he incidence of group B Streptococci in pregnant women varies significantly by location, ranging from 13.6% in Windhoek, Namibia, 21.2% in Malawi , 31.6% in Zimbabwe, 37% in South Africa , 1.8% in Mozambique, 15.7% in Ethiopia, 19% in Ivory coast, 22% in Gambia (Haimbodi et al., 2021).

1. INVESTIGATING THE PREVALENCE OF GROUP B
Streptococci AMONG PREGNANT WOMEN
ATTENDING ANTENATAL CLINIC AT EKITI STATE
UNIVERSITY TEACHING HOSPITAL, ADO-EKITI.
A PROPOSAL PRESENTATION
BY
OMOLAYO, ESTHER YETUNDE
22/PGC/SCI05/001
DEPARTMENT OF BIOLOGICAL SCIENCES
COLLEGE OF SCIENCES
AFE BABALOLA UNIVERSITY, ADO-EKITI.

2. OUTLINE
Introduction
Literature Review
Justification
Aims and Objectives
Scope of the study
Materials and Methods
References

3. INTRODUCTION
 Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is
a Gram- positive bacteria (Nguyen et al., 2021).
 it is a natural commensal found in healthy people's gastrointestinal and
genitourinary systems, is found in the vaginal tract of 20–60% of pregnant
women (Nguyen et al., 2021)
 GBS colonizes almost 20–40% of healthy women, and 50–70% of the
children born to these mothers get the disease. GBS mortality in neonates is
above 50%, and preterm infants are particularly at risk (Nguyen et al.,
2021).
 Premature labor and stillbirths are linked to vaginal colonization by GBS
during pregnancy (Nguyen et al., 2021).

4. INTRODUCTION CONTD
 In 2009, it was noted that 16% of pregnant women in Africa had the
median prevalence of GBS
 In Windhoek, Namibia, the frequency was 13.6% in 2016 (Jiao et al.,
2022).
 In addition, other Southern African nations recorded colonization rates
of 21.2% in Malawi in 2010 and 1.8% in Mozambique in 2008,
31.6% in Zimbabwe in 2000; 37% in South Africa in 2018 (Monyama
et al., 2018)
 While GBS does not affect healthy adults, it does represent a
considerable risk to certain populations, particularly pregnant women
and their newborns

5. INTRODUCTION CONTD
 GBS is recognized as a primary cause of newborn infections, including
sepsis, pneumonia, and meningitis, particularly within the first week of birth
(early-onset disease), and infrequently beyond the first week of life (late-
onset disease) (Dong et al., 2020).
 GBS infections in neonates can result in long-term neurological
impairments or death, making it a serious concern in perinatal healthcare
(Dong et al., 2020).
 GBS colonization occurs largely in the vagina of pregnant women,
potentially leading to maternal infections, urinary tract infections,
chorioamnionitis, postpartum endometritis and in rare cases bacteremia
(Puopolo et al., 2019).
 As a result, during the perinatal period, neonates become vulnerable to
invasive GBS infections (Hayes et al., 2020). The primary virulence factor for
GBS is the capsule.

6. INTRODUCTION CONTD
 Neonatal sepsis claims one million lives annually in
low-income nations during the first four weeks of
life, making up around 23% of all live births
According to the CDC (2016), Nigeria had a
newborn mortality rate of 35 per 1000 live births in
2015, with sepsis being the main cause
 Unfortunately, this threat that kills our newborns
has been discredited as a myth by numerous cultures
and tribes; in Yoruba, it is known as "ABIKU."
Neonatal deaths have a scientifically proven reason.

7. Group B Streptococcus (GBS) is emerging as the
primary cause of neonatal sepsis and meningitis,
according to studies in various sub-Saharan African
nations like Nairobi, Ghana, Gambia, and Cameroon
(Ezeonu & Agbo, 2014).
Nigeria, like many other low- and middle-income
countries, is struggling to develop routine GBS
screening programs and provide prompt and adequate
antibiotic delivery to avoid prenatal GBS illness
INTRODUCTION CONTD

8. LITERATURE REVIEW
 Streptococcus agalactiae, a faultative anaerobe known as group
B streptococcus or GBS, is beta-hemolytic and catalase-negative,
gram-positive coccus (Carreras-Abad et al., 2020).
 Rebecca Lancefield distinguished Streptococcus agalactiae, now known as
group B Streptococcus (GBS), from other streptococci for the first time in
the 1930s(Carreras-Abad et al., 2020).
 Lancefield observed the colonization of GBS in asymptomatic women's
vaginal tracts, but human pathogenicity was not described until 1938, when
three accounts of fatal postpartum infection were published (Carreras-Abad
et al., 2020).

9. LITERATURE REWIEW CONT.
Lancefield observed the colonization of GBS in
asymptomatic women's vaginal tracts, but human
pathogenicity was not described until 1938, when three
accounts of fatal postpartum infection were published
(Carreras-Abad et al., 2020).
According to Seale et al. (2017) systematic study, GBS
was projected to have resulted in 319 000 instances of
invasive newborn GBS illness and 90 000 fatalities
worldwide in 2015. As a result, at least 57,000
stillbirths and up to 3.5 million premature births may
potentially be linked to GBS (Seale et al., 2017)

10. LITERATURE REVIEW CONT.
S. agalactiae infections, can lead to endocarditis,
pneumonia, bacteremia, and urinary tract infections,
and are significantly more common in adults with
chronic diseases like diabetes, cancer, and HIV
Graux et al., 2021).
 it has been classified into ten serotypes (Ia, Ib, II,
III, IV, V, VI, VII, VIII, IX) based on changes in the
capsular polysaccharides ( Hsu et al., 2021).

11. LITERATURE REVIEW CONTD
 it is a primary cause of newborn sepsis, pneumonia, and meningitis,
primarily affecting infants under the age of three months (Goncalves et al.,
2022).
 GBS can induce invasive infections in nonpregnant individuals, particularly
those with underlying health issues, in addition to having a significant
impact on neonates (Bianchi-Jassir et al., 2020).
 Recent studies have found an increase in the prevalence of invasive GBS
disease in adults, highlighting the importance of continued surveillance and
study (Zwietering et al., 2020; Bianchi-Jassir et al., 2020).

12. LITERATURE REVIEW CONT
Early-onset disease can occur in neonates within the
first week of life, while late-onset disease can occur
within the first three months of life (Goncalves et
al., 2022).
Resistance to antibiotics, particularly penicillin and
erythromycin among GBS strains offers
considerable hurdles in the treatment of GBS
infections (Elsherif et al., 2020)

13. LITERATURE REVIEW CONT.
Clinical implication of GBS include;
 Chorioamnionitis
 Endometritis

14. LITERATURE REVIEW CONT
Risk factors associated with GBS include;
• Maternal Age,
 Gestational diabetes,
 Prior GBS colonization,
 Genital partinfection
 Nulliparity,
 Prolonged Rupture of Membranes,
 Intrapatum fever (De Seta et al., 2022).
 It is been transmitted from infected mother to child during delivery
(Kwatra et al., 2016).

15. LITERATURE REVIEW CONT.
GBS infections that are invasive, such as bacteremia
and meningitis, require immediate and appropriate
antibiotic treatment (Zhu and Lin, 2021).
Based on the clinical circumstances, high-dose
intravenous penicillin, ampicillin, or ceftriaxone are
routinely utilized.

16. SCOPE OF THE STUDY
 This study will examine pregnant women attending the
antenatal clinic of Ekiti State University Teaching Hospital,
Ado-Ekiti, Ekiti State Nigeria for Streptococcus agalactiae.
 Sample collections and analyses will be run simultenoeusly
through the period. The prevalence of S. agalactiae and the
outcome of delivery among pregnant women will be
investigated in this study.

17. JUSTIFICATION
 The incidence of group B Streptococci in pregnant women varies
significantly by location, ranging from 13.6% in Windhoek, Namibia,
21.2% in Malawi , 31.6% in Zimbabwe, 37% in South Africa , 1.8% in
Mozambique, 15.7% in Ethiopia, 19% in Ivory coast, 22% in Gambia
(Haimbodi et al., 2021).
 In Nigeria, 8.3% in Ibadan, 11.3% in Ile-Ife, 4.3% in Ogun, 15.8% in
Enugu, 8.6% in Zaria, 9.0% in Calabar (Akadri et al., 2019).
 However, information of group B Streptococcus in Ekiti is scanty and the
molecular identification of the organism has not been reported in the state.
It is therefore germane to assess this condition and its risk factors in Ekiti,
in order to provide guideline in our local environment and contributes to the
body of scientific knowledge

18. RESEARCH OBJECTIVES
The Objectives of the study are to:
 determine the prevalence of group B Streptococcus among pregnant women attending
antenatal clinic in EKSUTH
 identify the risk factors associated with Group B Streptococcus infection among
pregnant women
 isolate and identify Streptococcus agalactiae associated with the pregnant women
 determine the antibiotic susceptibility profiles of Streptococcus agalactiae
 carry out molecular Identification of Streptococcus agalactiae strains prevalent among
the pregnant women

19. MATERIALS AND METHODS
Study Design
 The prevalence of (GBS) among pregnant women attending prenatal clinics
at Ekiti State University Teaching Hospital, Ado Ekiti, will be determined
using a cross-sectional research methodology
 This methodology will enable data to be collected at a given period
in time, offering insights into the prevalence of GBS in the research
population.

20.  Sample size Determination
 This will be determined according to the method described by Leslie Kish
(N = Z2pq/d2) for a single percentage with an absolute error of 5% allowed
and prevalence of 11.3%
 The finite population adjustment will be determined using the formula n =
n0/(1+[n01]/N) for a small population (1300 deliveries/year in this case).
Where n0 is the minimum sample size = 154, n is the finite sample size,
and N is the population size = 1300. n = 138 for a finite sample size
 With 15% added for attrition, the final sample size will be 159. But 160
expectant mothers will be enlisted. Once the sample size is reach, the study
participants will be sequentially recruited
 The Ekiti State University Teaching Hospital pregnancy clinic will serve as
the pretest location for the questionnaire, which will be conducted there by
qualified research assistants
 The patient's biodata (age, parity, level of education, and occupation), the
time of last menstruation, and the use of antibiotics during the index
pregnancy will all be required.

21. Study Area and Ethical Clearance
 The study will be conducted in Gynecology and obstetrics Department of
the Ekiti State University Teaching Hospital (EKSUTH) Ado –Ekiti, Ekiti
State, Nigeria. A tertiary healthcare facility that serves a large population in
the region. The hospital has the required facilities and skills to carry out the
research efficiently
 The approval will be obtained prior to the commencement of the research
work from the Research Ethics committee of Ekiti State University
Teaching Hospital Ado-Ekiti, in order to ensure research ethical code of
conduct is maintain.

22. Inclusion and Exclusion Criteria
 Inclusion Criteria
 All expectant mothers delivering at EKSUTH or referred to
this facility from other private facilities or traditional birth
facilities
 Signed written informed consent
 Exclusion Criteria
 pregnant women who had previously scheduled an elective
cesarean section, and
 pregnant women who refused to consent.

23. Data Collection
 Questionnaires/Surveys
 A standardized questionnaire will be created to collect demographic,
clinical, and behavioral information from participants
 The questionnaire will ask about past GBS testing, GBS awareness,
antibiotic use, and risk factors linked with GBS colonization
 During their antenatal clinic appointments, the questionnaires will be
administered by trained interviewers to the participants, guaranteeing
complete data collection.

24. DATA COLLECTION CONTD
 The following are the questionnaires composition
 Sociodemographic Information
 Age, Educational Level, Occupation, and Socioeconomic Status, Marital Status, Type of
Marriage, Husband Occupation, Husband’s Level of Education, are all factors to
consider under the Sociodemographic Information
 Obstetrics Information/History
 Gravidity, Parity, Gestational Age, and Past Unfavorable Pregnancy Outcomes,
Last Menstrual Period, Expected Date of Delivery, Number of Children Alive,
Previous History of Preterm Delivery, Past History of Vagina Discharge in Current
Pregnancy, The Color if Yes, Any Association Itching, Any Offensive Odour,
History of Fever, Any Associated Lower Abdominal Pains, Any Treatment
Received, Any History of Vagina Douching, How Often if Yes and Do you Smoke
are all factors or questions to consider under the Obstetrics Information.

25. Specimen Collection
 Specimen will be collected from pregnant women attending
antenatal clinic at EKSUTH by consultant gynecologist to avoid
contamination and disqualification of samples in the study.
 Specimen that will be collected include high vaginal swap (HVS).
The high vaginal swap specimen will be collected with the aid of a
disposable speculum and sterile swab stick
 The specimen will be placed in a cooler containing ice packs and
will be transported to the Microbiology Laboratory of Afe Babalola
University Ado-Ekiti.

26. Specimen Culturing
 The samples will be inoculated in 5% sheep blood agar supplemented
with colistin (8 g/mL) and nalidixic acid (15 g/mL) and incubated at
35–37oC for 18–24 hours in ambient conditions (CO2)
 The sheep blood agar culture plates will be checked after incubation
for the presence of big, whitish-grey, translucent colonies with a
limited zone of -haemolysis.
 Group B Streptococci will proliferate and manifest as beta haemolytic
organisms.
 Gram staining and catalase testing, will next be used to validate the
identification of GBS colonies

27.  Gram Reaction
 Principle
Gram staining is based on the ability of the bacterial cell wall to retain the crystal violet dye following
solvent treatment. Gram-positive microbes have more peptidoglycans, while gram-negative organisms
have more lipids (Purkaystha & Megha, 2022).
 Procedure.
 A thin smear of each organism isolate from the culture plate will be created by emulsifying in a
drop of normal saline on a clean grease-free glass slide, air drying, and gently heating fixed by
passing through flame. After that, the glass slide will be placed on a staining rack and soaked with
crystal violet for 60 seconds before being rinsed in water and treated with Lugol's iodine for 60
seconds before being rinsed in water. The stain will be temporarily decolored with acetone before
being rinsed with water. After counterstaining with safranin for 60 seconds, the smear will be
washed with water. After cleaning and drying the back of the slide, a drop of immersion oil will be
applied on the stained smear and it will be inspected with an x100 objective lens (oil immersion)
under a microscope. Gram-positive bacteria will turn purple, while Gram-negative bacteria will
turn pink (Purkaystha & Megha, 2022).

28. Catalase Test
 Principle: Catalase functions as a catalyst in the breakdown of
hydrogen peroxide to oxygen and water. Catalase synthesis in an
organism is tested by exposing it to hydrogen peroxide. If the
organism generates catalase, it emits oxygen bubbles.
Procedure
 A tiny amount of colony growth will be applied on the surface of
a clean, dry glass slide with the use of a cover-slip tip loop and
emulsified in a drop of 3% H2O2 in the glass slide. The
evolution of oxygen bubbles will be observed (Kebede et al.,
2021). Immediate bubbling implies a favorable result, whereas
no bubbling suggests a negative result.

29.  Antibiotic Susceptibility Testing
 The disc diffusion method, as suggested by the Clinical and Laboratory Standards Institute
(CLSI, 2020), will be used for antibiotic susceptibility testing.
 A combination of common antibiotics relevant to GBS will be used. The zones of inhibition will
be measuredand evaluated in accordance with predetermined parameters.
 In order to test for antibiotic susceptibility, inoculums will be made from a suspension of the
organism, which will be created by selecting two or three colonies of the organism and
emulsifying them in peptone water. It will then be compared to a turbidity standard (0.5
McFarland standard) for this suspension.
 Growth at this point is anticipated to be in the logarithmic phase. The broth culture will be
injected into Mueller-Hinton agar using a sterile swab stick.
 A multi-antibiotic impregnated disc containing cefuroxime (30 mg), ampicillin (5 mg),
amoxicillin-clavulanate (10 mg), erythromycin (10 mg), gentamicin (10 mg), ciprofloxacin (10
mg), and ofloxacin (5 mg) will be placed on the surface of the agar after about 3 minutes. The
disc will then be incubated at 35–37°C for 24 hours.
 The zones of inhibition's diameter will be measured using a calibrated meter rule to ascertain the
results, and standard Clinical and Laboratory Standard Institute (CLSI) charts will be used to
interpret the data.(Gajic et al., 2022).

30.  Molecular Analysis
 DNA Extraction
 DNA extraction will be performed using established commercial kits in accordance with the manufacturer's guidelines to gain a better knowledge of the
genetic characteristicsand variety of the isolated GBS strains (Hsu et al., 2021).
 PCR Amplification
 To target specific GBS genes of interest, PCR amplification will be performed.
 The PCR mixture will include extracted DNA, primers that target the desired genes, and the necessary PCR reagents. Thermal
cycling conditions will include an initial denaturation at95°C, followed by denaturation, annealing, and extension cycles.
 GBS will be confirmed using a primer pair from Inqaba Biotechnical Industries (Pty) Ltd. (Pretoria, South Africa) that
targeted the scpB gene and was 5′-ACAACGGAAGGCGCTACTGTTC-3′ (forward primer) and 5′
ACCTGGTGTTTGACCTGAACTA-3′ (reverse primer) (Jones et al., 2022).
 This is how the PCR reaction mixture will include the following:
 One Taq® Quick load® 2X master mix in a final volume of 29.5 ml with addition of 12 ml of standard buffer, 10.5 ml of
nuclease-free water, 1 ml of forward and reverse primer, and 5 ml of DNA template.
 The following PCR conditions will be used: one cycle at 94 °C for four minutes for initial denaturation, 35 cycles at 93 °C for
one minute for denaturation, 57.6 °C for one minute for annealing, 72 °C for one minute for elongation, one cycle at 72 °C
for seven minutes for further elongation, and a four-degree dwelling temperature (Hsu et al., 2021).

31.  Purification of PCR Products and Sequencing of PCR
Products
 PCR products will be filtered to remove superfluous primers and
nucleotides. Purification will be done using commercial purification
kits and following the manufacturer's recommendations (Haimbodi
et al., 2021)
 Purified PCR products will be sequenced to reveal the genetic
sequence of the targeted GBS genes.
 The findings of the sequencing will next be evaluated to discover
the genetic characteristics of the GBS strains (Mohamed et al.,
2020).

32. Statistical Analysis
 SPSS-23 Statistical Analyses packages will be used to
analyse the data obtained in this study.
 One-way ANOVA, Pearson regression analysis will
be used to determine association between variables.
 A value P < 0.05 will be considered statistically
significant

33.  Expected Contribution to Knowledge
 This study is expected to contribute the following to the existing
knowledge:
i. the prevalence of group B Streptococcus among pregnant women
attending antenatal clinic in EKSUTH;
ii. the risk factors associated with Group B Streptococcus infection
among pregnant women;
iii.isolate and identify Streptococcus agalactiae associated with the
pregnant women;
iv. the antibiotic susceptibility profiles of Streptococcus agalactiae and
molecular identification of Streptococcus agalactiae strains
prevalent among the pregnant women.

34. REFERENCES
 Haimbodi, E. L., Mukesi, M., & Moyo, S. R. (2021). Prevalence and molecular characterization of group B
streptococcus in pregnant women from hospitals in Ohangwena and Oshikoto regions of Namibia. BMC
microbiology, 21(1), 1-9.
 Hsu, S., Ferrieri, P., Martin, I., Demczuk, W., McGeer, A., Fittipaldi, N., ... & Dewar, K. (2021). Emergence of serotype
IV group B streptococcus adult invasive disease in Manitoba and Saskatchewan, Canada, is driven by clonal sequence
type 459 strains. Journal of Clinical Microbiology, 59(1), e01497-20
 Jiao, J., Wu, W., Shen, F., Liu, Z., Zhou, H., Fan, G., & Zhou, Y. (2022). Clinical Profile and Risk Factors of Group B
Streptococcal Colonization in Mothers from the Eastern District of China. Journal of Tropical Medicine, 2022
 Jones, C. E., Naidoo, S., De Beer, C., Esser, M., Kampmann, B., & Hesseling, A. C. (2022). Maternal HIV infection and
antibody responses against vaccine-preventable diseases in uninfected infants. Jama, 305(6), 576-584.
 Mohamed, A. M., Khan, M. A., Faiz, A., Ahmad, J., Khidir, E. B., Basalamah, M. A., & Aslam, A. (2020). Group B
Streptococcus colonization, antibiotic susceptibility, and serotype distribution among Saudi pregnant women. Infection
& chemotherapy, 52(1), 70.
 Nguyen, L. M., Omage, J. I., Noble, K., McNew, K. L., Moore, D. J., Aronoff, D. M., & Doster, R. S. (2021). Group B
streptococcal infection of the genitourinary tract in pregnant and non‐pregnant patients with diabetes mellitus: An
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35. THANK YOU