MICROBIOLOGICAL PATTERN

IN CASES OF PRETERM PREMATURE RUPTURE

OF FETAL MEMBRANES (PPROM)

BY

DR. JERRY OSA UWAIFO MBBS (BENIN)

SUBMITTED TO THE NATIONAL POSTGRADUATE MEDICAL

COLLEGE OF NIGERIA IN PART FULFILLMENT OF THE

REQUIREMENTS FOR THE

FELLOWSHIP OF THE COLLEGE

2007

i

ACKNOWLEDGEMENT

I wish to express my profound thanks to Professor A. U. Oronsaye for all

his assistance right from the beginning to the final stage of this work. I also give

special thanks to Dr M. E. Aziken who was never tired of correcting, advising

and directing me in doing this work in order to come out with a good paper.

Many thanks to Dr Daniel Ugbomoiko, the microbiologist who was so

committed in the laboratory to ensure that all samples collected were properly

analysed. I also thank the resident Doctors especially Dr Ebunum and the

nursing staffs of central Hospital, ST. Philomena Hospital and Faith Medical

Centre, Benin City for their contributions even with very minimal motivation.

Special thanks also go to my dear wife Mrs Abiodun Uwaifo, and my

children Jerry(Jnr), Stephany, Walter and Divine for their love and emotional

support.

I remain ever grateful to Deacon Uwadiae Uwaifo(M.B.E) and Mrs

Hannah Uwaifo, my parents of blessed memory for the legacy of education they

gave to me. I will always write their names in gold.

Finally, I thank God Almighty for his provisions, continuous Grace and

protection up to this time of my life.

ii

CENTRAL HOSPITAL

BENIN CITY

ETHICS AND RESEARCH COMMITTEE

CLEARANCE CERTIFICATE PROJECT TITLE: MICROBIOLOGICAL PATTERN IN CASES OF PRETERM

PREMATURE RUPTURE OF FETAL MEMBRANES (PPROM)

PRINCIPAL INVESTIGATOR: DR JERRY OSA UWAIFO

DEPARTMENT/ INSTITUTION: OBSTETRICS AND GYNAECOLOGY, CENTRAL

HOSPITAL, BENIN CITY.

DATE CONSIDERED: 23RD NOVEMBER 2006

DECISION OF THE COMMITTEE: APPROVED

CHAIRMAN:

DECLARATION BY INVESTIGATOR(S)

To be completed in four and three copies returned to the secretary, Ethics and Research

Committee, Clinical Services and training Division, Central Hospital, Benin City.

I/We fully understand the conditions under which I am/We are authorized to Conduct

the above mentioned research and I/We undertake to re-submit the protocol to the

Ethics and Research committee.

Signature………………………………….. Date…………………….

iii

CERTIFICATION

We hereby certify that this work was carried out by DR. JERRY OSA

UWAIFO of the Department of Obstetrics and Gynaecology, Central Hospital, Benin

City under our supervision.

Supervisors:

Professor A.U. Oronsaye (FRCOG, FMCOG, FWACS)

Consultant Obstetrician and Gynaecologist

Department of Obstetrics and Gynaecology

University of Benin Teaching Hospital, Benin City

Dr. M.E. Aziken (FWACS, FMCOG, FICS, MPH)

Consultant Obstetrician and Gynaecologist

Department of Obstetrics and Gynaecology

University of Benin Teaching Hospital, Benin City

iv

CONTENTS

Acknowledgement ……………………………………………………………….…i

Ethical Committee Certificate ……………………………………………………....ii

Certification………………………………………………………………………… iii

Contents…………………………………………………………………………….. iv

Abstract……………………………………………………………………………...1

Introduction…………………………………………………………………………. 2

Literature Review………………………………………………………………….. 4

Justification for study……………………………………………………………… .8

Aims and Objectives………………………………………………………………... 8

Hypothesis………………………………………………………………………….. 8

Materials and Methods……………………………………………………………… 9

Results………………………………………………………………………………11

Tables……………………………………………………………………………….12

Discussion…………………………………………………………………………..17

References…………………………………………………………………………..22

1

ABSTRACT

Preterm premature rupture of fetal membranes(PPROM) is a significant contributor to

poor obstetric outcome. The need for a continuous search for the aetiological factors to enable its

prevention and proper management if it occurs cannot be overemphasized.

This was a case control study (73 cases versus 73 controls with on going pregnancy

without rupture of membranes) aimed at determining the microbiological pattern of of PPROM

in our environment. Endocervical culture of cases and controls were compared. The cases of

PPROM had significantly more positive cultures than controls( 97.3% versus 5.5% ; p <0.001).

Escherischia coli(32.8%) was the commonest organism isolated. Other organisms of statistical

significance isolated include Proteus(21.9%), Bacteroides(15.1%), and Klebsiella(9.6%). These

organisms showed very good sensitivity to Ciprofloxacin(90.4%), Ceftriaxone(89%),

Ceftazidime(86.3%), Co-amoxiclav(80.8%), Erythromycin(73.9%), Cefuroxime(73.9%) and

Gentamicin(65.8%). Clindamycin(45.2%) only showed moderate sensitivity but Co-trimoxazole

Ampicillin, Amoxicillin and Cloxacillin all had low sensitivity.

A significant number of PPROM(58.9%) occurred at 31 – 33 weeks gestation(P. value

0.014). Low level of education or low socioeconomic class was significantly associated with

PPROM(82.2%, P <0.001)

A combination of Cefuroxime and Erythromycin given intravenously for the first 48

hours followed by oral combination of these two drugs for a further 5 days is suggested based on

the sensitivity pattern of the isolated organisms in this study. It is recommended that

prophylactic use of antibiotics in the management PPROM should be based on the

microbiological pattern and their sensitivity in any environment.

2

INTRODUCTION

Premature rupture of membranes (PROM) defined as rupture of fetal membranes before the onset

of labour, is a common obstetric event occurring in 10% of pregnancies1. Most of the time, this occurs at

or beyond 37 weeks gestation with only 10% of PROM occurring at less than 37 weeks gestation1, 2.

The natural course of preterm premature rupture of membranes (PPROM) is preterm labour and

this places the mother and fetus at increased risk of short term and long term morbidity and mortality.

Preterm delivery continues to occur in 5 – 10% of all births with a perinatal mortality rate of between

50% and 80%.2, 3, 4 PPROM is the most common identifiable cause present in 20 – 30%3.

The causes implicated in the multifactorial aetiology of PPROM include cervical incompetence,

lower genital tract infections, smoking, sexual intercourse, amniocentesis or other invasive prenatal

procedures. History of PROM in prior pregnancy and low placental insertion have also been linked with

PPROM 6,22,48.

In recent years, the role of infection with lower genital tract organisms in precipitating PPROM

and preterm labour has come under considerable study. Although the etiology of PPROM is

multifactorial,22, 38 increasing evidence regarding clinical risk factors, membrane histology, membrane

culture and amniotic fluid microbiology shows a strong association with infection4, 5, 6. Not only can

infection complicate PPROM but there is evidence that many organisms can penetrate intact fetal

membranes7, 8. Several studies indicate that women from the lower social class not only have a higher

incidence of PPROM, they are also more likely to develop chorioamnionitis as a complication of

PPROM9.

For women who develop PPROM before 34 weeks gestation, there is need for conservative

management to ensure fetal lung maturity. The major risk of conservative management of PPROM is

3

the development of maternal and fetal infection9, 18, 20, 22. Recommended management strategy include

use of corticosteroids, early tocolytic and antibiotics for known infections9, 10, 11. Early studies also

suggest that prophylactic antibiotics can be beneficial for women with idiopathic PPROM and preterm

labour. Such treatment offers a reduction of chorioamnionitis, prolongation of latency and a possible

reduction of neonatal infections and gestational age dependent morbidity in the setting of PPROM

remote from term.10, 11, 12, 18, 28 Amniotic fluid culture, culture of endocervical and high vaginal swabs

with the sensitivity pattern of isolated organisms in cases of PPROM are done in order to institute

appropriate antibiotic therapy.

Women from the lower socio-economic group have a markedly higher risk of preterm delivery

which is especially pronounced for PPROM. 9,15 This finding makes this study relevant in this

environment. In particular, the lack of laboratory support for the confirmation of clinical diagnosis in

many developing countries inevitably leads to the use of antibiotics inappropriately.23 Consequently,

there is a high potential for the abuse of antibiotics, a situation that currently poses serious public health

problems in many developing countries. Broadspectrum antibiotics are often prescribed in these

situations without microbiological studies. The dearth of microbiological studies in cases of PPROM

and the lack of good laboratory backup in our environment creates the need to study the microbiological

pattern and their sensitivities in order to rationally suggest a treatment regimen. Such treatment regimen

may become invaluable in health institutions where there are no laboratories.

4

LITERATURE REVIEW

Preterm delivery is currently the leading cause of perinatal morbidity and mortality and preterm

premature rupture of fetal membranes (PPROM) is the most common easily identifiable cause, present

in 20 – 30% of preterm births.3, 6 Although the etiology of PPROM is multifactorial,5, 26, 38 a wealth of

data is emerging linking subclinical genital tract infection with preterm birth and PPROM particularly

prior to 34 weeks gestation.2, 4, 5, 6 Conversely, preterm PROM and preterm birth that occurs closer to

term is less likely to be associated with genital tract infection.4

Lower genital tract organisms do not only contribute to the aetiology of PPROM, they cause

maternal and fetal infection and therefore account for the majority of perinatal mortality and morbidity

especially in the setting of PPROM occurring remote from term where conservative management have

to be employed8, 9, 12, 18, 37. Mothers who deliver prematurely have a higher incidence of endometritis46

and their infants have a higher incidence of neonatal infection.47 Graham et al37 showed that

conservative management resulted in a significant decrease in perinatal mortality and morbidity in an

American population. Improved understanding of the link between genital tract infection and preterm

birth now provides an exciting potential for the development of sensitive new markers to identify

women at risk and effective interventions to prevent preterm PROM/birth.

The patient with PPROM remote from term can benefit from conservative management where

the use of antibiotics and serial evaluation of maternal and fetal wellbeing, offer significant potential for

the reduction of perinatal mortality and morbidity.11, 12

Genital tract organisms have been found to penetrate intact fetal membranes8 and subsequently

result in PPROM through mechanisms that have been studied but not clearly understood26, 33. This

implies that intra-amniotic infection is often present before the occurrence of PPROM and it has been

5

estimated that at least one of every four cases of PPROM/preterm birth occur in the presence of intra-

amniotic or amniotic fluid infection7, 8, 39, 40.

The presence of periodontal disease has also been linked with PPROM and preterm births17. Oral

opportunistic pathogens and/or their inflammatory products may also have a role in preterm

birth/PPROM via a haematogenous route. Fusobacterium nucleate, a common oral species, is the most

frequently isolated species from amniotic fluid cultures among women with preterm labor and intact

membranes7, 17, 45.

Understanding the pathophysiologic mechanism by which genital tract infection leading to

PPROM is initiated will give us a better insight to the role of infections and so help tailor treatment in

order to reduce the perinatal mortality and morbidity associated with PPROM13. The identification of

several factors such as interleukin–1, neutrophil activating peptide–1/interleukin–8, and tumour necrosis

factor (TNF) in patients with idiopathic preterm labour/PPROM may allow us to determine the

pathophysiologic mechanism that lead to PPROM13, 17. However, an improved understanding of this

pathophysiologic mechanism remains one of the greatest challenges in obstetric care in this decade14.

Evidence of subclinical infection as a cause of preterm PROM and preterm labour is raised by

finding elevated maternal serum c-reactive protein and abnormal amniotic fluid organic acid levels in

some patients with preterm labour26. Biochemical mechanisms for preterm labour in the setting of

infection are suggested by both in vitro and in vivo studies of prostaglandins and their metabolites,

endotoxins and cytokines26, 33. The high concentration of potentially pathogenic microorganisms in the

vagina and cervix of pregnant women with bacterial vaginosis may increase the possibility of an

ascending infection via the cervix, decidua, fetal membranes, maternal placenta, and amniotic fluid33.

Some of the bacteria associated with bacterial vaginosis such as Bacteroides sp. are particularly

virulent. Certain bacteria produce enzymes that potentially could affect the fetal membranes or maternal

6

decidua. Bacteroides sp. and group B streptococcus produce proteases31,33,34,36. Protease enzymes

reduce the chorioamniotic membrane strength in vitro. It is even possible that a high concentration of

bacteria in the lower genital tract could produce enough proteases to weaken the fetal membrane

strength, causing premature rupture of the membranes. Bacterial lipases could also produce tissue

injury. Lysosomes within fetal membrane cells contain phospholipase A2 in high concentrations.

Phospholipase A2 is a precursor of prostaglandin synthesis and the destruction of lysosomes within

deciduae or chorioamnion cells may induce prostaglandin synthesis resulting in uterine contractions17, 22,

25, 33, 34.

In some studies, a high rate of phospholipase A2 production was found to be associated with

Bacteriodes sp, anaerobic Streptococci, Fusobacterium sp, and Gardnerrela Vaginalis. It was also

demonstrated that bacterial products of group B Streptococci, Viridans Streptococci, Escherichia Coli

and Bacteroides fragilis but not lactobacillus sp. increase the synthesis of prostaglandins in the

membranes33. Thus, selected bacteria, including some closely related to bacterial vaginosis may play a

role in the initiation of uterine contractions by stimulating prostaglandin synthesis26, 33, 34. In an

alternative mechanism, either the release of prostaglandin in the membrane or uterine contraction could

cause microbreaks of the membrane that allow bacterial colonization of the membrane33.

The microorganisms mostly associated with PPROM and preterm labour have been mentioned

above. Of all these organisms of bacterial vaginosis complex, group B Streptococci, E. coli, Bacteroides

sp, Viridans Streptococci and an unusual organism fusobacterium are frequently implicated12, 17, 24, 28, 31,

33, 40.

The Bacterial vaginosis complex include organisms such as Gardnerella Vaginalis, anaerobic

species primarily among Prevotella, Peptostreptococcus and Mobiluncus, Mycoplasma Hominis and

Ureaplasma Urealyticum17, 24, 28. It is interesting that candida sp, common in the vagina, is an unusual

7

pathogenic cause of preterm labour/PPROM7 and Haemophilus influenza has also been reportedly found

in women presenting with PPROM27.

The role of Chlamydia trachomatis and viruses in PPROM/preterm labour remains to be

determined28. Use of molecular microbiology techniques to diagnose intrauterine infection may uncover

the role of fastidious microorganisms that have not yet been discovered.

In a study in Saudi Arabia, Bahar et al found a variety of aerobic or anaerobic organisms or both

in cases of PPROM41. Shaarawy et al found Mycoplasma Hominis and Ureaplasma Urealyticum in

addition to aerobic and anaerobic bacteria in Egypt 42. This was similar to the findings of Abd EL

Kareim et al in another study in Egypt 44. In a study in Brazil, Silva et al isolated Staphylococcus aureus

in addition to a wide diversity of microorganisms 43. They found an intense inflammatory infiltrate in

the membranes which they linked to the rupture of membranes.

A search of the literature revealed that not much work has been done on this subject matter in our

local environment even though PPROM is often seen and management problems often arise. The need

to study the pattern in this environment cannot be over emphasized.

Antibiotic treatment for women with PPROM especially remote from term, offers significant

benefit with respect to pregnancy prolongation of the interval from rupture of membranes to delivery

and improvement in neonatal outcome19, 29, 30, 49 with reduced risk of maternal infection. The best

evidence supports the choice of an extended-spectrum agent or combination administered intravenously

for 2 days followed by an extended-spectrum or combination of oral agents for several days30. Despite

the effectiveness of antimicrobial therapy in this setting, the potential risk of systemic antibiotic

administration, such as allergic reactions, overgrowth of commersal organisms, and emergence of

resistant pathogens must always be kept in mind. Nevertheless, in the majority of cases, assuming the

8

patient is a good candidate for expectant management, the benefits of antibiotic therapy outweigh the

risk.

JUSTIFICATION FOR STUDY

Preterm premature rupture of fetal membranes is a significant cause of preterm

delivery which accounts for about 60% of perinatal mortality rate. Available evidence indicate

that genital tract infection contribute significantly to the aetiology of PPROM which in turn

predisposes to further infections. A combination of PPROM, Preterm delivery and Infections

would obviously worsen both maternal and fetal outcome.

In a low resource setting like Nigeria, there is need to be proactive in our

interventions to improve maternal and perinatal outcome in our practice.There is therefore need

to determine the microbiological pattern of infections and their sensitivities amongst patients

with PPROM in our environment.

AIMS AND OBJECTIVES 1. To determine the common microbial isolates in cases of PPROM.

2. To determine the antibiotic sensitivity pattern of these organisms.

3. To suggest from the findings, an antibiotic regimen in these cases that may be useful where

laboratory facilities are not available.

HYPOTHESIS

Patients with Preterm Premature Rupture of Membranes have significantly more

cultures of pathogenic organisms than patients matched for age and gestational age

without PPROM .

9

MATERIALS AND METHODS

This is a case control study conducted in the Department of Obstetrics and Gynaecology of the

Central Hospital, Benin City. This study included patients recruited from the antenatal clinics, antenatal

wards and labour wards of Central Hospital, St Philomina Hospital and Faith Medical Centre, Benin

City, with ruptured membranes before 37 weeks gestation and less than 24 hours duration of rupture of

membranes. The control group was made up of selected women from the antenatal clinic with on going

pregnancy without ruptured membranes matched for age(+ or - 3 years) and gestational age(+ or - 2

weeks) at 28 to 36 weeks gestation.

Patients excluded from this study include:

1. Those with ruptured membranes at less than 28 weeks or after 37 completed weeks

gestation.

2. Patients who have taken antibiotics within the past 7 days.

3. Patients with PPROM > 24 hours duration

4. Those with PPROM and temperature up to 380 C and above

5. Patients with PPROM and medical disorders e.g hypertension, diabetes mellitus, renal

disease etc.

The study comprised 73 patients with preterm premature rupture of membranes and 73

matched controls for age and gestational age without rupture of membranes. PPROM was

confirmed by pool of amniotic fluid in the vagina, its leakage through the cervix and/ or a

positive colour change of nitrazine stick from brown to deep blue. Endocervical swabs were

taken from all cases and from the control group in a similar manner. All samples were collected

personally by me and sent to the laboratory for microbiological studies. A well qualified

microbiologist handled and analysed all the samples collected at Central Hospital laboratory.

10

Each sample was innoculated immediately after collection into dried chocholate agar, two blood

agar, mac conkay agar and sabouraud dextrose agar. One of the blood agar plate was incubated

anaerobically using the anaerobic jar. They were all incubated at 37o C for 24-48 hours. Few

drops of saline were added to each swab after inoculation, put on a slide and examined

microscopically. Few drops of 10% Potassium Hydroxide(KOH) were also added separately to

specifically test for Gardnerella Vaginalis.(Whiff test).

Information on age, parity, gestational age, and level of education (socio-economic status)

were obtained from both cases and control.

Sample Size

The sample size for this study was calculated based on a PPROM incidence of 5% using the

formula by D.W. Taylor.

N = pq 0

(E/1.96)2

Where N = sample size

1.96 is a constant

p = known prevalence of the disease

q = 1 – p (proportion of persons free from the disease)

E = error margin allowable

N = 0.05 x 0.95 0

(0.05/1.96)2

= 72.998

73.

Ethical Consideration

Approval for this study was obtained from the ethical Committee of the Central Hospital, Benin

City.(see attached ethical committee certificate )

11

Also, the study was carefully explained to the patients and their informed consent obtained before

being recruited into the study. The rights of patients to participate or not was respected.

Data Management and Analysis

All information obtained were recorded on a data collection sheet designed for the study. The

results were first presented as percentages and further analysis was done in the computer using the Epi-

info programme. Chi square ( x 2) test and Fischers exact test where appropriate were used to test for

significant difference in observed proportion between the cases and controls.

P value < 0.05 was taken as significant.

RESULTS Seventy three(73) samples collected from cases of PPROM and controls respectively, making a total of

one hundred and forty six(146) samples were sent to the laboratory for microbiological analysis. Seventy one(71)

samples taken from cases of PPROM had positive cultures of various organisms. This gives a positive culture

rate of 97.3% ( P.value < 0.001** ). Only four(4) samples out of seventy three(73) taken from the control group

had positive cultures(5.5%).

An interesting finding in this study is that most of the cases of PPROM (82.2% , P. value < 0.001**)

occurred in women with no formal or primary education and this group largely belong to the lower socio-

economic class. Majority of the cases of PPROM (58.9%) occurred at 31 to 33 weeks gestation. Analysis of

these figures show they are statistically significant(**).

12

Table 1 : Socio Demographic Characteristics of the study Population

** Statistically Significant

Characteristics Case{N=73(%)} Control{N=73(%)} X2 P value P. value

Age

< 20

20 – 29

30 – 39

40

.

7 ( 9.59 )

40 ( 54.79 )

23 ( 31.51 )

3 ( 4.11 )

9 ( 12.32 )

35 ( 47.95 )

24 ( 32.88 )

5 ( 6.85 )

1.105

0.777

Parity

0

1

2-4

5

7 (9.59)

17(23.29)

46 (63.01)

3 (4.11)

12 ( 16.44 )

26 ( 35.62 )

30 ( 41.10 )

5 ( 6.88 )

7.068

0.070

Level of Education

No formal Educ

Primary Educ

Secondary Educ

Tertiary Educ

33 (45.21)

27 (36.99)

7 (9.59 )

6 (8.22 )

13 (19.17 )

16 (21.92 )

32 (43.84 )

11 (15.10)

27.99

<0.001**

Gestational Age

28 – 30

31 – 33

34 – 36

3 (4.11 )

43(58.90)

27(36.99)

12 (16.44)

29 (39.72)

32 (43.84)

8.546

0.014**

13

TABLE 11: FREQUENCY OF ISOLATION OF VARIOUS ORGANISMS

ORGANISMS

CASES

CONTROL

P. VALUE

N=73

%

N=73

%

Escherischia Coli

24

32.8 - - <0.001**

Staphylococcus aureus

6 8.2 2 2.7 0.275

RR=3

Proteus

16 21.9 - - <0.001**

Klebsiella

7 9.6 - - 0.013**

B-haemolytic strept

2 2.7 - - 0.497

RR=2

Streptococcus viridans

3 4.1 2 2.7 1.000

RR=1.5

Bacteroides

11 15.1 - - 0.001**

Haemophylus Influenza

2 2.7 - - 0.497

RR=2

Total positive culture

71 97.3 4 5.5 <0.001**

14

Candida albicans was isolated in 9(12.3%) of the cases but 20(27.4%) in the control group. P.value

0.037**(RR=0.5)

Gardnerella vaginalis was demonstrated microscopically and whiff test positive in 4(5.5%) cases of

PPROM but non in the control group. P. value 0.120(RR=3.5)

Trichomonas vaginalis was seen microscopically in 10(13.7%) cases of PPROM but 3(4.11%) in the

control group. P. value 0.078(RR=3.3)

These three occurred mixed with some of the positive bacterial cultures.

15

TABLE 111: SENSITIVITY PATTERN OF ISOLATES

Drug

NUMBER SENSITIVE AND PERCENTAGE

E. coli

24(%)

Proteus

16(%)

Staph

aureus

6(%)

Klebsiella

7(%)

B-haemo

Lytic strept.

2(%)

Strept.

Viridans

3(%)

Bacteroides

11(%)

Haemophy-

lus influenza

2(%)

Nil

culture

2(%)

Total

number

73

(100%)

COTR

12(50.0)

6(37.5) 2(33.3)

2(28.6) 1(50) 1(33.3)

2(18.2)

2(100) -

28(38.4)

AMP

4(16.7) 3(18.8) 1(16.7) 0 1(50) 1(33.3)

0

0 -

10(13.7)

CLOX

2(8.3) 0 4(66.7) 1(14.3) 1(50) 0

1(9.1)

1(50) -

9(12.3)

ERY

22(91.6) 12(75.0) 5(83.3) 5(71.4) 1(50) 1(33.3)

6(54.6)

2(100) -

54(73.9)

CIPR

23(95.8) 16(100) 5(83.3) 7(100) 1(50) 3(100)

9(81.8)

2(100) -

66(90.4)

AUG

20(83.3) 14(87.5) 6(100) 5(71.4) 2(100) 2(66.7)

8(72.7) 2(100) - 59(80.8)

CLIND

12(50.0) 6(37.5) 3(50.0) 3(42.9) 1(50) 1(33.3)

6(54.6) 1(50) - 33(45.2)

CEFTR

23(95.8) 12(75.0) 6(100) 7(100) 2(100) 3(100) 10(90.9) 2(100) - 65(89.0)

FORTU

24(100) 11(68.8) 6(100) 6(85.7) 2(100) 3(100) 9(81.8) 2(100) - 63(86.3)

GENT

22(91.7) 6(37.5) 5(83.3) 5(71.4) 2(100) 2(66.7) 5(45.5) 1(50) - 48(65.8)

CEXM

23(95.8) 5(31.3) 5(83.3) 6(85.7) 1(50) 3(100) 9(81.8) 2(100) - 54(73.9)

AMOX

11(45.8) 8(50.0) 2(33.3) 2(28.6) 1(50) 1(33.3) 2(18.2) 0 - 27(37.0)

16

COTX=Co-trimoxazole, AMP=Ampicillin, CLOX=Cloxacillin, ERY=Erythromycin, CIPR=Ciprofloxacin,

AUG=Augmentin(Co-amoxiclav), CLIND=Clindamycin, CEFTR=Ceftriaxone,

FORTU=Ceftazidime, GENT=Gentamicin, CEXM=Cefuroxime(zinacef/zinnat), AMOX=Amoxicillin.

TABLE 1

This shows the age distribution in the case and control groups to be homogenous. The mean ages were

26.7 and 27.0 years for cases and controls respectively. The study population consisted mainly of multiparous

women( 63 and 41.1 percent for cases and control respectively ). No statistically significant difference was

seen in age and parity between case and control( P. value 0.777 and 0.070 respectively ).

A significant number of the cases of PPROM (58.9%) occurred at 31-33 weeks gestation followed by

those that occurred between 34-36 weeks (37.0%). Only 4.1% occurred between 28-30 weeks gestation. This

shows that the occurrence of PPROM is significantly associated with gestational age group 31-33 weeks

(P.value 0.014**).

Low level of education( low socio-economic class ) which accounted for 82.2% was significantly

associated with PPROM in this study(P.value < 0.001).

TABLE 11

This shows the prevalence of various organisms isolated. There were seventy one(71) and four(4)

bacteriological isolates in case and control groups respectively. Escherischia Coli was the commonest organism,

accounting for 32.8%. The other organisms isolated include Proteus (21.9%), Bacteroides(15.1%), Klebsiella

(9.6%), Staphylococcus aureus(8.2%), and Streptococcus Viridans(4.1%). B-haemolytic Streptococcus and

Haemophylus influenza each accounted for 2.7%.

Total positive cultures were 97.3% and 5.5% for case and control respectively(P.value < 0.001**). The

occurrence of E.Coli, Proteus, Bacteroides and Klebsiella were statistically significant. Candida albicans was

isolated microscopically in 9(12.3%) of the cases but 20(27.4%) in the controls giving a P.value of 0.037 and

RR 0.5. Gardnerella Vaginalis was demonstrated microscopically and whiff test positive in four(4) of

17

the cases(5.5%) but non in the control group( P.value 0.120, RR 3.5 ).

Trichomonas Vaginalis was seen microscopically in ten(10) of the cases(13.7%) and three(3) in the control

group

(4.1%)[P.value 0.078, RR 3.3]. Candida albicans, Gardnerella Vaginalis and Trichomonas Vaginalis all occurred

along with some of the positive bacterial cultures in a mixed fashion.

TABLE 111

This shows the antibiotic sensitivity pattern of the organisms isolated. The drug that showed the

highest sensitivity was Ciprofloxacin(90.4%). This was followed by Ceftriaxone(89%),Ceftazidime (86.3%), and

Co-amoxiclav(80.8%). Others that showed good sensitivity were Erythromycin(74%), Cefuroxime (73%) and

Gentimicin(65.8%).

Clindamycin(45.2%), Co-trimoxazole(38.4%), Amoxicillin(37%), Ampicillin(13.7%) and Cloxacillin

(12.3%) all showed low effectiveness( sensitivity less than 50% ). The commonest pathogens isolated in this

Study; E.coli, Proteus, Bacteroides and klebsiella, showed good sensitivity to Ciprofloxacin, Ceftriaxone,

Ceftazidime, Erythromycin, Co-amoxiclav and Cefuroxime. Proteus was particularly not very sensitive to

Gentamicin(37.5%).

DISCUSSION

Preterm premature rupture of fetal membranes (PPROM) is the most common easily identifiable

cause of preterm labour accounting for 20-30% of preterm birth and this is currently the leading cause of

perinatal morbidity and mortality3,6. The occurrence of PPROM also put the mother at increased risk of

morbidity46. A wealth of data is emerging linking genital tract infection with PPROM/Preterm birth

particularly prior to 34 weeks gestation2,4,5,6.

This study of seventy three(73) cases of PPROM revealed a positive culture rate of 97.3%. This

statistically significant finding suggests a strong link of genital tract infections with the occurrence of PPROM.

A significant number of the cases occurred at 31-33 weeks gestation(P.value 0.014**). The reason for this is not

clear but a possible explanation may be that the vaginal flora is altered in these patients and this situation leads to

growth of pathogenic organisns which may ascend to gain assess to the endocervix and trigger the process

18

leading to amniorhexis/preterm birth. It is possible that the effect of this process is maximal at 31-33 weeks

gestation and therefore highest rate of rupture of membranes at this period of pregnancy.

Low level of education(82.3%) was significantly associated with PPROM in this study. Level of

education approximately correlates with socio-economic class and this was used in this study to measure this

factor. It can therefore be said that the occurrence of PPROM is strongly associated with low socio-economic

class(P.value <0.001). This agrees with findings of previous studies by Okonofua et al9, and Savitz D.A et al15.

The association of PPROM with low socio-economic class could be a reflection of the personal hygiene of

women in this environment.

E. Coli was the commonest organism isolated(32.8%). This gram negative aerobic and facultative

anaerobic organism has been found in previous studies to penetrate intact fetal membranes, cause intra-amniotic

infection and subsequently amniorhexis occurs8,26. Proteus(21.9%) and Klebsiella(9.6%)

found in this order of prevalence in this study are gram negative aerobes and facultative anaerobes but

Bacteroides(15.1%) is a gram negative anaerobic organism. They all belong to the Enterobacteriaceae group of

organisms. These findings are similar to those reported in previous works by Bahar et al41 and Abd El Kareim

et al44. Shaarawy et al42 found Mycoplasma Hominis and Ureaplasma Urealyticum in addition to aerobic and

anaerobic bacteria. Mycoplasma Hominis and Ureaplasma Urealyticum was not tested for in this study because

of non availability of the materials needed to isolate them. The isolation of Staphylococcus aureus(8.2%) in this

study is also similar to the findings of Silva et al43 in their study with the isolation of this organism along with

a wide diversity of aerobic and anaerobic organisms.The finding of an intense inflammatory infiltrate in the

membranes in their study was linked with the rupture of membranes as the aetiologic factor.The isolation of

Streptococcus Viridans(4.1%), B-haemolytic Streptococcus(2.7%) and Haemophylus Influenza(2.7%)though not

statistically significant in this study may be the other organisms that contribute to the wide variety of microbes

found in association with PPROM cases in previous studies27,41,42,43,44.

The microscopic isolation of Candida spp in 20 out of the 73 controls(27.4%) as against 9 in the cases

19

(12.3%) is worthy of note in this study. There appears to be an inverse relationship between the presence of

Candida spp and the occurrence of PPROM. This finding is at variance with that previously documented by

Chaim W et al7. Gardnerella Vaginalis demonstrated in 4 cases(5.5%) and non in the control group all occurred

in the cases where Bacteroides spp were isolated. This is worthy of note as this organism which is part of the

bacterial vaginosis complex is often associated with anaerobic bacteria such as Bacteroides and their role

in the aetiology of PPROM is already well documented31,33,36,51,52.

The sensitivity pattern in this study revealed that Ciprofloxacin had the highest sensitivity(90.4%) with

almost all the isolated organisms sensitive to it. However, this drug which is a quinolone is not safe in

pregnancy. Other drugs that showed excellent sensitivity include Ceftriaxone(89%), Ceftazidime(86.3%),

Co-amoxiclav(80.8%), Erythromycin(74%) and Cefuroxime(73%). Although some of these drugs

are expensive, they are safe in pregnancy and readily available. Co-amoxiclav has been found to cause neonatal

necrotising enterocolitis in the ORACLE trial 200153. The overall sensitivity of Gentamicin(65%) was good and

particularly very effective against the commonest pathogens; E.coli(91.7%) and Klebsiella(71.4%) but showed

poor effectiveness against Proteus(37.5%) and Bacteroides(45.5%). The common antibiotics used in our general

practice; Ampicillin, Co-trimoxazole, Cloxacillin and Amoxicillin all showed very low effectiveness against the

bacterial isolates in this study. Clindamycin showed moderate sensitivity only in the cases of Bacteroides and

E.coli. Ugwumadu et al(2004)54 reported that oral Clindamycin eradicated bacterial vaginosis and intermediate

flora in 90% of women prior to 20 weeks gestation. How effective this is, in cases of PPROM in our

environment remains to be determined in a study. Carey J.C et al55 however demonstrated the effectiveness

of Metronidazole in preventing preterm labour/PPROM in women with asymptomatic bacterial vaginosis.

Metronidazole is generally used empirically and known to be an effective drug against gram negative

anaerobic bacteria, Gardnerella Vaginalis and related organisms of the bacterial vaginosis complex.

Metronidazole sensitivity was not tested in this study as the disc was not available.

Two of the largest studies that looked at the efficacy of antibiotic use in PPROM are the National

Institute of Child Health and Human Development-Maternal Fetal Medicine Units (NICHD-MFMU) study

20

of PPROM56 and the ORACLE trial53. In the NICHD-MFMU study, intravenous antibiotics : Ampicillin 2gms

6hourly and Erythromycin 250mg 6hourly were used for 48 hours. The patients were then placed on

oral Amoxicillin 250mg 8hourly and enteric-coated Erythromycin-base 333mg every 8hours to complete a

7-day course of antibiotic therapy. In this trial, the antibiotic group had a significantly longer duration of

latency than the control group. The antibiotic group was twice as likely to remain undelivered after 7 days.

The increased latency continued for up to 3 weeks after discontinuation of antibiotics. Composite and

individual morbidities for the neonates were lower in the antibiotic group. Incidence of chorioamnionitis

and neonatal sepsis, including group B Streptococcal sepsis was decreased. In the ORACLE trial where

Co-amoxiclav was used either alone or in combination with erythromycin, an increased risk of necrotising

enterocolitis was present and there was no significant difference in latency and morbidity between the antibiotic

group and controls.

Based on current evidence, 7 days of antibiotics as proposed by the NICHD-MFMU study

is recommended for PPROM cases that are being managed conservatively. In this study in Benin city however,

the sensitivity of most of the bacterial isolates to Ampicillin was very poor(13.7%).The sensitivity to Ceftriaxone

(89%), Ceftazidime(86.3%), Cefuroxime(73%) and Erythromycin(74%) were excellent and any of

these can be substituted for Ampicillin. The effectiveness of Amoxicillin was also poor(37%). Of the three

cephalosporins found to be very effective in this study, Cefuroxime is readily available in parenteral and oral

forms and can be used to replace Ampicillin and Amoxicillin.

The regimen suggested based on findings in this study is intravenous Cefuroxime 1.5gms

8hourly and Erythromycin 250mg 8hourly for 48 hours. Then oral Cefuroxime 250mg 8hourly and

Erythromycin 250mg 8hourly to complete a 7-day course. It is also suggested that intravenous Metronidazole

500mg or Clindamycin 300mg be added 8hourly for 48hours, then oral Metronidazole 400mg 8hourly or

Clindamycin 150mg 8hourly for the remaining five days especially in cases where microscopy reveals the

presence of Gardnerella Vaginalis with other gram negative anaerobic organisms.

21

In conclusion, genital tract infection is significantly related to the occurrence of preterm premature

rupture of fetal membranes(PPROM) and it is one of the major aetiologic factors in our environment.

Escherischia Coli is the commonest micro-organism implicated in this study. Other organisms which are mainly

gram negative aerobes and anaerobes include Proteus, Klebsiella and Bacteroides. These are significantly

involved as aetiologic factors in the cases of PPROM. Low socio-economic class is a significant risk factor

demonstrated in this study. Antibiotics of significant sensitivity and of good prospect in the expectant

management of PPROM include Ceftriaxone, Ceftazidime, Cefuroxime, Erythromycin and Co-amoxiclav.

However, use of Co-amoxiclav is discouraged because of the neonatal necrotizing enterocolitis it causes. A

combination of these other drugs intravenously for the first 48 hours followed by oral administration of this

combination to complete a 7-day course is suggested based on findings of this study. This regimen can be used

for cases of PPROM in places where there are no good laboratory facilities or while awaiting results of

microscopy, culture and sensitivity.

Finally, it is observed that improvement of the general socioeconomic condition of women is likely

to have a significant impact on the reduction of PPROM/Preterm birth and subsequently a reduction of maternal

and perinatal mortality and morbidity.

22

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