent emission of a fluorescent signal. When the probe binds to the target fragment, the action of polymerase cleaves the fluorophore from the probe, thereby separating the fluorophore from the quencher and allowing the fluorophore to emit a fluorescent signal. In this embodiment, the presence of a fluorescent signal indicates presence of the target fragment.

[0115] In some embodiments, the probe has a nucleic acid sequence comprising 5'-CGTTGGTAG-3' (SEQ ID NO: 25). In some embodiments, the probe has a nucleic acid sequence comprising 5'- AGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 26). In some embodiments, the probe has a nucleic acid sequence comprising 5'-AGAGAAGAACGTTGGTAGGAGTGGA-3' (SEQ ID NO: 27). In some embodiments, the probe has a nucleic acid sequence comprising 5'-AGAGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 28). In some embodiments, the probe has a nucleic acid sequence comprising 5'- AGAAGAACGTTGGTAGGAGTGGAAA-3' (SEQ ID NO: 29).

[0116] The invention also provides a method of detecting GBS in a sample, the method comprising: (a) contacting nucleic acid extracted from the sample with a probe having a nucleic acid sequence comprising 5'-CGTTGGTAG-3' (SEQ ID NO: 25); and (b) detecting binding of the probe to the fragment, wherein binding of the probe to the fragment is indicative of the presence of GBS in the sample. In some embodiments, the sample is a biological sample obtained from a patient.

[0117] Probes having a nucleic acid sequence comprising 5'-CGTTGGTAG-3' (SEQ ID NO: 25) bind to nucleic acid fragments comprising residues 432-440 of SEQ ID NO: 30. Advantageously, the inventors found that probes which bind to residues 432-440 of SEQ ID NO: 30 or the complement thereof accurately detect GBS, even in the presence of other closely related Streptococcal strains, including S. pneumoniae and S. pyogenes. In some embodiments, the probe has a nucleic acid sequence comprising 5'- AGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 26). In some embodiments, the probe has a nucleic acid sequence comprising 5'-AGAGAAGAACGTTGGTAGGAGTGGA-3' (SEQ ID NO: 27). In some embodiments, the probe has a nucleic acid sequence comprising 5'-AGAGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 28). In some embodiments, the probe has a nucleic acid sequence comprising 5'-AGAAGAACGTTGGTAGGAGTGGAAA-3' (SEQ ID NO: 29).

[0118] In some embodiments, the method is preceded by a step of contacting the nucleic acid extracted from the sample with reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene (SEQ ID NO: 30) under reaction conditions suitable for amplifying said DNA fragment.

[0119] In some embodiments, reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising 5'-ACGGAAGTCTGAC-3' (SEQ ID NO: 1). In some embodiments, reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising 5'-AATGGACGGAAGTCTGAC-3' (SEQ ID NO: 3). In some embodiments, the reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising: (a) 5'-ATGGACGGAAGTCTGAC-3' (SEQ ID NO: 5); (b) 5'-TGGACGGAAGTCTGAC-3' (SEQ ID NO: 6); (c) 5'- GGACGGAAGTCTGAC-3' (SEQ ID NO: 7); or (d) 5'-GACGGAAGTCTGAC-3' (SEQ ID NO: 8).

[0120] In some embodiments, reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene comprise a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2). In some embodiments, reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene comprise a reverse primer having a nucleic acid sequence comprising 5'-GTTAGTTACCGTCACTTGGTA-3' (SEQ I D NO: 4). In some embodiments, reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene comprise a reverse primer having a nucleic acid sequence comprising: (a) 5'-TTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 9); (b) 5'-TAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 10); (c) 5'-AGTTACCGTCACTTGGTA-3' (SEQ ID NO: 11); or (d) 5'-GTTACCGTCACTTGGTA-3' (SEQ ID NO: 12).

[0121] In some embodiments, the reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising 5'- GGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 13). In some embodiments, the reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising 5'-TAAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 15). In some embodiments, the reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising: (a) 5'- AAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 16); (b) 5'-AAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 17); (c) 5'-AAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 18) or (d) 5'-

[0122] AGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 19).

[0123] In some embodiments, the reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene comprise a reverse primer having a nucleic acid sequence comprising 5'- CAG l I I CCAAAGCGT-3' (SEQ ID NO: 14). In some embodiments, the reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene comprise a reverse primer having a nucleic acid sequence comprising 5'-TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20). In some embodiments, the reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene comprise a reverse primer having a nucleic acid sequence comprising (a) 5'- CCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 21); (b) 5'-CTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 22); (c) 5'- TCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 23); or (d) 5'-CCAGTTTCCAAAGCGT-3' (SEQ ID NO: 24).

[0124] In some embodiments, amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene is preceded by a step of contacting nucleic acid extracted from the sample with reagents for amplifying a first DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene (SEQ ID NO: 30) under reaction conditions suitable for amplifying said DNA fragment.

[0125] In some embodiments, the reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising 5'- GGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 13). In some embodiments, the reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising 5'-TAAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 15). In some embodiments, the reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising: (a) 5'- AAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 16); (b) 5'-AAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 17); (c) 5'-AAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 18) or (d) 5'-

[0126] AGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 19).

[0127] In some embodiments, the reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene comprise a reverse primer having a nucleic acid sequence comprising 5'- CAG l I I CCAAAGCGT-3' (SEQ ID NO: 14). In some embodiments, the reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene comprise a reverse primer having a nucleic acid sequence comprising 5'-TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20). In some embodiments, the reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene comprise a reverse primer having a nucleic acid sequence comprising (a) 5'- CCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 21); (b) 5'-CTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 22); (c) 5'- TCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 23); or (d) 5'-CCAGTTTCCAAAGCGT-3' (SEQ ID NO: 24).

[0128] It will be readily understood that absence of the fragment is indicative that the sample does not comprise GBS.

[0129] In some embodiments, the nucleic acid extracted from the sample is RNA and the method comprises a step of reverse transcribing the RNA to produce complementary DNA (cDNA). In some embodiments, reverse transcription is performed using a primer having a nucleic acid sequence comprising 5'- TCTAGGCCGGTCAGA-3' (SEQ ID NO: 31). In some embodiments, reverse transcription is performed using a primer having a nucleic acid sequence comprising 5'-TATCTCTAGGCCGGTCAGA-3' (SEQ ID NO: 32). In some embodiments, reverse transcription is performed using a primer having a nucleic acid sequence comprising 5'-AGCCTATCTCTAGG-3' (SEQ ID NO: 33). In some embodiments, reverse transcription is performed using a primer having a nucleic acid sequence comprising 5'- AGAAAGCCTATCTCTAGG-3' (SEQ ID NO: 34). In some embodiments, reverse transcription is performed using a primer having a nucleic acid sequence comprising 5'-CAG l I I CCAAAGCGT-3' (SEQ ID NO: 14). In some embodiments, reverse transcription is performed using a primer having a nucleic acid sequence comprising 5'-TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20). Reverse transcription may be performed using any suitable method known in the art. For example, RNA template may be incubated with primer, reverse transcriptase, deoxynucleotide triphosphates (dNTPs), and ribonuclease inhibitor. Suitable reverse transcription reaction conditions may include an initial denaturation and annealing step (e.g. ~65 °C for 5min), DNA polymerisation (e.g. ~53 °C for 5min), and enzyme deactivation (e.g. 80 °C for lOmin). Reaction conditions will vary depending on the RNA template, primer and reverse transcriptase used.

[0130] It will be appreciated that reference herein to "nucleic acid extracted from the sample", and the like, includes cDNA which has been reverse transcribed from RNA in the sample.

[0131] In some embodiments, the method further comprises a step of extracting the nucleic acid from the sample, typically prior to amplification. Methods of extracting nucleic acids are known in the art and include, but are not limited to, solid-phase extraction, magnetic bead-based purification, and phenolchloroform extraction. RNA may be extracted using a commercially available extraction kit, e.g. RNeasy Plus Mini Kit supplied by Qiagen (catalogue number 74134). In some embodiments, the sample is a crude sample that has undergone minimal processing, e.g. cells present in the sample have been lysed but nucleic acid has not been purified.

[0132] The skilled person can readily determine suitable reagents and reaction conditions for amplifying the target fragment. Reagents for amplifying a fragment of the DNA or cDNA typically comprise primers, DNA polymerase (e.g. Tag DNA polymerase) and dNTPs. Reaction conditions for amplifying a DNA or cDNA fragment may include an initial melting step (e.g. 95 °C for 20-180 seconds) followed by multiple cycles (e.g. 15-50 cycles) of melting (e.g. 95 °C for 5-20 seconds), annealing (e.g. 48-50 °C for 10-60 seconds), and extending (e.g. 60-68 °C for 20-60 seconds), followed by a final incubation (e.g. 68 °C for 3 min). It will be immediately apparent to the skilled person that the melting, annealing and extending time and temperature used will vary depending on the primers, template and fragment of interest.

[0133] In some embodiments, the method comprises identifying a neonate at an increased risk of neonatal sepsis, wherein the sample is a sample of placenta and the neonate is identified as being at an increased risk of neonatal sepsis if GBS nucleic acid is detected in the sample.

[0134] In some embodiments, the sample of placenta is a placental biopsy obtained after delivery of a neonate. In some embodiments, the sample of placenta is obtained within an hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours or within 6 hours of delivery of the neonate.

[0135] In some embodiments, the sample of placenta is a placental biopsy obtained prior to delivery of a neonate. In some embodiments, the placental biopsy is obtained at 35 weeks or more gestational age, e.g. at 36 or more, 37 or more, 38 or more or 39 or more weeks gestational age. In some embodiments, the placental biopsy is obtained during or after the intrapartum period.

[0136] In some embodiments, the mother of the neonate has previously tested positive for GBS following a vaginal / rectal swab.

[0137] In some embodiments, the sample of placenta comprises placental terminal villi. In some embodiments, the sample of placenta is obtained from separate lobules of the placenta, e.g. at least 2, at least 3, or at least 4 separate lobules.

[0138] It will readily be understood that the nucleic acid detection methods described herein are not limited to use with placental samples. Given the high levels of sensitivity and specificity, the methods described herein may be used to screen for GBS in any sample suspected of containing GBS. In some embodiments, the sample is obtained from a vaginal or rectal swab sample. In some embodiments, the sample is a neonatal blood or swab sample, e.g. a neonatal skin swab.

[0139] In some embodiments, the sample is an endometrial biopsy. In some embodiments, the endometrial biopsy is obtained from a patient who is not pregnant. An endometrial biopsy is a medical procedure which involves taking a sample from the lining of the uterus.

[0140] In some embodiments, the method of screening for an increased risk of neonatal sepsis according to the invention comprises performing the nucleic acid-based detection method of the invention on the sample of placenta.

[0141] The invention also provides a composition comprising a forward primer having a nucleic acid sequence comprising 5' ACGGAAGTCTGAC-3' (SEQ ID NO: 1) and / or a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2). In some embodiments, the composition comprises a forward primer having a nucleic acid sequence comprising 5' ACGGAAGTCTGAC-3' (SEQ ID NO: 1). In some embodiments, the composition comprises a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2). In some embodiments, the composition comprises a forward primer having a nucleic acid sequence comprising 5' ACGGAAGTCTGAC-3' (SEQ ID NO: 1) and a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2).

[0142] In some embodiments, the composition comprises a forward primer having a nucleic acid sequence comprising 5'-AATGGACGGAAGTCTGAC-3' (SEQ ID NO: 3). In some embodiments, the composition comprises a forward primer having a nucleic acid sequence comprising: (a) 5'-ATGGACGGAAGTCTGAC- 3' (SEQ ID NO: 5); (b) 5'-TGGACGGAAGTCTGAC-3' (SEQ ID NO: 6); (c) 5'-GGACGGAAGTCTGAC-3' (SEQ ID NO: 7); or (d) 5'-GACGGAAGTCTGAC-3' (SEQ ID NO: 8).

[0143] In some embodiments, the composition comprises a reverse primer having a nucleic acid sequence comprising 5'-GTTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 4). In some embodiments, the composition comprises a reverse primer having a nucleic acid sequence comprising: (a) 5'-TTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 9); (b) 5'-TAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 10); (c) 5'-AGTTACCGTCACTTGGTA-3' (SEQ ID NO: 11); or (d) 5'-GTTACCGTCACTTGGTA-3' (SEQ ID NO: 12).

[0144] The invention also provides a composition comprising a forward primer having a nucleic acid sequence comprising 5'-GGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 13) and / or a reverse primer having a nucleic acid sequence comprising 5'-CAG l I I CCAAAGCGT-3' (SEQ ID NO: 14). In some embodiments, the composition comprises a forward primer having a nucleic acid sequence comprising 5'- GGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 13). In some embodiments, the composition comprises a reverse primer having a nucleic acid sequence comprising 5'-CAG l I I CCAAAGCGT-3' (SEQ ID NO: 14). In some embodiments, the composition comprises a forward primer having a nucleic acid sequence comprising 5'-GGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 13) and a reverse primer having a nucleic acid sequence comprising 5'-CAG l I I CCAAAGCGT-3' (SEQ ID NO: 14).

[0145] In some embodiments, the composition comprises a forward primer having a nucleic acid sequence comprising 5'-TAAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 15). In some embodiments, the composition comprises a forward primer having a nucleic acid sequence comprising: (a) 5'- AAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 16); (b) 5'-AAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 17); (c) 5'-AAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 18) or (d) 5'-

[0146] AGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 19).

[0147] In some embodiments, the composition comprises a reverse primer having a nucleic acid sequence comprising 5'-TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20). In some embodiments, the composition comprises a reverse primer having a nucleic acid sequence comprising (a) 5'- CCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 21); (b) 5'-CTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 22); (c) 5'- TCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 23); or (d) 5'-CCAGTTTCCAAAGCGT-3' (SEQ ID NO: 24).

[0148] In some embodiments, the composition further comprises a probe having a nucleic acid sequence comprising 5'-CGTTGGTAG-3' (SEQ ID NO: 25). In some embodiments, the probe has a nucleic acid sequence comprising: (a) 5'-AGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 26); (b) 5'- AGAGAAGAACGTTGGTAGGAGTGGA-3' (SEQ ID NO: 27); (c) 5'-AGAGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 28); or (d) 5'-AGAAGAACGTTGGTAGGAGTGGAAA-3' (SEQ ID NO: 29).

[0149] The invention also provides a composition comprising a probe having a nucleic acid sequence comprising 5'-CGTTGGTAG-3' (SEQ ID NO: 25). In some embodiments, the probe has a nucleic acid sequence comprising: (a) 5'-AGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 26); (b) 5'- AGAGAAGAACGTTGGTAGGAGTGGA-3' (SEQ ID NO: 27); (c) 5'-AGAGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 28); or (d) 5'-AGAAGAACGTTGGTAGGAGTGGAAA-3' (SEQ ID NO: 29).

[0150] The invention also provides a kit comprising reagents for amplifying a DNA fragment comprising residues 370-452 of the GBS 16S rRNA gene (SEQ ID NO: 30); and a probe for detecting the fragment, wherein: (a) the reagents for amplifying a DNA fragment comprising residues 370-452 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising 5'- ACGGAAGTCTGAC-3' (SEQ ID NO: 1) and a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2); and / or (b) the probe has a nucleic acid sequence comprising 5'-CGTTGGTAG-3' (SEQ ID NO: 25).

[0151] In some embodiments, the reagents for amplifying a DNA fragment comprising residues 370-452 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising 5'-AATGGACGGAAGTCTGAC-3' (SEQ ID NO: 3). In some embodiments, the reagents for amplifying a DNA fragment comprising residues 370-452 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising: (a) 5'-ATGGACGGAAGTCTGAC-3' (SEQ ID NO: 5); (b) 5'-TGGACGGAAGTCTGAC-3' (SEQ ID NO: 6); (c) 5'-GGACGGAAGTCTGAC-3' (SEQ ID NO: 7); or (d) 5'-GACGGAAGTCTGAC-3' (SEQ ID NO: 8).

[0152] In some embodiments, the reagents for amplifying a DNA fragment comprising residues 370-452 of the GBS 16S rRNA gene comprise a reverse primer having a nucleic acid sequence comprising 5'- GTTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 4). In some embodiments, the reagents for amplifying a DNA fragment comprising residues 370-452 of the GBS 16S rRNA gene comprise a reverse primer having a nucleic acid sequence comprising: (a) 5'-TTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 9); (b) 5'-TAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 10); (c) 5'-AGTTACCGTCACTTGGTA-3' (SEQ ID NO: 11); or (d) 5'-GTTACCGTCACTTGGTA-3' (SEQ ID NO: 12).

[0153] In some embodiments, the kit further comprises reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene (SEQ ID NO: 30).

[0154] In some embodiments, the reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising 5'- GGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 13). In some embodiments, the reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising 5'-TAAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 15). In some embodiments, the reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising: (a) 5'- AAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 16); (b) 5'-AAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 17); (c) 5'-AAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 18) or (d) 5'-

[0155] AGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 19).

[0156] In some embodiments, the reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene comprise a reverse primer having a nucleic acid sequence comprising 5'-CAG l I I CCAAAGCGT-3' (SEQ ID NO: 14). In some embodiments, the reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene comprise a reverse primer having a nucleic acid sequence comprising 5'-TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20). In some embodiments, the reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene comprise a reverse primer having a nucleic acid sequence comprising (a) 5'- CCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 21); (b) 5'-CTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 22); (c) 5'- TCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 23); or (d) 5'-CCAGTTTCCAAAGCGT-3' (SEQ ID NO: 24).

[0157] In some embodiments, the kit further comprises a primer having a nucleic acid sequence comprising: 5'-TCTAGGCCGGTCAGA-3' (SEQ ID NO: 31), optionally a primer having a nucleic acid sequence comprising 5'-TATCTCTAGGCCGGTCAGA-3' (SEQ ID NO: 32). In some embodiments, the kit further comprises a primer having a nucleic acid sequence comprising: 5'-AGCCTATCTCTAGG-3' (SEQ ID NO: 33), optionally a primer having a nucleic acid sequence comprising 5'-AGAAAGCCTATCTCTAGG-3' (SEQ ID NO: 34).

[0158] The invention also provides a kit comprising reagents for amplifying a cDNA fragment corresponding to residues 370-452 of the GBS 16S rRNA gene (SEQ ID NO: 30); and a probe for detecting the fragment, wherein: (a) the reagents for amplifying a cDNA fragment corresponding to residues 370-452 of the GBS 16S rRNA gene comprise a primer having a nucleic acid sequence comprising: 5'- TCTAGGCCGGTCAGA-3' (SEQ ID NO: 31), optionally a primer having a nucleic acid sequence comprising 5'-TATCTCTAGGCCGGTCAGA-3' (SEQ ID NO: 32), and / or a primer having a nucleic acid sequence comprising: 5'-AGCCTATCTCTAGG-3' (SEQ ID NO: 33), optionally a primer having a nucleic acid sequence comprising 5'-AGAAAGCCTATCTCTAGG-3' (SEQ ID NO: 34); and / or (b) the probe has a nucleic acid sequence comprising 5'-CGTTGGTAG-3' (SEQ ID NO: 25).

[0159] In some embodiments, the probe comprises a fluorescent tag, optionally wherein the probe further comprises a quencher which is configured to be cleaved from the probe after the probes has hybridised to the fragment.

[0160] In some embodiments, the probe has a nucleic acid sequence comprising 5'- AGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 26). In some embodiments, the probe has a nucleic acid sequence comprising 5'-AGAGAAGAACGTTGGTAGGAGTGGA-3' (SEQ ID NO: 27). In some embodiments, the probe has a nucleic acid sequence comprising 5'-AGAGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 28). In some embodiments, the probe has a nucleic acid sequence comprising 5'-AGAAGAACGTTGGTAGGAGTGGAAA-3' (SEQ ID NO: 29).

[0161] In some embodiments, the kit further comprises reverse transcriptase. In some embodiments, the kit comprises dNTPs. In some embodiments, the reagents for amplifying the fragment of DNA comprise DNA polymerase (e.g. Taq DNA polymerase) and dNTPs.

[0162] Table 1. Nucleic acid primer and probe sequences. "Inner" primers are suitable for amplifying a DNA fragment comprising residues 370-452 of the GBS 16S rRNA gene and "outer" primers are suitable for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene. Said fragments do not include the primer sequences.

[0163] The nucleic acid sequence of GBS 16S rRNA gene is represented by NCBI reference: NR_040821.1:

[0164] GACGAACGCTGGCGGCGTGCCTAATACATGCAAGTAGAACGCTGAGGTTTGGTGTTTACACTAGACTGATGA

[0165] GTTGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTCATAGCGGGGGATAACTATTGGAAACGATAGCTAA

[0166] TACCGCATAAGAGTAATTAACACATGTTAGTTATTTAAAAGGAGCAATTGCTTCACTGTGAGATGGACCTGCG

[0167] TTGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGG CCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGG

[0168] AAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGAGAAGAAC

[0169] GTTGGTAGGAGTGGAAAATCTACCAAGTGACGGTAACTAACCAGAAAGGGACGGCTAACTACGTGCCAGCA

[0170] GCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTCTTTA

[0171] AGTCTGAAGTTAAAGGCAGTGGCTTAACCATTGTACGCTTTGGAAACTGGAGGACTTGAGTGCAGAAGGGGA

[0172] GAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCT

[0173] GGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCG

[0174] TAAACGATGAGTGCTAGGTGTTAGGCCCTTTCCGGGGCTTAGTGCCGCAGCTAACGCATTAAGCACTCCGCCT

[0175] GGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGT

[0176] TTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTCTGACCGGCCTAGAGATAGGCTTTCTCT

[0177] TCGGAGCAGAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCA

[0178] ACGAGCGCAACCCCTATTGTTAGTTGCCATCATTAAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCGG

[0179] AGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGT

[0180] ACAACGAGTCGCAAGCCGGTGACGGCAAGCTAATCTCTTAAAGCCAATCTCAGTTCGGATTGTAGGCTGCAAC

[0181] TCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGT

[0182] ACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGC CTAAGGTGGGATAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGG (SEQ ID NO: 30).

[0183] In some embodiments, the method comprises reverse transcription of RNA. The nucleic acid sequence of "Candidate Primer 1" (see Example 3) is:

[0184] 5'-TATCTCTAGGCCGGTCAGA-3' (SEQ ID NO: 32)

[0185] The 3' region of SEQ ID NO: 32 is provided by SEQ ID NO: 31:

[0186] 5'-TCTAGGCCGGTCAGA-3' (SEQ ID NO: 31).

[0187] The nucleic acid sequence of "Candidate Primer 2" (see Example 3) is:

[0188] 5'-AGAAAGCCTATCTCTAGG-3' (SEQ ID NO: 34)

[0189] The 3' region of SEQ ID NO: 34 is provided by SEQ ID NO: 33:

[0190] 5'-AGCCTATCTCTAGG-3' (SEQ ID NO: 33).

[0191] EXAMPLES

[0192] The invention will be further clarified by the following examples, which are intended to be purely exemplary of the invention and are in no way limiting.

[0193] Example 1

[0194] Placental GBS and the risk of neonatal morbidity: discovery and validation

[0195] Data from the dataset described in de Goffau, M. C. et al. Nature 572, 329-334 (2019) in which GBS was quantified in the placenta from 537 pregnancies was analysed. In this discovery dataset the presence of GBS DNA was defined using either deep sequencing or PCR of the bacterial 16S rRNA gene followed by amplicon sequencing, as described in de Goffau, M. C. et al. (2019). In this analysis, cases were defined as admission of the infant to the neonatal unit (NNU) within 48 hours of birth and for 48 hours or more (see Sovio, U. et al., Lancet 386, 2089-2097 (2015)). Given the high rates of NNU admission of preterm infants, the analysis was confined to 436 births at term (41 cases and 395 controls) (Fig. 1 and Table 2). Controls were defined as any term birth which did not fulfil the case criteria. Of the 436 infants born at term, 7 / 30 (23.3%) with placental GBS DNA and 34 / 406 (8.4%) without placental GBS DNA were cases (odds ratio [OR] = 3.3, 95% confidence intervals (95% Cl) = 1.3- 7.8, p=0.007). The association was similar following adjustment for maternal characteristics (adjusted OR = 3.1, 95% Cl = 1.2-8.0, p=0.02).

[0196] To validate this finding, a case control study was performed using non-overlapping subjects drawn from the same cohort. Cases were defined as pregnancies where the infant was admitted to the NNU without time limits for admission or duration of stay. Controls were infants who were not admitted to the NNU. We excluded preterm births and cases and controls which had already been included in the discovery study described above, leaving 239 cases and 686 controls (Fig. 1 and Table 3). A power calculation indicated >99% power to replicate the original finding with this sample size. Placental GBS DNA status was determined using an ultrasensitive PCR-qPCR assay targeted at a GBS-specific region of the 16S rRNA gene (Fig. 2 and 3). Consistent with the results of the discovery study, 16 / 36 (44.4%) with placental GBS and 223 / 889 (25.1%) without GBS were admitted to the NNU (OR = 2.4, 95% Cl = 1.2-4.6) and the association was similar following adjustment for maternal characteristics (Table 4).

[0197] Comparing septic and non-septic phenotypes of neonatal morbidity

[0198] The inventors hypothesised that the risk of neonatal unit admission was causally explained by the presence of GBS in the placenta. This hypothesis predicts that the association would be progressively stronger as the evidence for neonatal sepsis and inflammation was stronger. Hence, secondary analyses of the validation case control study was performed in relation to associated evidence for sepsis. First, cases of NNU admission were classified by clinical evidence, with classification performed blind to placental GBS DNA status. The infant's clinical record was reviewed and admissions were classified on an ordinal scale: no evidence of sepsis, possible sepsis, probable but culture negative sepsis, or proven GBS sepsis, with proven sepsis being defined on the basis of a positive postnatal culture result from a septic screen (Table 5). There was no association between placental GBS and the risk of NNU admission with no evidence of sepsis or only possible evidence (Table 4). However, there were strong associations with NNU admission with probable but culture negative sepsis (OR = 4.8, 95% Cl = 2.2-10.3) and proven GBS sepsis (OR = 66.6, 95% Cl = 7.3-963.7). Admissions were then classified on the basis of histopathological examination of the fetal membranes and a cross section of the umbilical cord (Fig. 4), again assessed blind to placental GBS DNA status. Inflammation was defined on the basis of a previously described ordinal scale (Stage 0, 1, 2 or 3) for chorioamnionitis and fu nisitis, respectively, and in both cases samples with Stage 2 or 3 findings were defined as positive (see Khong, T. Y. et al. Arch Pathol Lab Med 140, 698-713 (2016)). There was no association between placental GBS and the risk of NNU admission without evidence of chorioamnionitis or funisitis (Table 4). However, there were strong associations between GBS DNA in the placenta and NNU admission with chorioamnionitis (OR = 5.3, 95% Cl = 2.0-13.4) and funisitis (OR = 6.7, 95% Cl = 12.5-17.7). All associations were similar following adjustment for maternal characteristics (Table 4).

[0199] Comparing rates of placental GBS by genital tract colonisation

[0200] Of the 925 participants in the second case control study, 467 (50%) had a report of a high vaginal swab (HVS) during the pregnancy. GBS DNA was detected in the placenta in 7 / 57 (12%) of participants with a positive HVS culture for GBS and 8 / 410 (2%) of those with a negative HVS culture for GBS. Of those who had no HVS culture performed during the pregnancy 21 / 458 (5%) had placental GBS DNA detected. There was an excess of GBS positive placentas in the first group when compared with the second and third (p<0.0001 and 0.02, respectively, Fig. 5a).

[0201] Validating the GBS DNA signal

[0202] The possibility that GBS DNA was detected in the placenta due to contamination was tested. The design and results of the no-template controls from the discovery case control study were reported in de Goffau, M. C. et al. Nature 572, 329-334 (2019). In relation to the validation case control study, there was no temporal relationship between the presence of GBS DNA in the placenta and the week of delivery and there were no clusters of positive cases (Fig. 6). The proportion of infants delivered by caesarean section were compared, where the placenta is delivered abdominally precluding contamination during vaginal birth. A greater proportion of participants with a GBS DNA positive placenta were delivered by caesarean section compared with those who were GBS DNA negative (47% vs 28%, respectively, p=0.01) (Table 6). The proportion of caesarean delivery was even higher (63%) in the GBS DNA positive cases where the infant experienced neonatal morbidity related to sepsis (Table 7). This pattern is the opposite of what would have been predicted if GBS DNA was present in the placenta due to contamination at the time of vaginal birth. The strength of association between having a positive high vaginal swab in the antenatal period and the risk of the outcomes of interest was compared. There were either no statistically significant associations or the associations were weaker than observed with the presence of GBS DNA in the placenta (Table 8). Hence confounding by contamination of the placenta with GBS during vaginal birth is not a plausible explanation for these findings. This interpretation is consistent with the fact that the biopsy was taken from the interior of the placenta and was washed to remove surface contamination (see Methods).

[0203] Next, the inventors considered the potential for contamination during sample analysis. There was also no evidence of batch effects in relation to the conduct of the PCR-qPCR assay for placental GBS (Fig. 7 and Table 9). Moreover, all of the qPCR plates contained multiple negative controls, which were included at every step of the experimental workflow, and none of these (0 / 129 wells) were positive for GBS. To test the validity of the GBS DNA signal further, additional samples were analyses from the 36 placentas which were positive for GBS DNA and from 36 GBS DNA negative placentas using a novel RT-qPCR assay for GBS 16S ribosomal RNA (Fig. 8). RNA, unlike DNA, is highly prone to degradation and is much less likely to be an environmental contaminant. Moreover, this assay did not require to be nested because of the greater amount of template (i.e. rRNA versus DNA) per organism. Despite the fact that the majority of samples had not been collected using a protocol optimised for RNA preservation, the level of agreement in classification of GBS by the presence of 16S RNA and DNA was 87% and this was highly statistically significant (kappa=0.72, p=3.5xl010). Moreover, the case control study was re-analysed and GBS status re-defined based on the presence of both GBS DNA and rRNA, all of the previously identified associations remained highly statistically significant and the associations with sepsis- and inflammation-related outcomes became stronger (Table 10).

[0204] Placental GBS and fetal cytokine storm

[0205] Umbilical cord serum was available from 11 samples from term births across both studies where the placenta was positive for GBS DNA. The levels of four pro-inflammatory cytokines which have previously been associated with GBS were analysed, namely, interleukin (IL)-ip, IL-6, IL-8 and tumour necrosis factor (TNF)-alpha and 11 GBS positive cases were compared with 129 cord serum samples from infants with GBS negative placentas where the infant was not admitted to the NNU. Umbilical cord blood levels of IL-ip, IL-6 and IL-8, but not TNF-alpha were more likely to be elevated in the group where GBS had been detected in the placenta (Fig. 5b). Fetal cytokine storm was defined as two or more pro-inflammatory cytokines which were elevated >10 times the median level in controls. Cytokine storm was observed in 4 / 11 (36%) cases where the placenta was GBS positive and 5 / 129 (4%) placentas negative for GBS DNA: odds ratio = 14.2, 95% Cl 3.6-69.8, p=0.002 (Fig. 5c). There was no association between the presence of GBS DNA in the placenta and levels of pro-inflammatory cytokines in placental protein lysates obtained from the same biopsies (Fig. 5d) or in maternal blood prior to the onset of labour and near term (Fig. 5e). Clinical features of cases of proven GBS sepsis and cases of probable but culture negative sepsis plus the presence of GBS DNA in the placenta

[0206] There was a total of 3,689 term births where placental sampling had been performed. Among this group there were 3 cases of proven GBS sepsis (8.1 [95% Cl 1.7 to 23.7] per 10,000 births), which were all admitted to the NNU. In addition, there were 13 cases of NNU admission with strong clinical or histopathological evidence of sepsis where GBS DNA was detected in the placenta but not postnatal culture of the infant (35.2 [95% Cl 18.8 to 60.2] per 10,000 births) - this is referred to herein as probable but culture negative sepsis. Hence, approximately one birth in 200 had either proven GBS sepsis or had probable but culture negative sepsis plus GBS DNA in the placenta. This is approximately 10 times greater than the currently estimated rate of early neonatal GBS disease following term birth. Clinical characteristics for the 16 cases are tabulated separately by whether there was proven GBS sepsis or probable but culture negative sepsis plus the presence of GBS DNA in the placenta (Table 7). Key observations were that only 3 of the 16 cases had GBS colonisation detected in the antenatal period and only 3 had antibiotics in labour. The majority of the cases were delivered by caesarean section for fetal distress. Interestingly, none of the three cases of proven GBS sepsis had histopathological evidence of inflammation whereas this was present in the majority of cases of probable but culture negative sepsis plus the presence of GBS DNA in the placenta.

[0207] Discussion

[0208] The inventors performed discovery and validation studies and demonstrated that the presence of GBS DNA in the placenta is associated with an increased risk of neonatal morbidity, as evidenced by NNU admission. When analysed by the cause, the association was explained by increased rates of admission with probable but culture negative sepsis and with culture positive, proven GBS sepsis. The validity of these findings was further supported by the fact that there were similar associations when NNU admission was classified on the basis of blinded histopathological examination of the fetal membranes and umbilical cord. Furthermore, the likely mechanism of the observed morbidity in a proportion of these infants is cytokine storm, which was evident in utero. It is plausible that the presence of a pathogen in a normally sterile site would be causally associated with symptoms, signs and biomarkers of sepsis. The associations were similar when GBS was detected by the presence of GBS rRNA and the presence of GBS RNA suggests the presence of live, albeit rare, bacteria. Moreover, the inventors observed that 10-15% of infants who were born with symptoms, signs or laboratory tests suggestive of sepsis had GBS DNA present in the placenta even though the organism was not cultured from the neonate. The inventors concluded that GBS causes about 10 times the number of cases of neonatal morbidity than is currently recognized and that this morbidity is associated with bacterial invasion of the placenta and extreme activation of the fetal innate immune system prior to birth.

[0209] The current analysis demonstrated that only 3 / 16 cases affected by GBS (i.e. proven GBS sepsis or probable but culture negative sepsis where GBS DNA was present in the placenta) received antibiotics in labour. Given the efficacy of IAP to prevent neonatal sepsis, these results suggest that identification of these cases and treatment with antibiotics in labour may have prevented a number of the adverse outcomes.

[0210] The current findings are of clinical relevance in a number of areas. First, the study highlights that analysis of the placenta immediately after birth may allow detection of intrauterine invasion by GBS and thus infants who are at increased risk of neonatal morbidity could be identified. Second, as GBS was clinically undetectable in the neonate in the majority of cases of neonatal morbidity associated with the presence of GBS DNA in the placenta, and as demonstrated previously GBS DNA is present in the placenta in very small amounts, the data indicate that some neonatal morbidity may be due to an exaggerated inflammatory response to a burden of pathogen which is too low to be detected as a clinical infection. Experiments in neonatal mice have demonstrated that administration of small numbers of GBS colony forming units results in an increased risk of neonatal death which can be prevented by immune blockade (genetic or pharmacological).

[0211] The findings described herein indicate that the incidence of adverse neonatal outcome that might be prevented by interventions is much greater than previously thought, being ~1 in 200 as opposed to ~1 per 2,000 births at term. Identifying all cases of neonatal morbidity caused by GBS could make it easier to demonstrate the clinical effectiveness of interventions. Moreover, the potential clinical impact of these interventions may be far greater than estimated based on previous assessments of the burden of disease.

[0212] The strong association between placental GBS and genital tract colonization is consistent with the possibility that the infection originates from the genital tract. However, only a minority of cases with placental GBS DNA had chorioamnionitis which suggests that placental infection by GBS is not invariably due to passage of the organism through the fetal membranes. The timing of invasion is likely to be prior to the onset of labour in a large proportion of affected placentas as the proportion of placentas which were positive for GBS DNA did not vary between women delivered before versus after labour onset and membrane rupture. It is also possible that invasion precedes pregnancy as it is known that GBS can infect the non-pregnant endometrium. Hence it is possible that the GBS detected in the placenta originated from the decidua, i.e. placental infection could occur by implantation of the conceptus in GBS-infected decidua, followed by local invasion of the placenta by decidual GBS. Further studies will be required to address this point. It is of particular interest that none of the three cases of proven GBS sepsis had histopathological features indicating that infection ascended during labour, whereas two out of the three cases had GBS present in the placenta. Although the numbers are small, these findings indicate that antenatal invasion by GBS may be a particular feature of culture proven GBS EOD.

[0213] Strengths of the current study include the fact that it was prospective, that there were hypothesis generating and hypothesis testing elements, and that extensive clinical data were combined with comprehensive biological samples. The latter allowed objective classification of inflammation in 99% of NNU admissions using histopathology. Moreover, collection of cord blood allowed the mechanisms underlying the associations to be addressed. There was a high level of agreement for the detection of GBS analysing different samples from the same placentas using two different methods.

[0214] Table 2. Characteristics of cases and controls from discovery study (n=436). Data are expressed as median (inter-quartile range) or n (%), as appropriate. For fields where there is no category labelled 'missing', data were 100% complete. Maternal age was defined as age at recruitment. All other maternal characteristics were defined by self-report at the 20 weeks questionnaire, from examination of the clinical case record, or linkage to the hospital's electronic databases. Socio-economic status was quantified using the Index of Multiple Deprivation (IMD) 2007, which is based on census data from the area of the mother's postcode and classified into study specific quartiles. FTE denotes full time education; BMI denotes body mass index; CS denotes Caesarean section.

[0215] Cases (n=41) Controls (n=395)

[0216] Characteristic

[0217] Age, years 31 (28 to 34) 30 (27 to 34)

[0218] Age stopped FTE, years 21 (17 to 23) 21 (18 to 23)

[0219] Missing 2 (5) 12 (3)

[0220] BMI, kg / m2 24 (23 to 28) 25 (23 to 28)

[0221] Smoker 6 (15) 25 (6)

[0222] Any alcohol consumption 0 (0) 18 (5)

[0223] Deprivation score quartile

[0224] 1 (lowest) 17 (41) 100 (25)

[0225] 2 7 (17) 78 (20)

[0226] 3 9 (22) 101 (26)

[0227] 4 (highest) 7 (17) 88 (22)

[0228] Missing 1 (2) 28 (7)

[0229] Ethnicity

[0230] Non white 3 (7) 18 (5)

[0231] White 38 (93) 369 (93)

[0232] Missing 0 (0) 8 (2)

[0233] Married 28 (68) 271 (69)

[0234] Induction of labor 19 (46) 137 (35)

[0235] Mode of delivery Unassisted vaginal 12 (29) 174 (44)

[0236] Assisted vaginal 11 (27) 75 (19) Intrapartum CS 9 (22) 74 (19) Pre-labor CS 9 (22) 72 (18)

[0237] Gestational age at delivery, 39.1 (38.0 to 40.3) 40.1 (39.3 to 41.1) weeks

[0238] Birth weight, g 2750 (2375 to 3205) 3375 (2910 to 3660)

[0239] Table 3. Characteristics of cases and controls from validation study (n=925). Data are expressed as median (inter-quartile range) or n (%), as appropriate. For fields where there is no category labelled 'missing', data were 100% complete. Maternal age was defined as age at recruitment. All other maternal characteristics were defined by self-report at the 20 weeks questionnaire, from examination of the clinical case record, or linkage to the hospital's electronic databases. Socio-economic status was quantified using the Index of Multiple Deprivation (IMD) 2007, which is based on census data from the area of the mother's postcode and classified into study specific quartiles. FTE denotes full time education; BMI denotes body mass index; CS denotes Caesarean section.

[0240] Cases (n=239) Controls (n=686)

[0241] Characteristic

[0242] Age, years 31 (2.7 to 35)

[0243] Age stopped FTE, years 21 (.18 to 2.3)

[0244] Missing 8 (3)

[0245] BMI, kg / m2 25 (22 to 28)

[0246] Smoker 11 (5)

[0247] Any alcohol consumption 1.3 (5)

[0248] Missing 0 (0)

[0249] Deprivation score quartile

[0250] 1 (lowest) 45 (19) 157 (23)

[0251] 2 65 (27) 160 (23)

[0252] 3 65 (27) 157 (2.3)

[0253] 4 (highest) 55 (23) 182 (27)

[0254] Missing 9 (4) 30 (4)

[0255] Ethnicity

[0256] Non white 15 (6) 45 (7)

[0257] White 223 (93) 628 (92)

[0258] Missing 1 (cl) 13 (2)

[0259] Married 165 (69) 476 (69)

[0260] Induction of labor 108 (45) 204 (30)

[0261] Mode of delivery Unassisted vaginal 77 (32.) 36.1 (53)

[0262] Assisted vaginal 65 (27) 154 (22)

[0263] Intrapartum CS 81 (34) 109 (16)

[0264] Pre-labor CS 16 (7) 61 (9)

[0265] Missing 0 (0) 1 (cl)

[0266] Gestational age at delivery, 40.4 (39.0 to 4.1.4) 40.4 (39.4 to 41.1) weeks Birth weight, g 3490 (3180 to 3840) 3435 (3155 to 3740) Table 4. Association between the presence of GBS DNA in the placenta and the risk of neonatal unit admission, classified by clinical assessment or histopathology. For univariable analysis, unadjusted odds ratio (OR) with Baptista-Pike mid-p 95% confidence interval (Cl) and chi-square test p value are presented. Due to small numbers, Fisher's exact test p value is given for proven GBS sepsis. For multivariable analysis, OR, 95% Cl and p values were estimated using logistic regression analysis adjusted for maternal characteristics (age, body mass index, smoking and marital status). Due to small numbers, multivariable analysis is omitted for proven GBS sepsis. Chorioamnionitis and funisitis were both present in 4 cases where the placenta was GBS DNA positive and in 19 of the cases when it was negative. The diagnosis for six cases of neonatal unit admission could not be confirmed due to missing information.

[0267] Placental GBS Univariable analysis Multivariable analysis

[0268] Positive n (%) Negative n (%) OR 95% Cl p OR 95% Cl p

[0269] Neonatal unit admission

[0270] Not admitted (N=686) 20 (2.9) 666 (97.1)

[0271] All admissions (N=239) 16 (6.7) 223 (93.3) 2.4 1.2 to 4.6 0.009 2.3 1.4 to 4.5 0.02

[0272] By diagnosis

[0273] No sepsis (N=60) 2 (3.3) 58 (96.7) 1.1 0.3 to 4.6 0.85 1.0 0.2 to 4.4 0.99

[0274] Possible sepsis (N=90) 2 (2.2) 88 (97.8) 0.8 0.2 to 2.9 0.71 0.7 0.2 to 3.2 0.67

[0275] Probable sepsis (N=80) 10 (12.5) 70 (87.5) 4.8 2.2 to 10.3 <0.0001 4.5 2.0 to 10.1 0.0003

[0276] Proven GBS sepsis (N=3) 2 (66.7) 1 (33.3) 66.6 7.3 to 963.7 0.003

[0277] By histopathology

[0278] No inflammation (N=186) 9 (4.8) 177 (95.2) 1.7 0.7 to 3.6 0.19 1.5 0.6 to 3.3 0.36

[0279] Chorioamnionitis (N=44) 6 (13.6) 38 (86.4) 5.3 2.0 to 13.4 0.0002 5.3 2.0 to 14.1 0.0008

[0280] Funisitis (N=30) 5 (16.7) 25 (83.3) 6.7 2.5 to 17.7 0.0001 5.9 2.0 to 17.3 0.001

[0281] Table 5. Ordinal scale for the clinical classification of neonatal unit admission in relation to evidence supporting a diagnosis of sepsis. FBC denotes full blood count; CRP denotes C-reactive protein test. Laboratory evidence for probable sepsis were leucocytosis (white blood cell [WBC] count > 20 x 109 / l_), leucopenia (WBC count < 5 x 109 / l_), thrombocytopenia (platelet count < 150 x 109 / l_) and / or CRP > 10 mg / L. Radiological evidence for probable sepsis were presence of pulmonary infiltrates, consolidation and / or intestinal dilatation.

[0282] Table 6. Association between mode of delivery and placental GBS. Data are expressed as n (%). The proportion of C-sections was higher among patients with placental GBS DNA (47% vs. 28%; OR=2.3 [95% Cl = 1.2-4.4]; Pearson's X2test P=0.01). Mode of delivery was unknown for 1 patient of the validation study.

[0283] Placental GBS

[0284] No Yes Total

[0285] Mode of delivery

[0286] Vaginal 638 (72) 19 (53) 657 (71)

[0287] C-section 250 (28) 17 (47) 267 (29)

[0288] Total 888 (100) 36 (100) 924 (100)

[0289] Table 7. Clinical characteristics of neonatal unit admissions associated with proven GBS sepsis and probable but culture negative sepsis plus the presence of GBS DNA in the placenta. Proven GBS sepsis was defined as an infant admitted to the neonatal unit with cultured GBS from the septic screen. Probable but culture negative sepsis with GBS DNA in the placenta was defined as an infant admitted to the neonatal unit where GBS DNA was detected in the placenta by PCR / qPCR and there was clinical evidence of sepsis or histopathology was positive, but no organism was cultured from the septic screen. Note: (%) is omitted for the outcome proven GBS sepsis as there were only three cases. Positive GBS culture was a report of a high vaginal swab (HVS) confirming the presence of GBS where the result was reported prior to the onset of labour. No HVS indicates that the woman did not have a culture result of a vaginal swab in the pregnancy prior to the onset of labour. Duration of membrane rupture is the interval from the time of membrane rupture to the delivery of the infant, measured in hours. IAP = intrapartum antibiotic prophylaxis, which is benzyl-penicillin in non-allergic women. Other IP antibiotics excludes cases where a single dose of antibiotic was given immediately prior to caesarean delivery as routine prophylaxis. Pyrexia was defined as any measurement of temperature in labour of >38°C, leucocytosis was defined as a maternal blood white cell count greater than 15 X109 / L within 12 hours of delivery, and fetal tachycardia was defined as a fetal heart rate >160 for 10 minutes duration. Acidosis was defined on the basis of either umbilical venous or arterial blood. Chorioamnionitis and funisitis were defined as described in the Methods.

[0290] Proven Probable

[0291] GBS sepsis but culture negative sepsis

[0292] (n=3) plus placental GBS DNA

[0293] (n=13)

[0294] Antenatal

[0295] GBS culture (HVS) Positive, n (%) 1 2 (15)

[0296] Negative, n (%) 0 3 (23)

[0297] No HVS, n (%) 2 8 (62)

[0298] Membrane rupture to <24 hours, n (%) 3 9 (69) delivery interval 24-48 hours, n (%) 0 3 (23)

[0299] >48 hours, n (%) 0 1 (8)

[0300] Intrapartum

[0301] IAP given n (%) 0 0

[0302] Other IP antibiotics n (%) 0 3 (23)

[0303] Indicators of infection Pyrexia, n (%) 0 5 (38)

[0304] Leukocytosis, n (%) 2 10 (77)

[0305] Fetal tachycardia, n (%) 0 6 (46)

[0306] Operative delivery n (%) 1 6 (46) for fetal distress

[0307] Delivery

[0308] Gestational age Median (IQR) 40.1 40.7

[0309] (39.0-41.9) (40.1-41.0)

[0310] Mode Vaginal, n (%) 1 5 (38)

[0311] Caesarean, n (%) 2 8 (62)

[0312] Infant

[0313] Apgar <7 1 minute, n (%) 0 5 (38)

[0314] 5 minute, n (%) 0 1 (8)

[0315] 10 minute, n (%) 0 1 (8)

[0316] Acidosis (pH <7.00) n (%) 0 2 (15)

[0317] Histopathology Chorioamnionitis n (%) 0 7 (54)

[0318] Funisitis n (%) 0 6 (46) Table 8. Association between HVS (GBS positive versus negative swab) and the risk of neonatal unit admission, classified by clinical assessment or histopathology. For univariable analysis, unadjusted odds ratio (OR) with Baptista-Pike mid-p 95% confidence interval (Cl) and chi-square test p value are presented. Due to small numbers, Fisher's exact test P value is given for proven GBS sepsis. For multivariable analysis, OR, 95% Cl and p values were estimated using logistic regression analysis adjusted for maternal characteristics (age, body mass index, smoking and marital status). Due to small numbers, multivariable analysis is omitted for proven GBS sepsis. Chorioamnionitis and funisitis were both present in 6 positive and in 9 negative HVS. The diagnosis for six cases of neonatal unit admission could not be confirmed due to missing information. HVS denotes high vaginal swab.

[0319] HVS Univariable analysis Multivariable analysis

[0320] Positive n (%) Negative n (%) OR 95% Cl p OR 95% Cl p

[0321] Neonatal unit admission

[0322] Not admitted (N=348) 36 (10.3) 312 (89.7)

[0323] All admissions (N=119) 21 (17.6) 98 (82.4) 1.9 1.0 to 3.4 0.04 1.8 1.0 to 3.3 0.05

[0324] By diagnosis

[0325] No sepsis (N=26) 4 (15.4) 22 (84.6) 1.6 0.6 to 4.8 0.42 1.6 0.5 to 5.0 0.42

[0326] Possible sepsis (N=50) 10 (20.0) 40 (80.0) 2.2 1.0 to 4.6 0.05 2.1 1.0 to 4.7 0.06

[0327] Probable sepsis (N=36) 5 (13.9) 31 (86.1) 1.4 0.6 to 3.6 0.51 1.3 0.5 to 3.7 0.59

[0328] Proven GBS sepsis (N=l) 1 (100.0) 0 (0.0) N / A N / A 0.11

[0329] By histopathology

[0330] No inflammation (N=89) 14 (15.7) 75 (84.3) 1.6 0.8 to 3.1 0.15 1.6 0.8 to 3.1 0.18

[0331] Chorioamnionitis (N=28) 7 (25.0) 21 (75.0) 2.9 1.1 to 6.9 0.02 3.2 1.2 to 8.4 0.02

[0332] Funisitis (N=17) 6 (35.3) 11 (64.7) 4.7 1.6 to 14.0 0.002 4.9 1.6 to 14.8 0.005

[0333] Table 9. Distribution of samples by case / control-status and GBS-status in the qPCR plates. Cases and controls of the validation study are defined as described in the Methods and expressed as n (%) in each qPCR plate. GBS positive samples are expressed as n in each qPCR plate.

[0334] Controls

[0335] Plate Cases GBS positives

[0336] 150 (77) 2

[0337] 15 (23) 2

[0338] 240 (74) 0

[0339] 14 (26) 1

[0340] 357 (76) 0

[0341] 18 (24) 1

[0342] 460 (80) 2

[0343] 15 (20) 0

[0344] 27 (73) 3

[0345] 5a

[0346] 10 (27) 2

[0347] 5b & 9a55 (72)°

[0348] 21 (28) 0

[0349] 53 (70) 1

[0350] 6

[0351] 23 (30) 3

[0352] 58 (79) 2

[0353] 15 (21) 1

[0354] 849 (71) 2

[0355] 20 (29) 0

[0356] 9b27 (75) 2

[0357] 9 (25) 0

[0358] 1052 ,74) 1

[0359] 18 (26) 2 n 58 (70) 2

[0360] 25 (30) 2

[0361] 1258 (74) 2

[0362] 20 (26) 1

[0363] 1342 (72) 1

[0364] 16 (28) 1 tot 925 (100) 36

[0365] Table 10. Association between the presence of GBS DNA and RNA in the placenta and the risk of neonatal unit admission, classified by clinical assessment or histopathology. For univariable analysis, unadjusted odds ratio (OR) with Baptista-Pike mid-p 95% confidence interval (Cl) and chi-square test p value are presented. Due to small numbers, Fisher's exact test P value is given for proven GBS sepsis. For multivariable analysis, OR, 95% Cl and p values were estimated using logistic regression analysis adjusted for maternal characteristics (age, body mass index, smoking and marital status). Due to small numbers, univariable and multivariable analyses are omitted for no sepsis and multivariable analysis is omitted for proven GBS sepsis. Chorioamnionitis and funisitis were both present in 4 cases where the placenta was GBS positive and in 19 of the cases where it was negative. The diagnosis for six cases of neonatal unit admission could not be confirmed due to missing information.

[0366] Placental GBS Univariable analysis Multivariable analysis

[0367] Positive n (%) Negative n (%) OR 95% Cl p OR 95% Cl p

[0368] Neonatal unit admission

[0369] Not admitted (N=681) 16 (2.3) 665 (97.7)

[0370] All admissions (N=234) 13 (5.6) 221 (94.4) 2.4 1.2 to 5.1 0.02 2.3 1.1 to 4.9 0.03

[0371] By diagnosis

[0372] No sepsis (N=58) 0 (0.0) 58 (100.0)

[0373] Possible sepsis (N=88) 2 (2.3) 86 (97.7) 1.0 0.2 to 4.0 0.96 0.9 0.2 to 4.1 0.92

[0374] Probable sepsis (N=79) 9 (11.4) 70 (88.6) 5.3 2.2 to 11.9 <0.0001 5.1 2.1 to 12.0 0.0002

[0375] Proven GBS sepsis (N=3) 2 (66.7) 1 (33.3) 83.1 9.0 to 1201.9 0.002

[0376] By histopathology

[0377] No inflammation (N=181) 6 (3.3) 175 (96.7) 1.4 0.6 to 3.6 0.46 1.2 0.4 to 3.1 0.75

[0378] Chorioamnionitis (N=44) 6 (13.6) 38 (86.4) 6.6 2.5 to 18.0 <0.0001 7.1 2.6 to 19.6 0.0001

[0379] Funisitis (N=30) 5 (16.7) 25 (83.3) 8.3 3.1 to 23.5 <0.0001 7.9 2.6 to 24.1 0.0003

[0380] Table 11. Specificity of the PCR-qPCR 16S assay. Bacterial DNA mixes 1, 2 or 3 contained 900 genome copies / well of Streptococcus pyogenes, 900 genome copies / well Streptococcus pneumoniae and 90, 9, 0 genome copies / well of GBS (Streptococcus agalactiae), respectively. Each mix was tested in triplicate using the qPCR assays targeting the sip and 16S GBS genes. Undet, undetectable.

[0381] METHODS

[0382] Patient selection. The inventors used data and samples from a prospective cohort of unselected nulliparous women with a singleton pregnancy and attending the Rosie Hospital, Cambridge UK for antenatal care between 2008 and 2013. The rationale, design and conduct of the study have been described in Sovio, U. et al., Lancet 386, 2089-2097 (2015); Pasupathy, D. et al. BMC Pregnancy Childbirth 8, 51, (2008); and Gaccioli, F. et al., Placenta 59, S17-S25, (2017). In brief, women were recruited around the time of their dating ultrasound scan (typically at ~12 weeks) and were seen for research visits at 20, 28 and 36 weeks of gestational age. Following delivery, a team of technicians performed systematic sampling of the placenta, umbilical cord and fetal membranes, and samples were flash frozen for molecular analysis and fixed for microscopy (see below). Umbilical cord blood was collected from about a third of participants. Outcome data were obtained for all participants by individual examination of the medical record of the mother and by linkage to a range of electronic databases of clinical information, including the results of all microbial cultures performed during the pregnancy. Ethical approval for the study was given by the Cambridgeshire 2 Research Ethics Committee (reference number 07 / H0308 / 163) and all women gave written informed consent. This study is in compliance with all relevant ethical regulations.

[0383] Discovery case control study. Participants eligible for the first case control study were those included in de Goffau, M. C. et al. Nature 572, 329-334 (2019). In brief, these participants included two subgroups (i) participants delivered by planned caesarean section where GBS was detected in the placenta using deep sequencing, and (ii) participants where the presence of GBS DNA was determined by 16S rRNA amplicon sequencing, using DNA obtained from the same placenta using two different DNA extraction kits. The participants in subgroup one were all delivered by planned caesarean section and consisted of 20 women with a diagnosis of preeclampsia, 20 women delivering a small for gestational age infant and 40 controls. The participants in the second subgroup consisted of 100 women with a diagnosis of preeclampsia, 100 women delivering a small for gestational age infant, 100 preterm births, and 200 controls. The women in the second sub-group were not selected in relation to mode of delivery. As there was overlap between the two groups, the two sub-groups combined had data on placental GBS status of 537 participants. The inventors excluded preterm births and one sample failed analysis, resulting in a study group of 436 births at term. Case status was defined as admission of the infant to the neonatal unit (NNU) within 48 hours of birth and for 48 hours or more, which had previously been defined and employed as a marker of significant and immediate neonatal morbidity. Admissions were confined to episodes following delivery (i.e. the definition did not include infants who were discharged from hospital following delivery and subsequently readmitted). Controls were pregnancies where this did not occur. The characteristics of the cases and controls are tabulated (Table 2) and patient selection is presented in a flow diagram (Fig. 1).

[0384] Validation case control study. Participants eligible for the second case control study were pregnancies where delivery occurred at term, the placenta had been biopsied after the birth and the baby was live born. Cases were defined as any eligible participant where the infant was admitted to the neonatal unit without limit of timing or duration. The scope of neonatal admission was broadened as further analyses to define sepsis phenotypes was planned (see below) but, again, cases related solely to the delivery admission. Controls were selected in a ratio of two controls for each case. After selection of cases and controls, the inventors excluded any participants included in the first case control study. The characteristics of the cases and controls are tabulated (Table 3) and patient selection is presented in a flow diagram (Fig. 1).

[0385] The cause of neonatal unit admission was defined on the basis of clinical assessment of the infant's medical record and by histopathology of the fetal membranes and umbilical cord. Both forms of assessment were performed blind to placental GBS DNA status. Clinical assessment was performed by an experienced consultant neonatologist (HW) and admissions were categorised as being unrelated to sepsis (no clinical signs or laboratory tests suggestive of sepsis), possible sepsis (clinical signs of sepsis but no laboratory tests supportive of sepsis), probable sepsis (clinical signs of sepsis plus laboratory tests supportive of sepsis but with a negative septic screen) and proven sepsis (GBS cultured from a normally sterile site) (Table 5). Histopathological examination was performed by an experienced consultant perinatal pathologist (FJ). Membranes were prepared as a roll and cut in cross section, umbilical cord was also cut in a cross section where all cord vessels were included. Sections were stained using H&E and reported blind to placental GBS DNA status, on the basis of a classification system described in Khong, T. Y. et al. Arch Pathol Lab Med 140, 698-713 (2016), where stage 2 or stage 3 evidence was regarded as positive for chorioamnionitis or funisitis, and stage 1 or no evidence was regarded as negative.

[0386] Placental biopsy. Placentas were collected after delivery and the procedure has previously been described in detail in Pasupathy, D. et al. Study protocol. BMC Pregnancy Childbirth 8, 51 (2008). Placental sampling was confined to the placental terminal villi (fetal tissue). Villous tissue was obtained from four separate lobules of the placenta after trimming to remove adhering decidua from the basal plate, i.e. samples were from the interior of the placenta. Areas of the placenta to be biopsied were selected on the basis of no visible damage, hematomas, or infarctions. To remove blood and surface contamination the samples were rinsed in chilled sterile phosphate-buffered saline (Oxoid Phosphate Buffered Saline Tablets, Dulbecco A; Thermo Fisher Scientific dissolved in ultrapure water [ELGA Purelab Classic 18M0.cm]). After collection, all placental samples were frozen in liquid nitrogen and stored at -80°C until further processing. For DNA isolation, approximately 25 mg of villous tissue (combined weight obtained from fragments from all four biopsy collection points) was cut from the stored tissue. Tissue processing was carried out in a Class 2 biological safety cabinet.

[0387] DNA isolation and analysis in the discovery case control study. DNA isolation and the methods for detecting the presence of GBS DNA in the first case control study are described in detail in de Goffau, M. C. et al. Nature 572, 329-334 (2019). In brief, DNA was extracted using an MP Biomedical kit. Deep sequencing was performed using the Illumina XTen platform, 150 base pair, paired-end with an average of 424 million reads per sample. A sample was defined as positive for GBS if there was >1 read aligned to the GBS genome. 16S rRNA PCR was performed using degenerate primers and amplified the V1-V2 region of the gene. Amplicons were sequenced on the Illumina MiSeq platform using 250 base pair, paired-end sequencing. Samples were defined as GBS DNA positive if >1% of amplicons were derived from GBS. Analyses were replicated using the threshold >0.1%. The previous study reported a high level of agreement between deep sequencing and 16S rRNA amplicon sequencing (p=1.5xl0‘8). There were no cases where a sample was positive by 16S rRNA and negative by deep sequencing but there were 4 samples out of 79 which were positive by deep sequencing but negative by 16S rRNA amplicon sequencing. DNA isolation and analysis in validation case control study. DNA was isolated from placental tissue using the QIAamp 96 DNA QIAcube HT Kit (Qiagen, cat 51331) and the QIAcube HT instrument for automated high-throughput nucleic acid purification in 96-well format (Qiagen). The DNA isolation was carried out according to the manufacturer's instructions with the following initial steps. Placental biopsies were lysed in 200pl of lysis buffer, containing 20mM TrisHCI (pH=8) with 2mM EDTA (Sigma, cat T9285), 1.2% Triton X-100 (Sigma, cat T8787), Lysozyme lOmg / ml (Sigma, cat 10837059001), Mutanolysin 300U / ml (Sigma, cat SAE0092), and incubated at 37°C for 30 minutes with 400rpm shaking. After addition of 20pl of Proteinase K / sample (provided by the QIAamp 96 DNA QIAcube HT Kit), samples were incubated at 56°C overnight with 600rpm shaking. The following day the samples were transferred to the 96-well plate and 4pl of RNase A (lOOmg / ml) were added to each sample (Qiagen, cat 19101). After an incubation at room temperature for 5 minutes, the plate was loaded on the QIAcube HT instrument and run using the protocol for DNA isolation with the final elution in 200pl of elution buffer performed twice. Negative controls performed at this stage had tissue lysis buffer carried through the entire experimental workflow (n=17 DNA extraction blanks; Fig. 7). DNA concentrations were determined by Nanodrop Lite (Thermo Fisher Scientific) and samples were diluted to a concentration of 12.5ng / pl with IDTE (10 mM Tris, 0.1 mM EDTA, pH=8) (Integrated DNA Technologies, cat 11-05-01-05), using the PIPETMAX Liquid Handler (Gilson).

[0388] The presence of GBS DNA in placental samples was assessed using a nested multiplex PCR-qPCR assay targeting the GBS sip and 16S rRNA genes, and the human RNaseP gene (RPPH1). Briefly, the first- round (outer) PCR was performed using the Multiplex PCR 5X Master Mix (New England Biolabs, cat M0284S). The sip primers and probe were as previously described in de Goffau, M. C. et al. Nature 572, 329-334 (2019). The PCR-qPCR assay for the 16S rRNA gene utilized a GBS-specific region of the gene (Fig. 2). The following primers were employed for detection of the GBS 16S rRNA gene: forward (outer F) 5'-TAAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 15) and reverse (outer R) 5'- TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20); Amplification was performed in 50pl with primers at a final concentration of 0.2pM and 500ng of placental DNA per reaction. Negative and positive controls performed at this stage had H2O (n=25 reactions) or GBS gDNA (n=100 reactions with different GBS gDNA quantities, i.e. 4000, 400, 40, or 4 copies / well; ATCC, cat BAA-611DQ) as the reaction substrates, respectively (Fig. 7). The PCR amplification profile had an initial step of 95°C for 3 min followed by 15 cycles of 95°C (20 sec), 48°C (60 sec), and 68°C (60 sec), and a final incubation at 68°C for 3 min. The second-round (nested) qPCR was performed using the TaqMan Multiplex Master Mix (Thermo Fisher Scientific, cat 4461882); the sip Taqman assay as previously described with FAM dye / MGB probe, at a final lx concentration (Thermo Fisher Scientific, Assay ID: Ba04646276_sl); a custom-made Taqman assay for the GBS 16S rRNA gene at a final lx concentration: forward primer (inner F) 5'- AATGGACGGAAGTCTGAC-3' (SEQ ID NO: 3), reverse primer (inner R) 5'-GTTAGTTACCGTCACTTGGTA- 3' (SEQ ID NO: 4) and JUN dye / QSY probe 5'-AGAGAAGAACGTTGGTAGGAGTGGA-3' (SEQ ID NO: 27) (primers and probe by Thermo Fisher Scientific); and a human RNaseP TaqMan assay with VIC dye / MGB probe (Gene Symbol: RPPH1, Thermo Fisher Scientific, Assay ID: Hs04930436_gl) at a final 0.5x concentration. In each well, 6.1pl of the first-round PCR (or water in the no template control / blank wells) was used as the reaction substrate in a total volume of lOpl. Negative and positive controls performed at this stage had H2O (n=28 wells) or GBS gDNA (n=112 wells with different GBS gDNA quantities, i.e. 4000, 400, 40, or 4 copies / well) as the reaction substrates, respectively (Fig. 7). The qPCR amplification profile had an initial step of 95°C for 20 sec followed by 50 cycles of 95°C (5 sec), 50°C (10 sec) and 60°C (20 sec). The specificity of this assay was tested by amplifying GBS (Streptococcus agalactiae) DNA in presence of genomic DNA from Streptococcus pyogenes (ATCC, cat 700294DQ) and Streptococcus pneumoniae (ATCC, cat 700669DQ) (Table 11).

[0389] When samples were compared which had a detectable signal for GBS, there was a very strong correlation between the signals for 16S and sip when the Ct count was <30 and no correlation with higher values (Fig. 9). There were 8 placental biopsies which were positive using the 16S PCR-qPCR assay but negative with sip, and no samples were positive by sip but negative by 16S. Moreover, where a sample was positive using both methods, the Ct count was higher (i.e. a weaker signal) for the sip than for the 16S assay (mean difference = 2.4, 95% Cl = 1.0 to 3.7, p=0.0009). Fig. 10 illustrates a representative example. Given that the GBS genome has 7 copies of the 16S rRNA gene but only one copy of sip, is it assumed that there is more template for the 16S assay for a given number of colony forming units (CFU) than for the sip assay. The inventors concluded that the 16S PCR-qPCR assay was more sensitive and defined a positive sample simply on the basis of being positive by 16S PCR-qPCR.

[0390] Analysis of GBS 16S rRNA. Placental RNA was isolated from 72 samples (36 GBS DNA positive and 36 GBS negative placentas form the validation study) using the RNeasy Plus Mini Kit (Qiagen, cat 74134) according to the manufacturer's instructions with the following additional steps. Placental biopsies (approximately 15mg / sample including fragments from all four biopsy collection points) were lysed in 600pl of Buffer RTL Plus and homogenised by bead-beating (Lysing Matrix A tubes, 2 x 30sec, speed 6.0m / sec on a FastPrep-24, MP Biomedical). The subsequent steps were performed according to the manufacturer's instructions and RNA was eluted from the spin columns with 30pl of RNase-free H2O. Reverse transcriptase (RT) reactions were performed using lpg RNA / sample, O.lpM specific primer (outer reverse primer for GBS 16S rRNA described above) and Superscript IV (Invitrogen, cat 18090050) in a total volume of 25pl. Incubations were carried according to the instructions provided in the Superscript IV datasheet (65°C for 5min, ice incubation for 2min, 53°C for 5min and 80°C for lOmin). The RT setup included 2 types of negative controls (omitting the enzyme or the template RNA from the reaction) and 3 positive controls with decreasing concentrations of GBS RNA (100, 20, 4fg / well). The qPCR was performed using 3.6pl of cDNA, the custom-made GBS 16S Taqman assay described above and the TaqMan Multiplex Master Mix in a total volume of 12pl. The qPCR amplification profile had an initial step of 95°C for 20 sec followed by 50 cycles of 95°C (5 sec) and 60°C (25 sec). Samples were run in duplicate and were considered GBS positive if both wells had 16S signal. The qPCR setup included negative and positive controls with H2O or GBS gDNA (4000, 400, 40, or 4 copies / well as described above) as the reaction substrates, respectively (Fig. 7). Analysis of the controls performed at the various steps of the experimental workflow revealed the expected results: no 16S signal in the 20 wells corresponding to the negative controls and signal in 26 / 28 positive controls (signal was not detected in 2 / 4 wells corresponding to the RT positive controls obtained from the lowest GBS RNA quantity, i.e. 4fg / well).

[0391] Assessment of contamination. Assessment of contamination in the discovery case control study is described in detail in de Goffau, M. C. et al. Nature 572, 329-334 (2019). In the validation study an extensive series of control experiments were performed to determine whether the results might be affected by contamination, given that PCR-qPCR assays are highly sensitive and prone to contaminant dependent signals. First, the inventors compared rates of GBS DNA in the placenta in relation to mode of delivery and found that it was less common among infants delivered vaginally (Table 6). This is the opposite pattern to what would be expected if the placenta was usually positive due to contamination from the mother's genital tract. Second, the inventors compared rates of GBS DNA in the placenta in relation to week of delivery. The number of GBS positive placentas demonstrated no temporal association with the week of sampling (Fig. 6) whereas a temporal association with the contaminant signal Deinococcus geothermalis was previously demonstrated (de Goffau, M. C. et al. Nature 572, 329-334 (2019)). Third, all qPCR plates included analysis of positive and negative controls performed throughout the experimental workflow and each plate was designed to have similar numbers of cases and controls (Fig. 7 and Table 9). Analysis of signals by plate demonstrated no pattern in the distribution of positive results. Moreover, the signals from positive and negative controls demonstrated 100% agreement with the expected results. Finally, the inventors re-tested all of the GBS DNA positive placentas using different biopsies and detecting GBS by RT-qPCR of 16S ribosomal RNA, plus the same number of controls and assessed the level of agreement between the two assays and the associations with the clinical outcomes of interest (Table 10). Cytokine ELISAs. Cytokine levels were measured using targeted ELISAs in umbilical cord and maternal serum samples, and placental protein lysates using the Ella platform (Bio-Techne) and cartridges for the analysis of Interleukin-10, lnterleukin-6, lnterleukin-8 and Tumour Necrosis Factor alpha (Simple Plex Cartridge Kit for 32 samples, containing interleukin-10 / interleukin-lF2, interleukin-6 2nd gen, interleukin-8, TNFa 2nd gen, cat SPCKC-PS-003229, Bio-Techne). Positive controls (Human interleukin- 10 cat 894962, Human interleukin-6 cat 894968, Human interleukin-8 / CXCL8 cat 894950, Human TNFa cat 894977, Bio-Techne) were prepared according to the manufacturer's instructions. The inventors ran 145 unique cord serum samples from term pregnancies in 2 batches and cytokine levels were expressed as the multiple of the median (MoM) of GBS negative samples analysed in the same batch. In cases where the median was zero, the lowest detected value was used instead of the median in the calculation. GBS positives were defined as samples with 16S signal using the nested PCR-qPCR assay and / or with one or more GBS read identified by deep sequencing and / or GBS 16S rRNA amplicons >1% in the placenta. Positive samples which were weakly positive (i.e. GBS 16S rRNA amplicons >0% and <1%; n=5) were excluded from the primary analysis. However, the inventors performed a sensitivity analysis where the process was repeated defining GBS positivity as GBS 16S amplicons >0.1% of all reads (Fig. 11). Controls were pregnancies with GBS negative placentas on all tests completed and with infants not admitted to NNU. Therefore, the current analysis included 140 samples, 11 cases and 129 controls.

[0392] Cytokines were also assayed in maternal plasma and in placental biopsies from patients included in the current analysis using the targeted ELISAs. 55 maternal serum samples obtained at 36wkGA were assayed, which were randomly selected among patients with GBS positive (n=25) and negative (n=30) placenta. Cytokines were also measured in protein lysates obtained from 58 term placental samples, which were randomly selected among patients with GBS positive ( n=26) and negative ( n =32 ) placenta. GBS positives and negatives were defined as in the cord serum analysis described above.

[0393] Serum samples were diluted 1:2 with sample diluent (SD13) and run according to the manufacturer's instructions. Placental protein lysates were prepared from the same placental biopsies used for the detection of GBS DNA (5-10mg from each placenta). Tissues were homogenized in 400pl of lysis buffer (Phosphate Buffered Saline with 0.1% Triton X-100 and protease inhibitors (Complete mini-EDTA free protease inhibitor tablet; Roche, cat 04693159001)) by bead-beating in Lysing Matrix D tubes (MP Biomedical, cat 116913050-CF) for 10 seconds at a speed of 4m / second on a FastPrep-24 (MP Biomedical). Lysates were centrifuged twice at 10,000g for 10 minutes at 4°C and the supernatants were quantified using the Pierce BCA Protein Assay Kit (ThermoFisher, cat 23227). Placental lysates were not diluted with SD13 prior to loading them on the cartridges. Statistics. Proportions were compared using the chi-square test or Fisher's exact test, as appropriate, and Wilson 95% confidence intervals (Cl) calculated. Ninety five percent Cl for unadjusted odds ratios were calculated using the combination of the Baptista Pike method and the mid-p approach. Odds ratios adjusted for maternal characteristics (age, BMI, smoking and marital status) were estimated using multivariable logistic regression. The sample size for the POP study cohort was pre-determined by the total recruitment achieved. Power calculations were performed for all analyses using the available sample size to estimate the statistical power to detect a given strength of association based on alpha (two-sided) = 0.05. The power calculation for the primary outcome (admission of the infant to the neonatal unit) was based on observing the same relative risk and proportions of exposure and outcome in the validation study as observed in the development study. Power calculations for all the secondary outcomes (fetal cytokine storm, probable and proven sepsis, chorioamnionitis and funisitis) were based on the observed numbers of GBS positive and GBS negative placentas in the validation study, the observed proportion of the given outcome in the GBS negative group of the validation study, and a range of possible odds ratios. The results were plotted for each of the secondary outcomes (Fig. 12). All p values were two sided and all analyses were performed using Stata version 17 (StataCorp LLC) and GraphPad Prism version 9.2.0 (GraphPad Software LLC).

[0394] Example 2

[0395] Nested PCR / qPCR assay for detection of the Streptococcus agalactiae (Group B Streptococcus, GBS) 16S rRNA gene.

[0396] Aim: To test the specificity of a nested PCR / qPCR assay targeting the GBS 16S rRNA gene. The 16S rRNA gene has regions which are highly conserved among bacterial species and thus designing a species-specific assay is challenging. Seven copies of this gene are present in the GBS genome, thus enhancing the sensitivity of the assay.

[0397] Methods: Minor modifications were made to the nucleotide sequence of the primers used in Example 1 (outer PCR and inner qPCR) and probe (inner qPCR) (Table 12). The modified probes and primers were then tested in the PCR or qPCR assays using gDNA from GBS, Streptococcus pneumoniae and Streptococcus pyogenes as templates. Results:

[0398] 1) Modifications of 16S outer primers.

[0399] The inventors performed the PCR assay described in Example 1 using the original primers or a set of modified primers (outer forward primer with 5'-shift of 3 nucleotides and outer reverse primer with 5'-shift of 2 nucleotides; sequences listed in Table 12). These modified primers are less specific and allowed detection of the 16S gene from all three Streptococcus species tested. Results were similar when the PCR reaction was performed with a Ta of 48 °C or 43 °C (Fig. 13 and Table 13). Note, the original primers amplify both the GBS and S. pyogenes 16S genes, indicating that optimal specificity is achieved when both the outer PCR and inner qPCR are performed. Thus, the outer primers alone are not sufficient to confer specificity for GBS.

[0400] 2) Modifications of 16S inner reverse primer.

[0401] A modification of the 16S inner reverse primer (5'-sh ift of 6 nucleotides; sequence listed in Table 12) reduces the specificity of the PCR assay and allowed detection of S. pneumoniae 16S gene (Fig. 13 and 14, and Table 13). Primers with the original sequence only amplified the GBS 16S gene. Results are similar when the PCR reaction was performed with an annealing temperature (Ta) of 60 °C or 53 °C.

[0402] 3) Modifications of 16S probe.

[0403] The inventors performed the qPCR assay using the modified set of primers above (which detect both GBS and S. pneumoniae 16S genes) and two modifications of the probe (sequences listed in Table 12): i) 4 nucleotides shorter on the 3'end; ii) 3'-shift of 2 nucleotides. Both these modifications allowed detection of GBS but not S. pneumoniae and S. pyogenes 16S genes. Results are similar when the qPCR reaction was performed with a Ta of 60 °C or 53 °C. This result demonstrates that the probe is important for specificity. Therefore, it also contributes to the assay sensitivity allowing a nested approach to be performed and / or the number of PCR cycles to be increased. Furthermore, the probe tolerates some change in sequence at the ends and this points to the importance of the central portion of the probe. Table 12. 16S primers and probes.

[0404] Table 13. Summary of the signals obtained with the modified 16S primers and probes.

[0405] Example 3

[0406] Further improved detection of the Streptococcus agalactiae (Group B Streptococcus, GBS) 16S ribosomal RNA using a reverse transcription-nested PCR / qPCR assay.

[0407] Rationale: As noted above, the inventors have developed a DNA-based method which includes a nested PCR / qPCR assay and targets the GBS-specific regions of the 16S rRNA gene. As there are 7 copies of this gene per genome, this improves the assay sensitivity. The inventors have further developed an RNA-based version of the assay that detects the same region of the 16S ribosomal RNA (rRNA) itself and includes a reverse transcription (RT) step and a qPCR step. As there many thousands of ribosomes in a bacterium this approach further enhance the test sensitivity. However, this approach is extremely challenging because ribosomal RNAs are very related across species. Therefore, designing specific primers for the RT step and the PCR steps is critical. Despite these challenges, the inventors further developed an additional RNA-based assay which introduces a new GBS-specific primer in the RT reaction (located approximately 350 bases downstream the region targeted by the nested PCR / qPCR) followed by the specific nested PCR / qPCR assay used in the above-mentioned DNA-based assay. Moreover, as the RT step is now followed by a nested PCR / qPCR (rather than a qPCR), this approach provides stronger signals in the true positives (while the negatives remain negative), thereby further increasing robustness and reliability of the assay.

[0408] Aim: To test the sensitivity and specificity of the further developed RT-PCR / qPCR assay targeting the GBS 16S ribosomal RNA. The 16S rRNA has regions which are highly conserved among bacterial species. The inventors aimed to test whether an RNA-based assay could achieve further increased sensitivity while maintaining the same specificity of the above-mentioned DNA- and RNA-based assays.

[0409] 1) Design of the new RT primers targeting the GBS 16S rRNA.

[0410] The inventors identified and selected a region of the GBS 16S rRNA with high variability compared to other Streptococci species and other bacteria which are normally part of the vaginal microbiome. An overview of the design process is provided by Figure 15.

[0411] The inventors then developed two candidate RT primers which span in this highly variable region (also shown in Figure 15):

[0412] - Candidate Primer 1: 5'-TATCTCTAGGCCGGTCAGA-3' (SEQ ID NO: 32)

[0413] - Candidate Primer 2: 5'-AGAAAGCCTATCTCTAGG-3' (SEQ ID NO: 34) Methods: After designing the new RT primer candidates, the inventors tested the new experimental workflow using relevant clinical samples, i.e. high vaginal swabs (HVS). 2) Testing the performance of the further improved RT-PCR / qPCR assay.

[0414] The inventors extracted total RNA from clinical HVS samples (n=39), and the newly designed RT candidate primers were tested by performing the RT-PCR / qPCR assay on these RNA samples. Results are reported for Candidate Primer 2 as an example. As shown in Table 14, the inventors demonstrated that 5 samples were positive for GBS 16S rRNA using the above-mentioned RNA-based RT-qPCR assay. The same samples were positive when we performed the new RT-PCR / qPCR assay on the same RNA samples. The increased sensitivity is demonstrated by the fact that the Ct values are lower using the further improved assay (more than 5 Ct difference). The samples which were negative still had no signal, demonstrating that the assay specificity was maintained with the new workflow. Table 14. RT-PCR / qPCR assay performed on total RNA extracted from high vaginal swabs. Each sample was run in triplicated (technical replicate), nd: not detected; Ct: cycle threshold; SD: standard deviation.

Claims

CLAIMS1. A method of screening for risk of neonatal sepsis in a neonate comprising screening for Streptococcus agalactiae (Group B Streptococcus, GBS) in a sample of placenta, wherein the presence of GBS in the placenta indicates an increased risk of neonatal sepsis in the neonate.

2. The method of claim 1, wherein if GBS is detected in the placenta, the method further comprises screening the neonate for signs of infection, optionally wherein screening the neonate for signs of infection comprises one or more of:(a) culture from a blood sample and / or swab sample;(b) full blood count and differential;(c) C-reactive protein assay;(d) lumbar puncture; and(e) radiological tests.

3. The method of claim 1 or claim 2, wherein the method further comprises testing the level(s) of one or more inflammatory cytokines in the umbilical cord blood, plasma or serum, optionally wherein the one or more inflammatory cytokines comprise interleukin (IL)-ip, IL-6 and / or IL-8.

4. The method of any preceding claim, wherein the mother of the neonate has previously tested positive for GBS infection by vaginal / rectal swab.

5. The method of any preceding claim, wherein the mother of the neonate tested positive for GBS infection during a previous pregnancy and / or has previously given birth to a neonate who suffered neonatal sepsis.

6. The method of any preceding claim, wherein the sample of placenta is obtained after delivery of the neonate.

7. The method of any preceding claim, wherein the sample of placenta comprises placental terminal villi.

8. The method of any preceding claim, wherein the method comprises screening for GBS nucleic acid in the placenta, optionally wherein GBS nucleic acid is detected by polymerase chain reaction (PCR), optionally quantitative PCR (qPCR).

9. The method of any preceding claim, wherein the method comprises screening for GBS RNA in the placenta, optionally wherein GBS RNA is detected by reverse transcription PCR, optionally reverse transcription quantitative PCR (RT-qPCR).

10. The method of claim 8 or claim 9, wherein the nucleic acid is 16S rRNA.

11. A method of treating a neonate identified as being at an increased risk of neonatal sepsis by the method of any of claims 1-10, the method comprising administering antibiotics to the neonate.

12. A composition comprising an antibiotic for use in a method of treating a neonate identified as being at an increased risk of neonatal sepsis, wherein the neonate is identified as being at an increased risk of neonatal sepsis by the method of any one of claims 1-10.

13. The method of claim 11 or the composition for use according to claim 12, wherein the neonate exhibits one or more signs of infection and / or sepsis.

14. The method of claim 11 or claim 13 or the composition for use according to claim 12 or claim 13, wherein the antibiotic comprises penicillin and / or gentamicin.

15. The method of any of claims 11, 13 or 14 or the composition for use according to any of claims 12-14, wherein the antibiotic is administered to the neonate within 6 hours of birth.

16. A method for detecting GBS in a sample, the method comprising:(a) contacting nucleic acid extracted from the sample with reagents for amplifying a DNA fragment of GBS 16S rRNA gene under reaction conditions suitable for amplifying said DNA fragment, wherein the reagents comprise a forward primer having a nucleic acid sequence comprising 5'-ACGGAAGTCTGAC-3' (SEQ ID NO: 1) and a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2); and(b) detecting the presence of the fragment, wherein presence of the fragment is indicative of the presence of GBS in the sample.

17. The method of claim 16, wherein reagents for amplifying the DNA fragment comprise:(a) a forward primer having a nucleic acid sequence comprising 5'-AATGGACGGAAGTCTGAC- 3' (SEQ ID NO: 3); and / or(b) a reverse primer having a nucleic acid sequence comprising 5'- GTTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 4).

18. The method of claim 16 or claim 17, wherein the method comprises:(a) contacting nucleic acid extracted from the sample with reagents for amplifying a first DNA fragment of the GBS 16S rRNA gene under reaction conditions suitable for amplifying said first DNA fragment, wherein the reagents for amplifying the first fragment comprise a forward primer having a nucleic acid sequence comprising 5'-GGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 13) and a reverse primer having a nucleic acid sequence comprising 5'- CAGTTTCCAAAGCGT-3' (SEQ ID NO: 14); then(b) contacting the nucleic acid with reagents for amplifying a second DNA fragment under reaction conditions suitable for amplifying said second DNA fragment, wherein the second DNA fragment is a portion of the first fragment and wherein the reagents for amplifying the second fragment comprise a forward primer having a nucleic acid sequence comprising 5'- ACGGAAGTCTGAC-3' (SEQ ID NO: 1) and a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2); and(c) detecting the presence of the second fragment, wherein presence of the second fragment is indicative of the presence of GBS in the sample.

19. The method of claim 18, wherein:(a) the reagents for amplifying the first DNA fragment comprise:(i) a forward primer having a nucleic acid sequence comprising5'-TAAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 15); and / or(ii) a reverse primer having a nucleic acid sequence comprising5'-TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20); and / or(b) the reagents for amplifying the second DNA fragment comprise:(i) a forward primer having a nucleic acid sequence comprising5'-AATGGACGGAAGTCTGAC-3' (SEQ ID NO: 3); and / or(ii) a reverse primer having a nucleic acid sequence comprising5'-GTTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 4).

20. The method of any of claims 16-19, wherein detecting the presence of the fragment comprises contacting the nucleic acid with a probe configured to hybridize to the fragment and to emit a detectable signal when hybridised to the fragment.

21. The method of claim 20, wherein the probe has a nucleic acid sequence comprising 5'- CGTTGGTAG-3' (SEQ ID NO: 25); optionally wherein the probe has a nucleic acid sequence comprising:(a) 5'-AGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 26);(b) 5'-AGAGAAGAACGTTGGTAGGAGTGGA-3' (SEQ ID NO: 27);(c) 5'-AGAGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 28); or(d) 5'-AGAAGAACGTTGGTAGGAGTGGAAA-3' (SEQ ID NO: 29).

22. A method of detecting GBS in a sample, the method comprising:(a) contacting nucleic acid extracted from the sample with a probe having a nucleic acid sequence comprising 5'-AGAAGAACGTTGGTAGGAG -3' (SEQ ID NO: 26); and(b) detecting binding of the probe to the nucleic acid, wherein binding of the probe to the nucleic acid is indicative of the presence of GBS in the sample.

23. The method of claim 22, wherein the probe has a nucleic acid sequence comprising:(i) 5'-AGAGAAGAACGTTGGTAGGAGTGGA-3' (SEQ ID NO: 27);(ii) 5'-AGAGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 28); or(ill) 5'-AGAAGAACGTTGGTAGGAGTGGAAA-3' (SEQ ID NO: 29).

24. The method of claim 22 or claim 23, wherein the method comprises a step of contacting the nucleic acid with reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene (SEQ ID NO: 30) under reaction conditions suitable for amplifying said DNA fragment.

25. The method of claim 24, wherein the reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene comprise:(i) a forward primer having a nucleic acid sequence comprising 5'-ACGGAAGTCTGAC-3' (SEQ ID NO: 1), optionally wherein the forward primer has a nucleic acid sequence comprising 5'-AATGGACGGAAGTCTGAC-3' (SEQ ID NO: 3); and / or(ii) a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2); optionally wherein the reverse primer has a nucleic acid sequence comprising 5'-GTTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 4).

26. The method of claim 24 or claim 25, wherein contacting the nucleic acid with reagents for amplifying a DNA fragment comprising residues 422-452 of the GBS 16S rRNA gene is preceded by a step of contacting nucleic acid extracted from the sample with reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene (SEQ ID NO: 30) under reaction conditions suitable for amplifying said DNA fragment; optionally wherein the reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene comprise:(i) a forward primer having a nucleic acid sequence comprising 5'-GGAGCAATTGCTTCACTGTG- 3' (SEQ ID NO: 13), optionally wherein the forward primer has a nucleic acid sequence comprising 5'-TAAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 15); and / or(ii) a reverse primer having a nucleic acid sequence comprising 5'-CAGTTTCCAAAGCGT-3' (SEQ ID NO: 14), optionally wherein the reverse primer has a nucleic acid sequence comprising 5'-TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20).

27. The method of any of claims 20-26, wherein the probe comprises a fluorescent tag, optionally wherein the probe further comprises a quencher which is configured to be cleaved from the probe after the probe has hybridised to the fragment.

28. The method of any of claims 16-27, wherein the nucleic acid extracted from the sample is RNA and the method is preceded by a step of reverse transcribing the RNA, optionally wherein reverse transcription is performed using a primer having a nucleic acid sequence comprising:(a) 5'-TCTAGGCCGGTCAGA-3' (SEQ ID NO: 31), optionally a primer having a nucleic acid sequence comprising 5'-TATCTCTAGGCCGGTCAGA-3' (SEQ ID NO: 32);(b) 5'-AGCCTATCTCTAGG-3' (SEQ ID NO: 33), optionally a primer having a nucleic acid sequence comprising 5'-AGAAAGCCTATCTCTAGG-3' (SEQ ID NO: 34); and / or(c) 5'-CAG l I I CCAAAGCGT-3' (SEQ ID NO: 14), optionally a primer having a nucleic acid sequence comprising 5'-TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20).

29. The method of any of claims 16-28, wherein the sample is a sample of placenta.

30. The method of any of claims 16-28, wherein the sample is a biological sample obtained from a patient.

31. The method of claim 30, wherein the sample is selected from a sample obtained by vaginal and / or rectal swab and a neonatal blood sample.

32. The method of claim 30, wherein the sample is an endometrial biopsy.

33. The method of any of claims 1-10, wherein screening for GBS in the sample of placenta comprises performing the method of any of claims 16-29.

34. A composition comprising a forward primer having a nucleic acid sequence comprising 5'-ACGGAAGTCTGAC-3' (SEQ ID NO: 1) and / or a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2).

35. The composition of claim 34, wherein the forward primer has a nucleic acid sequence comprising 5'-AATGGACGGAAGTCTGAC-3' (SEQ ID NO: 3) and / or the reverse primer has a nucleic acid sequence comprising 5'-GTTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 4).

36. A composition comprising a forward primer having a nucleic acid sequence comprising 5'- GGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 13) and / or a reverse primer having a nucleic acid sequence comprising 5'-CAGTTTCCAAAGCGT-3' (SEQ ID NO: 14).

37. The composition of claim 36, wherein the forward primer has a nucleic acid sequence comprising 5'-TAAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 15) and / or the reverse primer has a nucleic acid sequence comprising 5'-TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20).

38. The composition of any of claims 34-37, wherein the composition further comprises a probe having a nucleic acid sequence comprising 5'-CGTTGGTAG-3' (SEQ ID NO: 25).

39. A composition comprising a probe having a nucleic acid sequence comprising 5'-CGTTGGTAG-3' (SEQ ID NO: 25).

40. The composition of claim 38 or claim 39, wherein the probe has a nucleic acid sequence comprising:(a) 5'-AGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 26);(b) 5'-AGAGAAGAACGTTGGTAGGAGTGGA-3' (SEQ ID NO: 27);(c) 5'-AGAGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 28); or(d) 5'-AGAAGAACGTTGGTAGGAGTGGAAA-3' (SEQ ID NO: 29).

41. The composition of any of claims 34-40, wherein the composition further comprises a primer having a nucleic acid sequence comprising:(a) 5'-TCTAGGCCGGTCAGA-3' (SEQ ID NO: 31), optionally a primer having a nucleic acid sequence comprising 5'-TATCTCTAGGCCGGTCAGA-3' (SEQ ID NO: 32); or(b) 5'-AGCCTATCTCTAGG-3' (SEQ ID NO: 33), optionally a primer having a nucleic acid sequence comprising 5'-AGAAAGCCTATCTCTAGG-3' (SEQ ID NO: 34).

42. A kit comprising reagents for amplifying a DNA fragment comprising residues 370-452 of the GBS 16S rRNA gene (SEQ ID NO: 30); and a probe for detecting the fragment, wherein:(a) the reagents for amplifying a DNA fragment comprising residues 370-452 of the GBS 16S rRNA gene comprise a forward primer having a nucleic acid sequence comprising 5'- ACGGAAGTCTGAC-3' (SEQ ID NO: 1) and a reverse primer having a nucleic acid sequence comprising 5'-TTACCGTCACTTGGTA-3' (SEQ ID NO: 2); and / or(b) the probe has a nucleic acid sequence comprising 5'-CGTTGGTAG-3' (SEQ ID NO: 25).

43. The kit of claim 42, wherein the reagents for amplifying a DNA fragment comprising residues 370-452 of the GBS 16S rRNA gene comprise:(a) a forward primer having a nucleic acid sequence comprising 5'-AATGGACGGAAGTCTGAC- 3' (SEQ ID NO: 3); and / or(b) a reverse primer having a nucleic acid sequence comprising 5'- GTTAGTTACCGTCACTTGGTA-3' (SEQ ID NO: 4).

44. The kit of claim 42 or claim 43 further comprising reagents for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene (SEQ ID NO: 30); optionally wherein the reagent for amplifying a DNA fragment comprising residues 205-609 of the GBS 16S rRNA gene comprise:(a) a forward primer having a nucleic acid sequence comprising 5'- GGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 13), optionally a forward primer having a nucleic acid sequence comprising 5'-TAAAAGGAGCAATTGCTTCACTGTG-3' (SEQ ID NO: 15); and / or(b) a reverse primer having a nucleic acid sequence comprising 5'-CAG l I I CCAAAGCGT-3' (SEQ ID NO: 14), optionally a reverse primer having a nucleic acid sequence comprising 5'- TCCTCCAGTTTCCAAAGCGT-3' (SEQ ID NO: 20).

45. The kit of any of claims 42-44, wherein the probe comprises a fluorescent tag, optionally wherein the probe further comprises a quencher which is configured to be cleaved from the probe after the probe has hybridised to the fragment.

46. The kit of any of claims 42-45, wherein the probe has a nucleic acid sequence comprising:(a) 5'-AGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 26)(b) 5'-AGAGAAGAACGTTGGTAGGAGTGGA-3' (SEQ ID NO: 27);(c) 5'-AGAGAAGAACGTTGGTAGGAG-3' (SEQ ID NO: 28); or(d) 5'-AGAAGAACGTTGGTAGGAGTGGAAA-3' (SEQ ID NO: 29).

47. The kit of any of claims 42-46, wherein the kit further comprises a primer having a nucleic acid sequence comprising:(a) 5'-TCTAGGCCGGTCAGA-3' (SEQ ID NO: 31), optionally a primer having a nucleic acid sequence comprising 5'-TATCTCTAGGCCGGTCAGA-3' (SEQ ID NO: 32);(b) 5'-AGCCTATCTCTAGG-3' (SEQ ID NO: 33), optionally a primer having a nucleic acid sequence comprising 5'-AGAAAGCCTATCTCTAGG-3' (SEQ ID NO: 34).

48. The kit of any of claims 42-47, wherein the kit further comprises reverse transcriptase.

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