NEONATAL SEPTICAEMIA AS SEEN IN THE SPECIAL

CARE BABY UNIT OF AHMADU BELLO UNIVERSITY

TEACHING HOSPITAL, ZARIA

A DISSERTATION SUBMITTED TO THE NATIONAL POSTGRADUATE MEDICAL COLLEGE OF NIGERIA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE FELLOWSHIP OF THE COLLEGE OF PHYSICIANS

NOVEMBER 2006

BY

ONALO, RICHARD. MBBS (ABU), 1997

DECLARATION

It is hereby declared that this work is original unless otherwise acknowledged. The work has not been presented to any other college, nor has it been submitted elsewhere for publication.

Signature:______ONALO, R.

ii CERTIFICATION

We certify that this work was carried out by Dr. R. ONALO of the Department of

Paediatrics, Ahmadu Bello University Teaching Hospital, Zaria under our supervision, from the beginning to the end.

______Prof. W. N. Ogala, FMC (Paed), FWACP Professor of Paediatrics Ahmadu Bello University Teaching Hospital Zaria – Nigeria.

______DR. G. O. Ogunrinde, FWACP Senior Lecturer and Head, Department of Paediatrics Ahmadu Bello University Teaching Hospital Zaria – Nigeria.

iii DEDICATION

This work is dedicated to my father, Hyacinth Onyekwe Onalo who led me into intelligent pursuits.

iv ACKNOWLEDGEMENT

I wish to express profound gratitude and indebtedness to my supervisors, Prof.

W. N. Ogala and Dr. G. O. Ogunrinde, who despite their very tight schedules diligently and patiently supervised this project. I appreciate the contribution of Dr. E. A. Ameh of

Paediatric Surgery unit and consultants of the Paediatric Department for useful suggestions given during the period of this work. My appreciation goes to Dr.S. Adama and the other fellow residents in the Department of Paediatrics who assisted me during sample collection. Special thanks to Dr. A.T. Olayinka, Head of Department of

Microbiology and Staff of Department of Microbiology for their efforts towards the success of this project. I thank Dr. T. S. Kene of the Department of Community

Medicine, ABU, Zaria, for his statistical assistance and Miss Oluwakemi Oguntolu for secretarial assistance.

I express my profound gratitude to my wife Deborah, who laboured along with me in prayers for the success of this project.

Finally, I am extremely grateful to my loving father-God for grace and unusual ability to stay awake late into the night to put this work together.

v DEFINITION OF TERMS

Out-born babies - Babies delivered outside Ahmadu Bello University Teaching

Hospital’s delivery suite.

In-born babies - Babies born at the Ahmadu Bello University Teaching Hospital’s delivery suite.

Birth asphyxia - One minute Apgar score of 6 or less.

Prolonged rupture of membranes - Rupture of the fetal membranes for 24 hours or more before delivery.

Prolonged labour - Active labour lasting for 24 hours or more.

Empirical antibiotics - Antibiotics prescribed based on previous observation or unit experience.

Early-onset septicaemia - Systemic infection occurring in a newborn infant within the first 48 hours of life.

Late-onset septicaemia - Systemic infection occurring in a newborn infant after 48 hours of life.

Nosocomial infection - Those infections that were not acquired or incubating at the time of admission of the neonate to hospital or transplacentally acquired but including those acquired in the birth process.

Preterm babies - Babies born before 37 completed weeks of gestation.

Term babies - Babies born at 37 completed weeks through 41 weeks of gestation.

Probiotics - Live bacterial supplements administered to induce gut mucosal resistance to pathogenic bacteria.

Macrosomic babies – Babies born with a birthweight of 4000 grams or more.

Low birthweight babies – Babies born with a birthweight of 2500 grams or less.

vi ABBREVIATIONS

1. ABUTH - Ahmadu Bello University Teaching Hospital.

2. ANC - Antenatal care.

3. CD - Cluster of Differentiation.

4. CFU - Colony forming unit.

5. CSF - Cerebrospinal fluid.

6. CONS - Coagulase negative staphylococci.

7. DAMA - Discharged against medical advice.

8. df - Degree of freedom.

9. GBS - Group B streptococcus.

10. Ig - Immunoglobulin.

11. IL - Interleukin.

12. kg - Kilogramme.

13. L/L - Litre per litre.

14. M:F - Male : Female.

15. mm3 - Cubic millimetre.

16. NADPH - Nicotinamide-adenine-dinucleotide-phosphate.

17. PROM - Prolonged rupture of membranes.

18. RNA - Ribonucleic acid.

19. RT - Rectal temperature.

20. SCBU - Special Care Baby Unit.

21. SD - Standard deviation.

22. Sep - Septicaemia.

23. SIRS - Systemic inflammatory response syndrome.

24. SPA - Suprapubic aspirate.

vii TABLE OF CONTENTS

Page

Title page i

Declaration ii

Certification iii

Dedication iv

Acknowledgement v

Definition of terms vi

Abbreviations vii

Table of Contents viii

List of Tables ix

List of Figure x

Summary xi

Introduction 1

Literature review 3

Justification of the study 42

Objectives of the study 44

Patients and methods 45

Results 53

Discussion 73

Conclusions 83

Recommendations 84

Limitations of the study 85

Future research 86

References 87

Appendix 99

viii LIST OF TABLES

Page

Table 1: Place of delivery and frequency of septicaemia among 211 neonates studied 54

Table II: Infection rate in relation to gestational age, postnatal age and sex 55

Table III: Age by sex distribution of the 211 neonates 56

Table IV: Maternal risk factors and rate of neonatal septicaemia in 211

neonates 57

Table V: Influence of host-related risk factors on infection rate 58

Table VI: Presenting symptoms in 211 neonates studied 59

Table VII: Distribution of signs of septicaemia among the study population 60

Table VIII: Mean haematological indices of the 211 infants studied 62

Table IX: Bacterial isolates in 75 septicaemic neonates 63

Table X: Distribution of bacterial isolates according to age at presentation 64

Table XI: Distribution of common organisms in relation to place of delivery 65

Table XII: Distribution of bacterial isolates from clinical specimens of the 75 neonates with culture-proven septicaemia 66

Table XIII: Gram-positive isolates and their in-vitro antibiotic sensitivity rates 68

Table XIV: Gram-negative isolates and their in-vitro antibiotic sensitivity rates 69

Table XV: Comparison of the antibiotic sensitivity profile in previous studies and present study. 70

Table XVI: Outcome of patients` management 71

Table XVII: Comparison of patterns of neonatal septicaemia in previous

studies and present study 72

ix LIST OF FIGURE Page

Figure 1: Bar Chart of the bacterial isolates and antibiotic sensitivity pattern 67

x SUMMARY

Neonatal septicaemia is associated with high morbidity and mortality, particularly in the tropics where risk factors for the development of the disease are prevalent. The epidemiologic, aetiologic and antibiotic sensitivity patterns of neonatal septicaemia vary from place to place and from time to time. Appropriate and prompt antibiotic therapy is pivotal to reducing complications that could arise from the disease, hence the need for a stand-by empirical treatment protocol. In order to determine the current pattern of neonatal septicaemia in this locality, a prospective study was conducted in

ABUTH, Zaria, between 25th May 2004 and 31st May 2005.

Babies (in-born and out-born) admitted in the SCBU of the hospital with the working diagnosis of septicaemia were studied. Each of the babies that fulfilled the inclusion criteria was evaluated clinically and a single venous blood sample obtained under aseptic conditions was inoculated into a thioglycolate containing bottle for culture before commencement of antibiotics. The blood samples were subjected to standard microbiologic culture technique. Isolated bacteria were identified using standard biochemical and enzymatic tests. Antibiotic sensitivity testing was done on each isolate using antibiotic discs manufactured by Oxoid and Abtek Biological Ltd.

Fourteen antibiotics (ampicillin, amoxicillin, Augmentin,® cloxacillin, cotrimoxazole, chloramphenicol, cefuroxime, ceftriaxone, ceftazidime, ciprofloxacin, erythromycin, gentamycin, tetracycline and ofloxacin) were employed. Relevant haematologic indices of infection were also determined on each blood sample. The patient’s data

(biodata, pregnancy and delivery history, clinical features and laboratory findings) were entered into a proforma and the babies were subsequently managed as per the unit routines.

Two hundred and eleven (211) infants were studied, consisting of 69 in-born infants and 142 out-born infants. The M:F ratio was 1.37:1. Seventy-five (35.5%) of them had proven septicaemia. Among those that had proven septicaemia, 21(28.0%) were in-born infants while 54(72.0%) were out-born infants. The prevalence of

xi septicaemia among in-born and out-born babies was 30.4% and 38.0% respectively

(p>0.05). The M:F ratio of the septicaemic neonates was 1.21:1. The total number of live births in the hospital during the study period was 1190, thus the incidence of neonatal septicaemia for in-born babies was 17.6 per 1000 live births.

The symptoms and signs of neonatal septicaemia were non-specific and there were no significant differences in the frequency of symptoms and signs in septicaemic and non-septicaemic neonates.

The perinatal risk factors for neonatal septicaemia encountered among the 211 neonates are lack of antenatal care 40 (19.0%), prolonged rupture of membranes 31

(14.7%), prolonged labour 18 (8.5%), intrapartum maternal fever 14 (6.6%), unsupervised delivery 79 (37.4%), preterm delivery 25 (11.8%), perinatal asphyxia 25

(11.8%), traditional uvulectomy 31 (14.7%), umbilical cord care without use of antiseptic agents 95 (45.0%) and presence of symptoms of maternal infections during pregnancy such as copious/offensive vaginal discharge, diarrhoea and dysuria 13

(6.2%). The frequency of maternal risk factors among neonates with proven septicaemia were generally higher than those in non-septicaemic neonates, though only the frequency of unsupervised antenatal period (p<0.05), prolonged labour

(p<0.05), prolonged rupture of membranes (p<0.05) and presence of symptoms suggestive of maternal infections during pregnancy (such as copious/offensive vaginal discharge, diarrhoea and dysuria) (p<0.05), reached statistical significance, especially in neonates with early-onset septicaemia. Among the host risk factors, only preterm delivery (p<0.05) and perinatal asphyxia (p<0.05) had statistically significant association with the occurrence of septicaemia.

Staphylococcus aureus was the commonest isolate, accounting for 33 (42.9%) of the total (77) isolates in the 75 septicaemic neonates, two of which had double isolates. The other common isolates were Escherichia coli 15 (19.5%), Streptococcus species seven (11.0%), Klebsiella pneumoniae six (7.8%), Proteus mirabilis five

(6.5%), Citrobacter species three (3.9%) and Acinetobacter species three (3.9%).

Staphylococcus aureus was also the commonest isolate among the out-born infants

xii (p<0.05) while Streptococcus species predominated among the in-born neonates with early-onset disease (p<0.05). Gram-negative bacteria were the commonest isolates among neonates (in-born and out-born) with early-onset septicaemia, while Gram- positive bacteria predominated among those with the late-onset type.

The sensitivity of the pathogens to commonly used antibiotics was on the decline when compared with those of previous studies. However, cefuroxime (85.0% sensitivity to Gram-positive, 32.4% to Gram-negative) and gentamycin (70.5% sensitivity to Gram-positive, 78.4% to Gram-negative) still retained good sensitivity rates. Chloramphenicol, third generation cephalosporins and quinolones also showed very high in-vitro activity.

It is concluded that the current aetiologic and epidemiologic patterns of septicaemia are similar to patterns earlier documented in our unit except for the higher incidence of the disease and the increasing prevalence of antibiotic resistant agents.

Therefore a combination therapy consisting of cefuroxime and gentamycin is recommended as the preferred empirical choice for the treatment of neonatal septicaemia in the unit. Preventive measures should be re-emphasised through aggressive health education, while the institution of an effective infection surveillance system is highly desired in order to combat the problems associated with the increasing prevalence of antibiotic resistant organisms.

xiii INTRODUCTION

Septicaemia has remained a major concern in medical practice both in developed and developing countries as the disease has been associated with high morbidity and mortality.1-9 In order to reduce the impact of septicaemia, there have been several advances in molecular biology, diagnosis and therapy.1 Despite this progress, septicaemia remains a serious bacterial disease, especially in the neonatal period. The newborn is highly vulnerable to bacterial infection because of deficient immunological system in the milieu of highly potent factors that enhance the multiplication of bacteria and introduction of bacteria into the body.1-3 In spite of the major advances made in neonatology in the last three decades, many newborn infants still develop life-threatening infections. Udoma et al10 observed a high rate of infection among newborn infants in Calabar. The authors observed that as high as 35.7% of newborn infants admitted in University of Calabar Teaching Hospital had septicaemia.10 In Shagamu,11 the disease was listed among the first four leading causes of neonatal mortality between 1989 and 1991. Ibrahim et al12 also observed and reported the leading role of neonatal septicaemia in neonatal mortality in Sokoto in a prospective study carried out between 1990 and 1991. In that study, 28.3% of the 60 neonatal deaths recorded were attributed to neonatal septicaemia.

In Zaria, Ogala et al13 evaluated the pattern of admission into the Special Care

Baby Unit and found that neonatal infections were the third commonest indication for admission among sick babies. Of the 650 sick newborn infants admitted during the period of study, 71 (10.9%) of them had infection, out of whom seven died. Earlier in the same unit, Winfred14 studied the incidence of neonatal bacterial infections between

1976 and 1979 and found bacterial infections in 445 (11.1%) of 4022 babies admitted during the study period. Of the 445 babies, 116 (26.1%) of them had proven neonatal septicaemia. Later in 1988, Amiebenomo et al15 described the pattern of neonatal septicaemia in the unit after a 10-month prospective study. The most recent work on 1 septicaemia in the unit was reported by Ogala et al.16 The authors described the antibiotic sensitivity pattern in the unit.16

From the aforementioned, it is evident that the disease is a common cause of neonatal morbidity and mortality in Nigeria. The reported incidence of neonatal septicaemia in this country ranges between 5 and 22.5 per 1000 live births.9,14,15,17-20

This figure is much higher than those reported from developed countries.5,21

A decline in mortality rate has been observed worldwide both in term and preterm babies.5,22 Nonetheless, approximately 1500 neonates in United States of

America die annually from systemic infection.5

The epidemiological and bacteriological patterns of neonatal septicaemia vary from place to place and from time to time. In 1981, Freeman et al23 observed that the change in aetiologic pattern of neonatal septicaemia in Yale occurred every decade.

The reasons for this changing aetiologic pattern are not known with certainty. This changing pattern has significant therapeutic implications hence the call for regular bacteriological surveillance of neonatal units to ensure timely detection of any deviation.7,8,23

Staphylococcus aureus, Escherichia coli and Klebsiella species predominate among the bacterial isolates in infected neonates in Nigeria and most parts of tropical

Africa7,17-19 in contrast to group B streptococcus and coagulase-negative staphylococci in developed countries.21-23

Analysis of accumulated data from the time Winfred reported his findings till date demonstrated a change in the aetiologic pattern of neonatal septicaemia in

Zaria.14-16 It is therefore necessary to conduct a study to determine the current status of neonatal septicaemia in Zaria in view of this changing pattern of epidemiology and bacteriology. The knowledge of its distribution and determinants (risk factors) will help in formulating contemporary preventive measures.

2 LITERATURE REVIEW

HISTORICAL BACKGROUND

The origin of the word septicaemia dated back to 1837, when according to

Bearn, the Frenchman Pierre Piory coined the word to describe a condition associated with pus formation.1 Later in 1925, Churchman, according to Johnson and Sell,24 described septicaemia as sequelae of a focal infection with systemic dissemination of micro-organisms. About the same time, Rulsion25 reported an outbreak of staphylococcal disease with septicaemia in newborn units in Europe. By 1933, Ethel

Dunham according to Schaffer and Avery26 called the attention of paediatricians to the ravaging effect of the disease in a communication which many considered as one of the important milestones in early clinical paediatrics.26 Since then, several workers have described the pattern of neonatal septicaemia and the burden of the disease both in America and various parts of the world including some African countries.5-9

DEFINITIONS

Septicaemia has been defined as a persistence of bacteria in the blood stream accompanied, as a rule, by a general reaction on the part of the host and by secondary manifestations at one or more sites distant from the original lesion and often overshadowing those of the initial focus or portal of entry.2 This is different from bacteraemia which is defined as recovery of viable bacteria from blood in the absence of clinical manifestation.2,3 Bacteraemia is usually an asymptomatic and a transient phenomenon.3 Some authors believed that it may not be necessary to differentiate bacteraemia from septicaemia in clinical neonatal practice since the complications that could arise from either condition may be serious.3 However, since a positive blood culture in a neonate could be due to contamination of the sample, it is desirable to

3 differentiate between a baby with clinical illness and one without clinical illness in the presence of a positive blood culture.

Neonatal septicaemia has been defined as “a disease of infants who are less than one month of age, who are clinically ill and who have positive blood cultures”.21

Neonatal septicaemia may be primary (if it occurred de novo) or secondary (if it follows surgery or an invasive procedure).9 Primary neonatal septicaemia can be further classified into “early-onset” and “late-onset”.9,21 “Early-onset septicaemia” has been defined differently by various workers. The term has been used to refer to neonatal septicaemia occurring from birth up to the seventh day of life.2,5,8,21 It has however been advocated that its use should be restricted to infections occurring within the first

72 hours of life, in which perinatal pathogenesis is adjudged most likely.3,4,9,21

Acquisition of bacteria before or during the birth process may lead to infection after an interval of 1 – 2 days.21 Hence neonates presenting with sepsis very early in life most likely acquired the infection either in utero or at the time of delivery and carry higher mortality. “Late-onset septicaemia,” on the other hand, refers to infections that occur between days 8 and 28 of life.21 Most community-acquired and nosocomial infections are of late onset.5,21 Many authors in Nigeria9,17,18 use 48 hours as the dividing line between early and late neonatal septicaemia in line with the recommendation of

Placzek and Whitelaw.4

The term sepsis has been used by various authors to imply different clinical conditions. Whereas some authors21,23,26 used the term interchangeably with septicaemia, others used it for all clinical conditions associated with the presence of pathogenic micro-organisms.5 To streamline its definition, a consensus conference of physicians was called where a term Systemic Inflammatory Response Syndrome

(SIRS) was coined to refer to the systemic response to tissue injury caused by infectious and non-infectious conditions such as ischaemia, trauma, haemorrhagic

4 shock and immunologic injury.21 When SIRS occurs as a result of infection, it is termed sepsis.21 In adults, the term SIRS is defined by the presence of two or more of the following: pyrexia or hypothermia, tachycardia, tachypnoea and abnormal white blood cell count or increase in immature neutrophils.21 While in the newborn population, the criteria for sepsis include the documentation of infection in a newborn infant with a serious systemic illness in which non-infectious explanations for the abnormal pathophysiologic state are excluded or unlikely.21 Hence the term probable sepsis may be use to refer to a clinical condition in which there is combination of clinical symptoms of infection and abnormal white cell indices even before the isolation of bacteria.9

INCIDENCE

The pattern of distribution of neonatal septicaemia has received much attention globally.5,6,9,27 Various reports have shown that the incidence of neonatal septicaemia is higher in developing countries than in developed countries.17,18,28 The incidence varies according to definition, from 1 - 4 per 1000 live births in the developed countries11,21 to 5 - 22.5 per 1000 live births in the under-developed countries. 3,8,9,15,17-

20 By the late 1970’s, a study carried out in Ibadan reported an incidence of 9 per 1000 live births.9 At about the same time, Omene19 reported an incidence of 6.1 per 1000 live births in Benin City. In the north, similar incidence rates were reported in Jos18 and

Ilorin.29

In the earliest series on neonatal infections from Zaria,14 an incidence of 9 per

1000 live births was reported in 1984. But in 1988, Amiebenomo et al15 reported a much lower incidence of 5 per 1000 live births from the same unit. In fact, the incidence rate reported by Amiebenomo et al15 has consistently remained the lowest of all the observed incidence rates in Nigeria.9,17-20 The reason for the low incidence in this report was not certain. However, the associated low blood culture positivity rate of

5 25.0%15 compared to 30.8-59.8% in other centres3,17,29 suggests the possibility of defective selection criteria such as inclusion of infected neonates who had received antibiotics before presentation in the study. It is possible that many of the mothers did not voluntarily divulge the history of antibiotic self-medication and hence the low blood culture positivity rate. In addition, laboratory and personnel factors might have played, at least, a minor role. In contrast to the low incidence rate in Zaria, the rates observed in Calabar (19.3 per 1000 live births)17 and Ile-Ife (22.5 per 1000 live births) 20 were exceptionally higher than the rates observed in most neonatal units in Nigeria.9,18,19

Differences in the selection criteria might have contributed to this significant difference in incidence rates. In addition, some of the blood samples used for the studies could have been obtained from umbilical vein in some of the neonates with umbilical catheter inserted for active resuscitation. Culture of umbilical venous blood obtained via umbilical catheterization has been shown to yield positive growth in neonates without septicaemia.30 The possibility of blood contamination, either at the time of sample collection or at the time of inoculation and incubation in the laboratory, cannot be excluded judging from the exceptionally high blood culture positivity rates of

55.5%17 and 59.8%20 reported from these studies.

From the aforementioned, it is obvious that the incidence of neonatal septicaemia varies from hospital to hospital. Apart from methodological inadequacies, personnel and laboratory factors, the other reasons for these hospital-to-hospital variations in incidence may be related to differences in rates of prematurity, prenatal care, conduct of labour and environmental conditions and practices in nurseries.21

The distribution of neonatal septicaemia varies with the gestational age at birth and the postnatal age at presentation.21,27,31 In Ibadan, 70.5% of neonates with proven septicaemia were preterm.9 Similarly the Calabar study17 showed a high frequency of septicaemia among preterm babies compared to term babies. Karpuch et al31 found a

6 relative risk of 47 among preterm compared to term neonates. The higher incidence of infections among preterm babies has been attributed to developmental deficiencies in virtually all arms of the immune system.21 The younger neonates are more prone to infections because of the immaturity of their immunological system.21 Hence neonatal septicaemia is commoner during the first postnatal week.7,17,18 In Jos, the mean age at presentation was 3.3 days,18 while in Calabar, 77.0% of cases of neonatal septicaemia were seen within the first week of life.17 In most of these neonates, the symptoms were noticed within the first 48 hours of life.7,8

Neonatal septicaemia is more common in males, with a sex ratio (M:F) of

2:1.4,7,9 Studies in Nigeria have consistently observed this male preponderance9,15,18,19 although the strength of the occurrence varies from study to study. In Ilorin29 the M:F ratio was 1.2:1, whereas in Jos18 and Benin City19 it was 1.8:1 and 2:1 respectively. A sex-linked factor has been proposed for this male predominance32 but the reasons for the observed differences in the sex ratios are not known. It may be attributed to observed differences in sex ratio at birth in the various institutions.33

Racial differences have been reported.34 Naeye and Blanc,34 in their study on the relationship of poverty and race to antenatal infection, found that neonatal infections were commoner in black newborn babies compared to the whites. This was partly attributed to higher incidence of prolonged rupture of membranes and poor utilization of antenatal and delivery services among black women.17,34

Seasonal variations in neonatal septicaemia have not been widely studied.

Nazer3 in Jordan reported two peaks of admission for neonatal septicaemia; one occurred during the cold months (December to March) and the other during the hot months (June to August). The reasons for this seasonal variation were not given. In

Nigeria, Alausa and Onile,35 in their study on epidemiological pattern of septicaemia in

University College Hospital, Ibadan, found a higher incidence of septicaemia in the hot

7 months of April to June (46.1 per 1000 hospital admissions) and the wet cold months of July to September (30.6 per 1000 hospital admissions) compared to the other months of the year (29.5 – 30.0 per 1000 hospital admissions) in 1978. During this period of apparent increase in incidence of septicaemia, there was a strict restriction of hospital admission and only very ill patients were hospitalized due to an acute shortage of funds and water supply to the hospital. This restricted admission could have probably modified the distribution of septicaemia across the months of the year.

From these reports, it is not certain that there is seasonal variation in the pattern of neonatal septicaemia.

CAUSATIVE AGENTS

In the past seven decades, a wide variety of bacteria have been observed to be associated with neonatal septicaemia. Most of these organisms, particularly Gram- negative enteric bacteria, normally inhabit the maternal genital and gastrointestinal tracts.5 The micro-organisms responsible for early-onset septicaemia differ from those responsible for late-onset infection. This difference is related to the pathogenesis of infection in the newborn.21 Whereas early-onset septicaemia commonly results from maternal genital and intestinal flora acquired in utero or at the time of delivery, the late- onset category usually involves bacteria acquired from the environment where the baby lives.21,36,38

The agents also vary from place to place and from time to time.5 In developed countries, the commonest agents of early-onset neonatal septicaemia in recent times are group B streptococcus (GBS), Escherichia coli, Klebsiella species, Listeria monocytogenes and Staphylococcus aureus, while the agents commonly isolated in the late-onset category are coagulase-negative staphylococci (CONS),

Staphylococcus aureus and Pseudomonas species.28,38,39 However, in some infants presenting very late in the neonatal period, their infections may be caused by

8 organisms that are implicated in post neonatal infections such as Haemophilus influenzae, Streptococcus pneumoniae and Neisseria meningitidis.40

In 1999, a national point-prevalence study of nosocomial infections in neonatal intensive care unit patients in America lent credence to the predominance of CONS in late neonatal septicaemia.39 In that study, CONS accounted for 48.3% of the isolates followed by Enterococci (15.5%), Pseudomonas aeruginosa (5.2%), Escherichia coli

(3.4%) and Staphylococcus aureus (3.4%). Similarly, a 10-year multi-centre study between 1991 and 2000 in Australia28 revealed CONS as the commonest agents of late-onset neonatal septicaemia, accounting for 57.1% of the bacterial isolates. The increased prevalence of CONS infections in neonatal intensive care units has been attributed to the increased survival of very low birthweight infants, prolonged hospitalization, prolonged exposure to broad spectrum antibiotics and use of invasive procedures for monitoring and treating unstable infants.21 In addition, the use of intravenous lipid emulsion has also been associated with an increased risk of CONS bacteraemia.28

Since the emergence of GBS in the 1970s, it has remained the leading cause of early-onset neonatal septicaemia in America and Europe.36,41 These organisms (GBS and CONS) that tend to dominate the causative agents of neonatal septicaemia in developed countries do not play such a prominent roles in the developing countries, except in very few places like Dubai in United Arab Emirates where GBS was reported to be the commonest agent of early neonatal septicaemia.42 The organisms commonly encountered in infected neonates in developing countries are Escherichia coli,

Klebsiella species, Staphylococcus aureus, Streptococcus species and Pseudomonas species.7,9,17 In Nigeria, the agents predominantly reported are Staphylococcus aureus, Escherichia coli and Klebsiella species, although the predominant agents differ from centre to centre.9,14,18,19 In Benin City, Omene19 reported Escherichia coli as

9 the leading cause of neonatal septicaemia in 1979, followed by Staphylococcus aureus, Klebsiella species and Pseudomonas species. About the same time,

Klebsiella species predominated, as agent of neonatal septicaemia in Ibadan, with

Escherichia coli and Staphylococcus epidermidis occupying the second and third positions respectively.9 In Ile-Ife in mid-1990s, Adejuyigbe et al20 found that

Pseudomonas species and CONS were the prominent organisms in their institution, accounting for 18.8% and 15.9% of the bacterial isolates respectively.

In northern Nigeria, the predominance of Staphylococcus aureus was observed.15,18,43 Staphylococcus aureus, Escherichia coli and Klebsiella species were the commonest bacterial agents isolated in Zaria.14-16 These three agents accounted for 65.5% to 81.3% of the bacterial isolates in previous studies in the same centre.14-16

The other bacterial agents reported in Zaria included Streptococcus pyogenes,

Salmonella species, Pseudomonas aeruginosa, Citrobacter species, Alcaligenes species, Streptococcus pneumoniae and GBS.14,15 In addition to these agents, Akpede et al,43 reported a high incidence of untyped Coliform species among 108 newborn infants with septicaemia in Maiduguri, accounting for 32.4% of total bacterial isolates.

The reasons for this regional variation are uncertain. The antibiotic policies of the various regions might have contributed. Use of antibiotics allows selection or induction of resistant organisms which in turn may rapidly spread to become the dominant bacterial agents of the region.41,44

Reports from various newborn units suggest that neonatal GBS septicaemia is rare in Nigeria.9,14,18 However, GBS constituted 2.0%, 2.5% and 5.0% of the reported isolates in Zaria,15 Calabar,17 and Benin City19 respectively. In 1980, the incidence of neonatal GBS disease in Ibadan was 0.4 per 1000 live births.45 The low incidence of neonatal GBS septicaemia occurred against the backdrop of high vaginal GBS colonization among pregnant women reported to be as high as 19.5% in Ibadan.45 The

10 reasons for the low incidence of neonatal GBS infection are not clear. The relatively free use and uncontrolled availability of antibiotics in this environment might have contributed. Prophylactic administration of ampicillin has been shown to decrease neonatal colonization during birth.46 Widespread use of antibiotics which are easily obtained over the counter without prescriptions could have decreased the rate of neonatal colonization and hence the incidence of GBS septicaemia.45

A changing pattern in the aetiologic agents of neonatal septicaemia has been noted globally especially in the industrialized nations.41,47,48 In the pre-antibiotic era,

Gram-positive organisms predominated. Before the 1940’s, the predominant organism causing neonatal septicaemia was group A beta haemolytic streptococcus.

Coincidental with the increasing use of penicillin and sulphonamides, group A streptococcal diseases in babies began to decline, with Staphylococcus aureus becoming the major cause. This shift was attributed to the introduction of antimicrobial therapy, but subsequent developments suggest that other factors may have been, at least, equally important.41

The predominance of Staphylococcus aureus was short-lived having soon been replaced by Gram-negative enterobacteria.41 Since this new shift did not coincide with the introduction of any significantly more effective antistaphylococcal agents or new nursery techniques, the reason for the change remains elusive.41 Similarly the reasons for the emergence of GBS in 1970 and CONS in recent time as the commonest agents of neonatal septicaemia in many newborn units in developed countries28,39,49 are not known with certainty. Increased survival of low birthweight infants with its attendant prolonged hospitalization, use of humidified units in specialized patients care, high frequency of antibiotic resistance, increasing use of invasive diagnostic and therapeutic measures and lowered incidence of breast feeding were among reasons advanced for the changing aetiologic pattern.50

11 In Nigeria, the changing aetiologic pattern of neonatal septicaemia has been observed in reports from various newborn units,47,51-54 although the typical switch from

Gram-positive predominance to Gram-negative preponderance was not observed in most of these studies.47,51-54 In Lagos, Ogunbi et al51 studied the pattern of bacterial infections in babies with neonatal jaundice between 1967 and 1973. The authors observed that Escherichia coli, Pseudomonas species and atypical Coliform, which formed the bulk of bacterial isolates in 1967, were found to be either completely absent or of reduced frequency in subsequent years. Instead Klebsiella species, which formed only 3% of bacterial isolates in 1967, contributed 10.0% of bacterial isolates in

1972.51

Similar changing pattern was also observed in reports from Ile-Ife.20,53 The study of Ako-Nai et al53 in 1999 and those of Adejuyigbe et al20 in 2001 demonstrated an increasing prevalence of Pseudomonal septicaemia, from the initial 3.0% in 1999 to

18.8% in 2001. In both series, however, Staphylococcus aureus maintained its leading role in neonatal septicaemia in the centre contributing 33.8% and 36.2% in 1999 and

2001 respectively. In Ibadan, the series of Dawodu and Ashiru52 on neonatal meningitis showed an increasing prevalence of Coliform and Klebsiella meningitis as against the predominant role of Salmonella species in the report of Barclay,55 both from the same unit. In addition, the frequency of Pneumococcal organism was twice as many as those reported by Barclay.55

The experience in Zaria in the last two decades revealed a gradual change in the predominant agent of neonatal septicaemia.14-16 Winfred series14 revealed the predominance of Staphylococcus aureus septicaemia (33.0%), with Escherichia coli and Klebsiella species contributing only 22.4% and 10.3% respectively. About nine years later, Amiebenomo et al15 recorded more cases of Klebsiella septicaemia than

Escherichia coli septicaemia with the frequency of Klebsiella species almost tripling

12 (29.0% versus 10.3%) that of Winfred report.14 By 1996, Klebsiella species was reported as the leading cause of neonatal septicaemia in the same unit, accounting for

29.2% of the bacterial isolates in Ogala et al series,16 while Staphylococcus aureus contributed only 13.0%. The reasons for this change in pattern of bacterial isolates in this unit are not clear. Differences in the place of delivery and the relatively free

(indiscriminate) use of common antibiotics in the community might have contributed.

Since most of the easily procurable, orally administered antibiotics have higher activity against Gram-positive agents, drug pressure in favour of Gram-negative bacilli is a possibility.44

The causative agents of neonatal septicaemia in Nigeria have also been shown to vary with age, even though most studies in Nigeria did not attempt relating the bacterial agents to the age at onset of illness.9,11,14,56 The few that attempted doing this, reported Gram-negative bacilli as the commonest cause of early neonatal septicaemia while Gram-positive cocci predominate among the agents of late-onset septicaemia.18,19,29 In Ilorin,29 the predominant organisms in the first 48 hours of life were Gram-negative bacilli, accounting for 70.0% of the isolates, but between the ages of three and seven days, Gram-positive cocci accounted for 60.0% of the isolates. The predominance of Gram-negative bacilli in early-onset septicaemia could have resulted from the fact that most of the organisms implicated in early-onset infections constitute the normal flora of the maternal genital and intestinal tracts,5 most of which are Gram- negative bacilli. In the late-onset category, the predominance of Gram-positive cocci especially Staphylococcus aureus suggests that the infections were mostly community-acquired.

The contribution of anaerobic organisms in the aetiology of neonatal septicaemia has been largely unexplored in Nigeria. Most authors15,17-19 did not culture for anaerobes. However, Ogala et al16 did isolate anaerobic Streptococcus in one of

13 the 41 neonates suspected of having septicaemia. Of the 24 bacteria isolated, 23 of them were aerobes while one (4.2%) was an anaerobe. This underscores the need to investigate for anaerobic bacteria in neonates presenting with features suggestive of neonatal septicaemia.

DEVELOPMENTAL IMMUNOLOGIC DEFICIENCY IN THE NEONATE.

The newborn resists infection by means of a number of integrated systems collectively known as the immune system. The immune system is divided into two parts based on the speed and specificity of their reaction.57 The innate immunity includes physical, chemical and micro-biological barriers as well as the element of the immune system (neutrophils, monocytes, macrophages, complements, cytokines and acute phase proteins) and it provides immediate host defence while adaptive immunity consists of antigen-specific reaction through T- and B-lymphocytes.57 Whereas the innate response is rapid but sometimes damages normal tissue through lack of specificity, the adaptive response is precise but takes several days or weeks to develop.57 The development of these two arms of immune system starts early in fetal life but required several years before reaching maturity.58 Adult levels of IgM, IgG and

IgA are achieved at ages 1 year, 5-7 years and 10-14 years respectively.58 This developmental immunologic deficiency has been observed in virtually all aspects of the neonatal immune system.21

Deficiency in innate (non specific) immunity

Deficiency in mechanical barrier: The skin, a normal protective barrier in humans, is more permeable in neonates, particularly those of preterm gestation.59

Breaks in skin integrity are common in newborns, creating portals of entry for infectious agents.59 The protective secretions, such as tears, saliva, mucus and laryngeal secretions are low in quantity in the early days of life.59,60 Ciliary action in the

14 respiratory tract is poor and this, coupled with poorly developed cough reflex predisposes the infant to respiratory infection.59

Deficiency in complement system: There is essentially no transplacental transfer of maternal complements.21 The fetus begins to produce complements as early as the first trimester.21 The complement level progressively increases with age, reaching 50-

70% of maternal complement level at birth.21 Both the classical and the alternative pathways of the complement system are deficient but the deficiency is more pronounced in the alternative pathway than in the classical pathway.21 At birth, the alternative pathway complement activity is usually 35%-60% of the corresponding maternal level.21 Because of the age-dependent increase in complement level during fetal life, the deficiency in complement activity is much more pronounced in preterm babies. Functionally, complements mediate bactericidal activity against certain organisms such as Escherichia coli and also serve as opsonins in the phagocytosis of bacteria such as GBS.21 Since complement activation is an amplification cascade, small early deficiencies may markedly diminish the formation of later components that are critical to granulocytes chemotaxis and bacterial opsonisation.5 Studies have shown that opsonisation of Staphylococcus aureus is normal in neonatal sera but impaired against GBS and Escherichia coli.21

Deficiency in polymophonuclear leukocytes system: Newborn babies have both quantitative and qualitative deficiencies in neutrophils.21 The neutrophil storage pool

(non circulating neutrophils) is markedly low.5 At birth the neutrophil storage pool is 20-

30% of that in adults.21 During the very early stages of infection, there is a release of cytokines from activated macrophages. Two of these, granulocyte- and granulocyte- macrophage colony stimulating factors,57 stimulate division of myeloid precursors in bone marrow, releasing millions of cells into the circulation and causing a characteristic neutrophil leucocytosis.57 But in newborns, the neutrophil progenitor

15 cells are unable to increase their proliferative rate during stresses such as infection, because it is already at its near maximum level.5 Hence it is, not surprising that relative neutropaenia is found in up to one third of premature infants.5

Qualitative neutrophil deficiency has also been demonstrated.61 Normally, neutrophils are recruited to the site of infection through a multi-step process involving pro-inflammatory mediators, adhesion molecules, chemoattractants and chemokines.

The recruited neutrophils phagocytose the organisms and bacterial killing is achieved by a combination of two mechanisms namely: oxygen-dependent response

(respiratory burst) and oxygen-independent response.57 The oxygen-dependent response involves the sequential reduction of oxygen by an NADPH oxidase leading to production of toxic oxygen metabolites such as hydrogen peroxide, hydroxyl radicals and singlet oxygen.57 The oxygen-independent response uses the highly toxic cationic proteins and enzymes such as myeloperoxidase and lyzozymes contained within the neutrophil cytoplasmic granules.57 In newborn infants, there is a quantitative defect in signal transduction and hence a delay in neutrophil response to infection.21 In addition, the number of receptors for opsonic proteins such as complement fragment C3b and

C3bi are low compared to adults and as such opsonization is inadequate.21

Neutrophils derived from stressed neonates have been shown to have decreased chemotaxis, impaired adherence and decreased bactericidal activity.61,62 Wright et al61 assessed leucocytes function in a case controlled study involving 40 stressed newborns, 12 well newborns and 23 normal adults. The authors found that leucocytes from 60.0% of the 40 stressed newborns had decreased in-vitro activity against

Staphylococcus aureus and Escherichia coli, while only 17.0% of the 12 well infants demonstrated such decrease in leucocytes activity. In that study, the mean bactericidal activity (percentage of organisms killed after two hours) of leucocytes from stressed newborns against Staphylococcus aureus was significantly less than those from the

16 well infant and adult control groups. In a similar study involving 10 severely asphyxiated babies in Zaria, Ogala et al62 demonstrated a significant impairment in neutrophil chemotaxis and bacterial killing ability in 1996. The principal defect against

Staphylococcus aureus was a killing defect whereas both phagocytosis and killing defects were observed against Escherichia coli.61 All these deficiencies result in delayed response to infection.

Deficiency in mononuclear phagocytic system: The number of circulating monocytes in neonatal blood is normal;59 their phagocytic and bacterial killing ability also approximates that of the adult.2,21 However, chemotaxic activity is impaired both in term and preterm babies.2,63 The chemotaxic defects play an important role in the newborn’s susceptibility to infections by diminishing the quality and rapidity of non- specific inflammatory reactions.61,63 The tissue macrophages in the reticuloendothelial system are also diminished both in mass and in function especially in the preterm infants.21

Deficiency in fibronectin: Fibronectin normally participates in the immunological clearance of injured tissues, complements and antibody-coated materials and numerous pathogenic organisms such as Staphylococcus aureus, Streptococcus species and some Gram-negative bacilli.2,64 It serves as opsonin for some of these bacteria and also assists with neutrophil adherence.64 Newborns have low plasma concentration of fibronectin.64 A direct correlation exists between its plasma level and the gestational age.21 The plasma level progressively increases until about the age of two months when the adult level is attained.64 This low level in the first weeks of life contributes to hypofunction of the neonatal reticuloendothelial system and predisposes the baby to bacterial infection.2,64

Deficiency in cytokines: Interferons are a major class of cytokines that have important roles in immunity. In the newborn, levels of type 1 interferons (α and β-

17 interferons) are normal but type 2 ( or immune) interferon synthesis is low.57,59

Interferon  plays an important role in neonatal immunity. It acts directly on the immune system to activate macrophage and neutrophil intracellular killing.57 It also stimulates natural killer cells function and enhances antigen presentation by increasing major histo-compatibility complex class 2 expression on antigen presenting cells.57

Tumour necrosis factor-α (TNF-α) levels are elevated in infants with septicaemia, but the response is less consistent than in adults.21,59 Interleukin-2 (IL-2) activity in cord blood from full term and preterm infants was reported to be higher than in the adult, but messenger RNA for the IL-2 receptor could not be detected in cells from premature infants.21 During infection, the blood levels of both pro-inflammatory cytokines (IL-2, IL-6, TNF-α, INF-) and anti-inflammatory cytokines (IL-4, IL-10) are increased.65 Despite the inconsistency in cytokines response in neonatal septicaemia, their levels have been used in prognostication.65 Persistently high plasma IL-6, IL-10 and TNF-α concentrations signify severe infection.65 Poor prognosis has also been associated with high IL-10/ TNF-α and IL-6/IL-10 ratios.65

Deficiency in the natural killer (NK) cells: Natural killer cells lyse cells that are coated with antibodies in a process called antibody-dependent cell-mediated cytotoxicity.21 In addition, NK cells secrete perforins and granzymes which cause induction of apoptosis of cells that do not have major histo-compatibility class 1 expression such as viruses and tumor cells.57 The level of NK cells in neonates is equivalent to that in adults, however they have decreased cytotoxic activity when compared with adult cells.21

Adaptive (specific) immune system

Deficiency in immunoglobulins: It is only immunoglobulin G (IgG) that is transferred transplacentally to the fetus.58,66 Its transfer occurrs maximally during the latter half of the third trimester. Hobbs and Davis67 reported a mean serum IgG of 18 1000mg/dl in term babies compared with 400mg/dl and less in preterm babies. The specificity of the IgG is dependent on the maternal antigenic experience and immunologic response, and so the newborn may be protected against those infections in which protection is IgG mediated such as tetanus and GBS. The IgM class, which contains bactericidal activity against enteric bacteria, is not transferred across the placenta, thus partly explaining the increased susceptibility of the newborn to Gram- negative septicaemia.66 In general, the newborn infant is deficient in antibody- mediated protection against Escherichia coli and other enterobacteriaceae.21

Deficiency in T-cell function: Although T-cell development begins as early as seven weeks of gestation, most of the circulating T-cells in the neonate immediately after birth have not been stimulated by antigen, hence there is deficiency of memory T- cells.66 The lack of memory T-cells is important because such cells produce a variety of cytokines.5 There is therefore a delay in the development of cytotoxic T-lymphocytes in neonates which increases their susceptibility to intracellular pathogens.5 In addition, neonatal T-cells express CD40 ligands poorly; this expression is required for cognate interaction with B-cells in the induction of antibody synthesis.39 Chandra68 in 1975 assessed T-cell function by means of 10% 2,4-dinitrochlorobenzene sensitisation and lymphocyte response to phytohaemaglutinin and found significant qualitative and quantitative deficiencies in T-cell function in small-for-gestational age infants.

These immunologic deficiencies make neonates highly prone to infections.

However, despite these defects, the rate of invasive infectious disease is low in the absence of obstetric, neonatal and environmental risk factors.21 The presence of a combination of these risk factors greatly increases the probability of infection. For example, the incidence of infection may increase to greater than 5% when two or more risk factors are present.5

19 PREDISPOSING FACTORS

Prominent among the host factors that predispose a newborn infant to infections are prematurity, male sex, black race, birth asphyxia and congenital malformations.7 The single most important risk factor for infection is prematurity.2,69

Infants born at less than 32 weeks gestation are 4 to 25 times more likely to develop early-onset septicaemia compared to their more matured counterparts.5 This was supported by the report of Geme et al70 which showed that prematurity increases the risk of infection 11-fold and if the baby was of the male sex, the risk was further increased to 25-fold. Similarly, many studies9,17,18 in Nigeria have documented a higher prevalence of infection in preterm infants compared to their term counterparts.

About 70.0% of neonates with proven septicaemia in Ibadan in late 1970s were preterm.9 The higher prevalence of infections in preterm babies is linked to the developmental immunodeficiency, which appeared exaggerated in them compared to the term infants.21 In addition, preterm infants are more likely to be resuscitated at birth, to have more invasive procedures and to have prolonged hospitalization.

Male sex also increases the vulnerability of the infant to infection in the neonatal period.69,70 A predominance of male sex is apparent in almost all studies on septicaemia in the newborn.18,19,34 The greater susceptibility of the male is even more evident in cases of septicaemia due to Gram-negative enteric bacilli than in septicaemia due to Gram-positive cocci.21 A sex linked factor has been suggested for this.32 According to Washburn et al,32 the gene responsible for immunoglobulin synthesis is located on X-chromosome with which the female is doubly endowed.

Rhodes et al71 supported Washburn’s hypothesis by demonstrating that serum IgM levels were directly related to the number of X-chromosomes present in abnormal

XXX-females, normal XX-females and normal XY males.

20 The work of Naeye and Blanc34 on antenatal infection suggests the occurrence of racial differences in the prevalence of neonatal septicaemia. The authors reported higher infection rate among black newborn infants compared to their white counterparts. This may be attributed to the higher incidence of prolonged rupture of membranes and poorer utilization of antenatal and delivery services among black women.17,34

Perinatal asphyxia has been shown to cause immune suppression and hence predisposition of severely asphyxiated newborn infants to infection.62 The integrity of the skin and mucous membrane could be compromised in the process of resuscitation thereby creating easy access of pathogenic bacteria to blood circulation. Similar mechanism explains the predisposition of babies with congenital malformations such as omphalocoele, spinal bifida cystica and gastroschisis to infection. The problems associated with multiple gestations such as preterm delivery, traumatic delivery (as a result of abnormal lie) and asphyxia (due to retaining of the second twin); all of which could predispose a newborn to infection, may explain the association of multiple births with development of neonatal infection.

Several maternal risk factors have also been identified, namely: low socioeconomic factor,69 maternal genital colonization with organisms such as GBS and Escherichia coli7 and maternal illnesses such as septicaemia and urinary tract infection.21 Maternal obstetric problems such as prolonged rupture of the membranes,72 prolonged second stage of labour,73 chorioamnionitis,70 intrapartum maternal fever74,75 and intrapartum haemorhage75 are other maternal risk factors.

Naeye and Blanc34 had reported that neonates born to women of low socio- economic status had higher incidence of infection than infants born to women of higher income groups. The authors observed that women from low income background were more likely to have inadequate antenatal care, delivery outside the hospital and

21 complicated labour.17,34 The authors also found that the amniotic fluid of women of low socio-economic status have reduced bactericidal activity with consequent higher frequency of amniotic fluid infection than in women of higher socio-economic status.34

Amniotic fluid infection is a major source of neonatal septicaemia especially of the early-onset type.34 A ten-fold increase in the risk of infection has been reported in babies born to women with chorioamnionitis.70 Similarly, the role of prolonged rupture of membranes in neonatal septicaemia was demonstrated by Airede.72 In that study,

44.0% of babies born after prolonged rupture of the membranes developed bacteriologically proven septicaemia.72 Prolonged rupture of the membranes allows ascension of the maternal genital flora resulting in infection of the fetus. In addition, frequent vaginal examinations before delivery increases the risk of infections.73

Other risk factors reported include therapeutic procedures such as umbilical catheterization76 and use of indwelling venous or arterial line.77,78 Introduction of a catheter may permit organisms to ascend along the catheter-blood vessel interface and may also release contaminated thrombi present in the lumen of the vessel.31 The incidence of bacteraemia in infants with umbilical catheter ranges between 2.7% and

8.0%,76 while that of non-catheterized infants is 3.4% in Balagtas et al76 series. In that series, however, Balagtas et al76 reported cases of systemic bacterial infection following umbilical catheterization. Six of the eighty-six newborns involved in that study developed septicaemia. It was however difficult to ascertain the exact role of the catheter in the development of infection. In three of these patients, the organisms isolated from the peripheral blood were also isolated from the cord blood samples taken at the beginning of catheterization. Unfortunately, no peripheral blood cultures were taken prior to catheterization, which would have settled the question on the origin of the bacteria in these cases.31 In most cases of intravenous lines-related infection,

Gram-negative organisms were the predominant isolates.77

22 Harmful traditional practices like uvulectomy, tribal markings, administration of traditional concoctions either orally or topically on the umbilical stump are among the risk factors identified.21,75

DIAGNOSIS OF SEPTICAEMIA

In both term and preterm infants, early signs and symptoms are often minimal, subtle, non-specific and can easily be misinterpreted as being due to non-infective causes.79 Although the onset of illness is often inconspicuous, the clinical course may be alarmingly fulminant leading to septicaemic shock, disseminated intravascular coagulation and death within hours of onset of definite clinical manifestations.79

Infected infants must therefore be promptly identified and antibiotics started without delay in order to achieve any therapeutic success.79,80 The difficulty in early identification of infected newborn infants has led to the evolution of many clinical scoring systems for prediction of neonatal septicaemia in high risk babies.

Takkar et al80 suggested a scoring system for the prediction of early neonatal infections using a combination of adverse perinatal factors. In this system, a score of 2 each was assigned for the presence of 1-minute Apgar score of  6, unclean vaginal examination before delivery, foul smelling liquor amnii and duration of labour exceeding 24 hours. A score of 1 each was given for prolonged rupture of membranes and a birthweight of  2.0 kg or gestation period of less than 37 weeks. The authors assessed the scoring system on 1000 consecutive deliveries and found that the incidence of neonatal infections was low (0.5%) for those with a total score of 0 – 3,

17.6% for those with a score of 4 – 5 and 85.7% for those with a score of 6 or more.

Among those with a score of  6, majority had systemic infections. A score of  6 is therefore highly predictive of septicaemia. Similarly, Singh et al73 evolved predictive clinical scores for the diagnosis of septicaemia of late-onset type. This scoring system

23 consists of seven signs. The presence of 3 or more of the signs was highly predictive of septicaemia. The signs include grunting, abdominal distension, increased pre-feed aspirate, tachycardia, pyrexia, chest retraction and lethargy. When these clinical signs were allotted weight proportionate to the relative magnitude of the positive likelihood ratio, a score of 2 each was assigned to grunting and abdominal distension, while a score of 1 each was assigned to increased pre-feed aspirate, tachycardia, pyrexia, chest retraction and lethargy. A total score of  2 had a positive predictive value of

65.0%. Thus the authors suggested the use of this seven-item weighted clinical score for clinical diagnosis of late-onset neonatal septicaemia.73

Use of perinatal factors as selection criteria may not be sensitive in some neonates that do not have such adverse perinatal factors hence reliance on Takkar et al scoring system may results in high number of false negative cases with consequent under treatment and hence increase morbidity and mortality. Similarly, non-infection clinical conditions like meconium aspiration syndrome could present with most of the features listed in Singh et al scoring system and this may result in over diagnosis of septicaemia in babies who otherwise are not infected.

Laboratory evaluation is therefore necessary for confirmation of infection.

Positive cultures of blood, cerebrospinal fluid or urine are the gold standards for confirming systemic infections.79,81 Ideally, all specimens for cultures should be obtained before initiation of antibiotic therapy. However, as many as 20.0% to 30.0% of American women in labour currently receive antibiotics before delivery.5 Thus an infant may have already been exposed to antibiotics at the time of birth. Therefore, in a considerable proportion of neonates at risk of infection, their culture results may be influenced by prior antibiotic exposure.

Blood is the principal fluid assessed for suspected septicaemia.81 The presence of bacterial growth may be influenced by several factors such as intermittency of

24 bacteraemia, size of the inoculum and previous antibiotics exposure.5,81-83 The optimal number and volume of blood culture samples to be obtained has received wide study.81,83,84 Two or more cultures obtained from different sites yielded the greatest amount of information in infants with early-onset infection, while at least 2 ml of blood is recommended.81

The yield of blood cultures in adults increased by 3.0% per millilitre of blood cultured,81 hence higher volume of blood is advocated for adult blood culture. This was partly explained by the fact that more than 50.0% of adult systemic infections were associated with low colony-count bacteraemia of less than 1cfu/ml81 in contrast to the high bacteria count in neonates with Escherichia coli septicaemia as was demonstrated by Dietzman et al84 who reported a colony-count of greater than 1000 cfu/ml of blood in 31.0% of 30 neonates with Escherichia coli septicaemia. In that study, as little as 0.2 to 0.5 ml of blood was adequate for isolation of the organism. On the basis of this report, a minimum of 0.5 to 1ml has been the recommended volume of blood for neonatal blood culture.81,83,84 Most true pathogens grow within 48 hours.

With current blood culture systems, at least 95.0% of positive cultures will show evidence of bacterial presence within 48 hours of inoculation into media.5,82 Therefore, growth after this length of time may represent contamination from non-virulent skin organisms.

The percentage of positive blood cultures among neonates with presumptive diagnosis of septicaemia varies from study to study. In late 1970s, Nazer3 screened

112 neonates admitted with presumptive diagnosis of septicaemia in the Jordan

University Hospital. As high as 62.5% of the blood samples yielded bacterial growth.3

In Nigeria, the reported blood culture positivity rates were much lower than that reported by Nazer.3 Mokuolu et al29 in Ilorin and Egri-Okwaji et al56 in Lagos reported

30.8% and 36.0% positivity respectively. Antia-Obong et al17 however reported a rate

25 as high as 59.8%. The reasons for these differences in observed blood culture positivity rates might have probably resulted from differences in patient selection criteria, volume of blood cultured, size of the inoculum, laboratory and personnel factors as well as the rate of indiscriminate use of antibiotics in the respective localities.

Urine should be cultured whenever an infant older than 72 hours is suspected of being infected since urinary tract infection is relatively uncommon in early-onset septicaemia but common with late-onset type.85 The low yield of urine cultures in early- onset septicaemia may be due to the fact that specimens are obtained in these infants before sufficient time has elapsed to establish the infection in the urinary tract.85

The cerebrospinal fluid should also be examined and cultured. Because of the rarity of meningitis in early-onset neonatal septicaemia, some authors86 have suggested that lumbar puncture should be limited to infants with early-onset septicaemia presenting with symptoms specific to the central nervous system and those infants with suspected or proven late-onset septicaemia.

Over the years, it has been popular to obtain culture specimens from such diverse sites as skin surfaces, gastric fluid, ear canal, umbilical cord surface, rectum and tracheal aspirates. However, most information in the medical literature in the late

1980s and early 1990s had indicated that such cultures are of limited value in confirming systemic infections.87-89 Additionally, it has been speculated that routine surveillance cultures would be of benefit in the early detection of hospital-acquired infections.90,91 In many nurseries, swabs or aspirates of the endotracheal tubes are cultured on a weekly basis.90,91 A child who then becomes sick is presumed to be infected with organisms that are cultured from the surveillance cultures.90,91 However, this hypothesis has been refuted by virtually all researchers that evaluated the practice.89,91,92 Any sterile endotracheal tube placed in a neonate may become

26 colonized within hours of initial placement. Secondly, there is little correlation between organisms colonizing the endotracheal tube and the organisms that are grown from blood of infants with nosocomial infections.91 Therefore some authors have concluded that surveillance cultures do not decrease morbidity or mortality, nor are they cost effective.89,90

Since microbiological culture results and antibiotic susceptibility data are not usually available until at least 48 hours after the specimen reaches the laboratory, early diagnosis of genuine septicaemia remains a major problem.79 Consequently, a wide variety of diagnostic markers of infection was investigated for the evaluation of clinical neonatal systemic infection.79 In the early- and mid-1980’s, neonatal clinicians relied mainly on haematological indices as adjunct indicators for early diagnosis of neonatal septicaemia.79 Total leucocyte count, total neutrophil count, immature to mature neutrophil ratio, morphological or degenerative changes in neutrophils such as vacuolation, Döhle’s bodies, intracellular bacteria, toxic granulation and platelet count have been studied either singly or in combination.93-96 Results of white cell counts and ratios varied widely across studies, with sensitivity and specificity ranging from 1.7% to

90% and 31% to 100% respectively.79 Findings that have been associated with culture-proven septicaemia include an increased immature-to-total granulocyte ratio

(I:T>0.20), a high band count (>2000/mm3), neutropaenia (total neutrophil count

<1750/mm3), exceptionally high (>25,000/mm3) or low (<5000/mm3) total white cell count and thrombocytopaenia (platelet count < 100,000/mm3).94-96

Okolo et al93 assessed the usefulness of leucocyte indices and micro erythrocyte sedimentation rate (mini ESR) in the diagnosis of neonatal infection among 32 infected newborns in Benin City. Using 20 non-infected newborns as control, the authors found a sensitivity of 100% and specificity of 92.3% when mini ESR was combined with leucocyte indices.93 In view of this, some authors93,96 suggested that combination of

27 these haematological indices is more sensitive in predicting neonatal infection than the use of individual indices alone. In line with this recommendation, Rodwell et al96 proposed a scoring system using these haematological indices. To each of the indicators, the authors assigned a score of 1. A total score of 3 or more was found to be highly suggestive of septicaemia, having a sensitivity of 96.0% but a disappointingly low positive predictive value of 31.0%.79 The indices used in the scoring system included:

1. Abnormal total leucocyte count either less than 5 x 10 9/l or greater than 25 x

109/l at birth, 30 x 109/l at 12-24 hours of life and 21 x 109/l at Day 2

onwards.

2. Abnormal total neutrophil count.

3. Elevated immature polymorphonuclear cell count.

4. Elevated immature-to-total granulocytes ratio (I:T > 0.20).

5. Elevated immature to mature polymorphonuclear cells.

6. Degenerative changes in neutrophils.

7. Thrombocytopaenia (platelet < 150 x 109/l).

This scoring system was not widely adopted because of its unfavourable diagnostic value, complexity of the scoring method and the fact that some of the tests were labour intensive and required a highly trained technician to produce an accurate result.79

Examination of buffy coat smear have also been suggested in making diagnosis of neonatal septicaemia.5 Microscopic examination of the buffy coat smears with Gram or methylene blue stain may reveal bacteria many hours before culture results are available.5,21 In addition, determination of the activity of several leucocyte enzymes such as leucocyte oxidase and leucocyte alkaline phosphatase through nitroblue tetrazolium test has been studied as an ancillary method in diagnosis of neonatal

28 septicaemia.97,98 Unfortunately, non of these tests had demonstrated consistent sensitivity or specificity to warrant their routine use.

The use of acute phase reactants became prominent in the late 1980’s and early 1990’s as it was recognised that haematological indices alone could not be confidently used as decision criteria for diagnosing neonatal infections.79 The most extensively used and investigated acute phase reactant is C-reactive protein (CRP).

This protein is synthesised within 6 to 8 hours of exposure to an infective process79 and has been shown to have higher sensitivity and specificity values than neutrophil counts and I:T ratio.99,100 However, the rise in the serum concentration of CRP in the initial phase of infective process is slow, making it less sensitive in diagnosing early- onset neonatal septicaemia.79 In addition, non-infection clinical conditions such as meconium aspiration syndrome have been associated with elevated level of CRP.79

These limitations led to the discovery of procalcitonin (PCT) as an acute phase reactant. Serum concentration of PCT begins to rise four hours after exposure to bacterial endotoxin, peak at 6 to 8 hours and remain raised for at least 24 hours.101

The half life of PCT is 25-30 hours, thus making it useful in detecting both early- and late-onset neonatal septicaemia.102 The main setback of PCT is the physiologic rise that occurred in the first 24 hours of life which was attributed to rapid bacterial colonization of the gastrointestinal tract with subsequent translocation of endotoxin through the bowel wall101,102 In spite of this limitation, PCT is useful, both in making diagnosis, in prognostication and in monitoring response to treatment.79 However, as non-infection clinical conditions such as respiratory distress syndrome, cardiac failure and severe trauma are associated with very high serum concentration of PCT, it was not recommended as a routine decision making test for neonatal septicaemia.102 Many other acute phase reactants such as fibronectin, haptoglobin, α1-antitrypsin, lactoferin and neopterin have also been found to rise with infection but they are not routinely

29 used clinically because of their limited diagnostic ability in the presence of other better tests.79

In a more recent report, granulocyte colony stimulating factor (G-CSF), a mediator produced by the bone marrow for facilitating the proliferation and differentiation of neutrophils was proposed as a reliable marker for early diagnosis of neonatal infections.103 A high sensitivity (95.0%) and negative predictive value (99.0%) was reported by Kennon et al.103 Despite this high sensitivity, there is need for further study before the routine use of G-CSF in diagnosis of neonatal infections could be advocated, because of lack of standardized, universally accepted cut-off value for diagnosis of septicaemia.

In the mid and late 1990’s, the role of chemokines, cytokines, adhesion molecules and complement activation products such as C3a-des Arg, C3bBbP and sC5b-9 in the diagnosis of neonatal septicaemia was extensively studied.100,104-108 The rationale for investigating this diverse group of intercellular messengers was that leucocyte indices and CRP were late markers and were not sensitive enough for early diagnosis of neonatal septicaemia. Of the many mediators studied, much attention has been focused on IL-6, IL-8 and TNF-. Interleukin-6 is an important marker of early host response to infection.79 Its concentration increases sharply after exposure to bacterial products and precedes the increase in CRP.79 At the onset of infection, IL-6 has the highest sensitivity (89.0%) and negative predictive value (91.0%) compared to other biochemical markers such as CRP, TNF-, IL-1 and E-selectin.100 Interleukin-6 however, has a very short half-life such that its serum level becomes undetectable within 24 hours of commencing antibiotic therapy.100 The measurements of IL-6 ( for early and sensitive marker of neonatal infection) in combination with CRP ( for late and specific marker of infection) in the first 48 hours of presumed infective episode have been shown to yield a better sensitivity than either marker alone.100,104 In addition,

30 Küster et al105 have shown that the combination of serum IL-6 with IL-1 receptor antagonists can predict neonatal septicaemia two days before clinical manifestation and this can result in earlier initiation of antibiotic therapy. IL-8 has also been shown to be highly sensitive and specific for neonatal infection,106 with a sensitivity ranging from

80.0% to 91.0% and a specificity ranging from 76.0% to 100%.106 Thus, the combination of IL-8 and CRP is useful in restricting unnecessary antibiotic use in newborn.107

In the late 1990s and early 2000s, the role of polymerase chain reaction and cell surface markers in diagnosing early and late neonatal systemic infection began to be explored.106,108 Neutrophil CD11b and CD64 were the most promising of all the cell surface markers studied. The expression of CD11b increases considerably within a few minutes after inflammatory cells come into contact with bacterial endotoxin.79 The sensitivity and specificity of CD11b and CD64 for diagnosing neonatal infections are very high.106 Whereas CD11b is highly sensitive (96.0% to 100%) for early-onset neonatal infection, CD64 was found to be more sensitive for late-onset neonatal infection with a reported sensitivity and specificity values of 97.0% and 90.0% respectively and a negative predictive value of 99.0%.106

Despite the favourable claims by these authors, most of the recent diagnostic markers fail to meet the stringent demands required for clinical practice because of their cost, inaccessibility of reagents and lack of standardized assay methods.79

TREATMENT

The standard management for suspected neonatal infection consists of early diagnosis, aggressive supportive care and appropriate use of antibiotics.9,59,109

Therefore it is not surprising that as many as 75.0% of all infants admitted to the

Neonatal Intensive Care Unit in United States of America received antibiotic treatment

31 in the first 48 hours of life.5 Treatment should begin immediately after cultures are obtained, even with relatively minimal indications for septicaemia. Estimates are that from 1 in 6 to 1 in 20 of infants who are treated with antibiotics for suspected septicaemia end up having positive blood, cerebrospinal fluid or urine cultures.5

General principles of antibiotic use in newborn: The initial choice of antibiotics should depend on the knowledge of the prevalent organisms responsible for infection within a geographical area as well as the pattern of specific antibiotic susceptibility.109

Owing to lack of adequate laboratory facilities in most hospitals in Nigeria, delay in getting laboratory results is not uncommon.109 This delay occurred against a backdrop of the fact that most systemic bacterial infections in neonates demand immediate therapeutic attention without which death may result. Hence, empirical treatment has been suggested.59,109 The drug use in empirical treatment should be broad spectrum, non-toxic when given within limits of therapeutic dosages, readily available and cheap with low cost-benefit ratios.109

The spectrum of initial antibiotics should be broad enough to cover for Gram- negative and Gram-positive bacteria since either group of organisms can cause infection in the neonatal period.36,109 Hence combination therapy is preferred to monotherapy during empirical treatment. Toxic antibiotics should, however, be avoided. Consequently, the use of systemic chloramphenicol is not favoured in neonates as it may cause ‘Gray baby syndrome’.109 It serves no useful purpose prescribing antibiotics that are not affordable and not readily available in the market.

For this reason, the third generation cephalosporins, even though, highly effective against a wide variety of bacteria cannot be use as first line drugs in resource poor country like Nigeria.109

In most studies on neonatal septicaemia in Nigeria,9,14,15,17,18 Escherichia coli,

Klebsiella species and Staphylococcus aureus dominated the bacteria isolates, hence

32 a combination therapy involving an aminoglycoside and penicillins have been suggested as the drugs of choice in the initial treatment of suspected cases of septicaemia.9,109 Therapeutic changes should be effected as soon as the results of the cultures as well as the sensitivity pattern are known.

Drugs for empirical treatment: In mid 1980s, Staphylococcus aureus and

Klebsiella species constituted the commonest bacterial agents of neonatal septicaemia in Zaria.15 These two agents accounted for 70.0% of the total bacterial isolates and

63.0% of cases with fatal outcome. These agents showed high degrees of resistance to commonly used antibiotics. However, 83.0% of Staphylococcus aureus were sensitive to cloxacillin and 67.0% of Klebsiella species were sensitive to gentamycin.15

Consequently, the authors recommended cloxacillin and gentamycin as the drugs of choice for empirical treatment of neonatal septicaemia in the unit. Similarly, Mokuolu et al 29 suggested ampicillin-sulbactam and gentamycin combinations as drugs of choice for empirical treatment of neonatal infections in Ilorin based on the sensitivity pattern observed in their unit. Airede18 however, suggested the use of gentamycin alone based on the finding that most of the organisms isolated in Jos were sensitive to gentamycin. A 12-year retrospective study in Sweden revealed high success rate with the use of ampicillin-aminoglycoside combinations, though a failure rate of 20.0% was encountered.110 From the aforementioned, it is obvious that most centres include an aminoglycoside in the initial treatment protocol in line with the recommendations of

Olowu109 and Onile.111

Multiple drug resistant infections are developing in many centres in

Nigeria.15,16,29,111 In the most recent report from Lagos,56 the sensitivity of Klebsiella species to cephalosporins was surprisingly low. Among the third generation cephalosporins, the percentage sensitivity of Klebsiella was 17.7% for ceftazidime and

29.4% for ceftriaxone. The aminoglycosides were not spared of this resistance. Only

33 11.8% of these agents were sensitive to gentamycin. All the strains were, however, sensitive to ofloxacin. In view of this, use of ofloxacin (in the absence of other effective drugs) in neonates with septicaemia was suggested by Onile111 for physician practising along the coastline where multi-drug resistant Klebsiella septicaemia are prevalent, while Egri-Okwaji et al56 recommended the substitution of kanamycin for gentamycin in Lagos.

Duration of treatment: Treatment for 7 to 10 days or at least 5 to 7 days after clinical response has occurred have been suggested.21 The practice recommended by

Wiswell5 was to treat for at least seven days after the first negative culture result. For infants with meningitis, the duration of treatment is 14 to 21 days depending on the aetiologic agent(s).112,113 Treatment of Gram-negative meningitis should last for 21 days or for at least 14 days after sterilization of the cerebrospinal fluid, whichever is longer.21 Most Gram-positive meningitis cleared after 14 days of treatment except for

GBS meningitis in which treatment may be as long as 21 days.112,113

Despite optimal antibiotics and supportive therapy such as oxygen therapy, meticulous nutritional and fluid management, many neonates still die from septicaemia. Consequently, many adjunctive therapeutic agents have been used in the management of neonatal septicaemia. The adjunctive therapies available are fresh whole blood exchange transfusion (EBT), granulocyte transfusion, intravenous immunoglobulins and administration of haematopoietic growth factors. The results of some studies suggest that some of these therapies might improve survival. 114-118

However, most of the trials were small or anecdotal. A meta-analysis of some non- randomized predominantly retrospective trials114,115 suggested that babies who received EBT survived better than their counterparts who were not treated with EBT.

These reports have been challenged by Freeman et al23 on the grounds of

34 methodological inadequacies. Apart from this, the potential hazards associated with

EBT preclude its routine use.

The use of granulocyte transfusion in infected babies was based on the demonstration of quantitative and qualitative circulating neutrophil deficiencies.58,60-64

Christensen et al117 conducted a study on seven neutropaenic septic babies who had granulocyte transfusion. These were matched against nine controls that also had neutropaenia but not granulocyte transfused. All of those who received granulocyte transfusion survived compared to 11.0% survivor in the control group. Granulocyte transfusion, however, can cause fluid overload, sequestration of neutrophils in the lungs, graft-versus-host disease and transmission of infectious agents,36,60 thus making it unsuitable for routine use.

Intravenous immunoglobulins (IVIG) can theoretically provide antibodies that bind to cell surface receptors on phagocytes, provide opsonic activity and activate complements.118,119 Haque et al118 studied the effect of immunoglobulin treatment in preterm babies and found that both morbidity and mortality from infections were low in them compared to the controls. However, a recent Cochrane119 review of the effectiveness of IVIG in reducing morbidity and mortality showed that the quality of various IVIG studies was generally poor. Moreover, the reviewers believed that the evidence of IVIG mitigating morbidity and mortality in these studies was insufficient.

The use of fibronectin concentrates as adjunctive therapy was suggested by

Gerdes et al64 in view of fibronectin deficiency and the resultant impairment of reticulo- endothelial system activity in neonates with infection. This also needs further investigation before it can be included in the routine treatment protocol.

Haematopoietic growth factors are cytokines that play an important role in the production and maturation of white blood cells in the bone marrow. These substances are produced by many cells during times of increased demand such as sepsis.57 Two

35 commercially available myeloid-stimulating factors are granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF). To date, there are only a few reports116,120 of their use in neonatal septicaemia either as prophylaxis or treatment. In the single largest study of GM-CSF as a prophylaxis in infection, it was not effective in reducing the incidence of nosocomial infections.5

However, in a case controlled study involving 20 non-neutropaenic preterm infants, the duration of hospital stay was shortened in those that were given 5 g/kg/day of recombinant G-CSF over three days period compared to those that did not receive the medication.120 In view of the conflicting reports on the efficacy of these haematopoietic factors, their use should only be under an investigational protocol or in the direst of circumstances following informed consent.

A recent study in the adult population121have indicated the usefulness of recombinant human activated protein C (rhAPC) and corticosteroids in the treatment of

SIRS associated with septicaemia. Recombinant human activated protein C is an anticoagulant that has been found to have anti-inflammatory activity. In a large scale, randomised, double blind international study of 1690 adult patients with SIRS and multi-organ dysfunction, Bernard et al121 demonstrated a 19.4% reduction in the relative risk of death following intravenous infusion of activated protein C at a dose of

24g/kg/hr. The authors observed a mortality rate of 30.8% in the placebo group as against a mortality rate of 24.7% in the group that received rhAPC. They also found that one additional life could be saved for every 16 patients treated with rhAPC.121

Activated protein C inactivates factor V and VIII in the clotting cascade, thereby preventing the generation of thrombin.121 The efficacy of an anticoagulant agent in patients with SIRS has been attributed to feedback between the coagulation system and inflammatory cascade. Inhibition of thrombin generation by activated protein C decreases inflammation by inhibiting platelet activation, neutrophil recruitment and

36 mast cell degranulation.121 In addition, activated protein C has direct anti-inflammatory properties which include: blocking of the production of cytokines by monocytes and cellular adhesion.121 A major risk associated with rhAPC is life threatening bleeding episode which was observed in 3.5% of patients in Bernard et al series.121

Consequently, the use of rhAPC was recommended only for patient with severe SIRS in which the likelihood of death is high.121

In the neonatal population, Rawicz et al122 recently reported the use of recombinant human activated protein C at a dose of 24g/kg/hr continuous four-day infusion in a neonate with severe septicaemia complicated by disseminated intravascular coagulopathy and multiple organ dysfunction with good therapeutic outcome and with no side effect observed. The positive outcome of this case indicates that rhAPC can be safely used in infants. However, there is need for a large-scale study of the effectiveness and safety of rhAPC.

Finally, researches123,124 have been made into the protective effect of breast milk. The anti-infective properties of breast milk make it highly favoured as the feed of first choice in babies with septicaemia124 except for those with contraindications for enteric feeding.

PROGNOSIS OF NEONATAL SEPTICAEMIA

The mortality rate of neonatal septicaemia has been high, greater than 95.0% in the pre-antibiotic era, decreased to 20.0% to 40.0% by the 1970s and 1980s and is now 10.0% to 15.0% in United States of America.5,36 In Africa, higher mortality rates are being observed.9,17,18 The earliest reports9,19 on neonatal septicaemia in Nigeria recorded higher mortality rate compared to more recent reports.17-19 In a retrospective review of 78 cases of neonatal septicaemia in Benin City, Omene19 reported an overall mortality rate of 41.5%. Similar study in Zaria14 in the late 1970s (but reported in

37 1984) recorded a mortality rate of 44%. More recently, much lower mortality rates are being observed in Nigeria. Amiebenomo et al15 (1986 in Zaria), Airede (1992 in Jos)18 and Egri-Okwaji et al (1995 in Lagos)56 reported mortality rates of 34.0%, 27.3% and

22.2% respectively. This decreasing mortality rate may be related to general improvement in medical care.

The mortality pattern is influenced by several factors including patient’s age, sex, infecting agents and associated complications. In Calabar,17 a mortality rate of

40.5% occurred in septicaemic neonates that presented within the first two days of life.

This reduced to 31.6% among those diagnosed at ages 3 _ 7 days and reduced further to 13.9% in those diagnosed between the ages of 8 – 28 days. Infants of lower gestation had higher mortality rate than their mature counterparts in Jos.18 Sex difference in mortality pattern has also been observed. Nazer3 reported higher mortality rates among male infants than female infants in Jordan in Asia. Mortality rate was higher in Gram-negative than in Gram-positive septicaemia.7 Among the Gram- negative agents, mortality rates were higher with lactose fermenting than non-lactose fermenting Coliforms, with the exception of Proteus and Pseudomonas infections which were found to be fatal.7 Those with complications such as meningitis carried higher mortality.

The exact cause of death in patients with septicaemia remains elusive. The theory that death from SIRS (a complication of septicaemia) results from over- stimulation of the immune system was based on findings from animal studies that did not seem to reflect the clinical picture in humans.125 These studies used large doses of endotoxin and bacteria.125 Consequently, levels of circulating cytokines such as TNF-α were exponentially higher in these animals than they were in patients with systemic infection. In these studies, the animals died from “cytokines storm.”125 Although, elevated cytokine levels have been observed in patients with SIRS, it has been shown

38 that the frequency of an exaggerated systemic inflammatory response is lower than it was originally thought to be.125 In addition, use of cytokine antagonist has been shown to increase mortality in patient with severe SIRS.125 The various organ changes seen in this syndrome might have been implicated as the cause of death, but in the observation of Hotchkiss and Karl125, adequate exogenous support to the damaged organs did not prolong life. Hence, the cause of death in neonatal septicaemia may not be attributed to organs changes alone.

PREVENTION OF INFECTION IN THE NEWBORN

Extensive effort has gone into the search for the best approach to the prevention of neonatal septicaemia in the developed countries. Most of these studies were targeted at GBS because of its leading role in early neonatal septicaemia in

United States of America.126-129 Several approaches to the prevention of early-onset

GBS infection in neonates have been suggested. One of these is universal treatment of all neonates with antibiotics (typically penicillins).129 The major draw back of this is the development of penicillin resistant pathogens. Active immunization of mothers with vaccines in the hope of inducing IgG production in sufficient quantities has also been tried.127-129 Transplacentally transferred IgG confers immunity against infection in the newborn. Passive immunization with intravenous immunoglobulins has also been suggested although there are scanty data assessing its effectiveness.129

Administration of antibiotics to the mother to prevent neonatal GBS disease has also been advocated.128,129 Currently, aggressive management of maternal chorioamnionitis with antibiotics before delivery, rapid delivery of the fetus and selective intrapartum chemoprophylaxis appear to have decreased the morbidity and mortality rates of neonatal bacterial infections.128

39 Several nursery practices have been tried with the aim of reducing the rate of spread of infection within the neonatal intensive care unit. Of utmost importance is meticulous attention to hand washing by caregivers (a 2-minute scrub before entering nursery, 15-second washing between patients).21 Following a recent outbreak of

Acinetobacter baumannii septicaemia in Taoyuan neonatal intensive care unit,130 an environmental culture survey that included bacterial culture of 94 environmental specimens and hand washings of all 43 health workers involved in the unit was carried out. Genotyping of the isolates was done. The genotype of the bacteria in 3 of the hand washing isolates was the same with the genotype of the isolates in 3 septicaemic neonates. It was concluded that this agent was probably transmitted via the hands of the health care workers.130 This observation lent credence to meticulous hand washing that has been advocated. The use of protective barriers such as gowns, gloves, face masks and caps became a common practice after the outbreak of Staphylococcal infections in Europe and America.21,41 Adequate staffing, avoidance of overcrowding and sterilization of equipments are essential preventive measures.21,59

An emphasis on early achievement of enteral nutrition, preferably with human milk, also helps by reducing unnecessary resort to central catheters and parenteral nutrition which predispose to infection.123 Recently the role of probiotics in the prevention of infection in preterm infants was described.131 Enteric feeding of live non- pathogenic bacteria (probiotics) results in changes in the pattern of gastro-intestinal tract colonization, with consequent reduction in the extent to which potentially pathogenic bacteria, such as enterococci, colonize the gut.131 The other effects of probiotic bacteria include increased mucosal barrier to translocation of bacteria and bacterial products, enhancement of immune response and improved enteral nutrition, leading to reduction in the use of intravenous feeding which is a major risk factor for bacterial infection in hospitalized patients.131 As far back as the 1960s, the ability of

40 probiotics in preventing infection was clearly demonstrated.132 Non-pathogenic strain of Staphylococcus aureus (strain 502A) was sprayed in newborn unit during outbreak of staphylococcal infections in the United States of America.132 Over 4000 infants were colonized and the outbreak was brought under control with no serious adverse outcome. Currently, there is paucity of data on the safety and efficacy of probiotics in newborn infants, hence there is need for clinical trials of sufficient size before the routine use of probiotics in newborn infant could be advocated.

41 JUSTIFICATION OF THE STUDY

The newborn infant is vulnerable to bacterial infections as a result of his immature immune status.58-64 Local sepsis commonly becomes generalized and follows rapid progression,36 culminating in death if not treated.10-14 Hence early identification, and appropriately intense and prompt therapy is paramount in the control of neonatal septicaemia.9,59,109

Bacterial infections often present a diagnostic challenge in the resource-poor setting of most developing countries, partly because the symptoms are similar to those of other common neonatal illnesses and also because most health care facilities lack adequate laboratory facilities for isolation of the infecting organisms.109 Hence empirical treatment protocol must be available if morbidity and mortality due to septicaemia may be controlled.9

It has also been well documented that the bacterial agents and their sensitivity to antibiotics tend to vary from place to place and from time to time in the same environment.47,48 It is therefore important that the bacterial isolates and their antibiotic sensitivity pattern be continually monitored, so as to timely detect any deviation from the earlier pattern and hence maintain good therapeutic outcome and avert possible sequelae.7,8,23 In spite of the availability of potent antibiotics for the treatment of neonatal septicaemia, prevention is the ultimate goal.59 Knowledge of the prevailing risk factors will assist in formulating preventive strategies necessary to curtail the incidence of the disease.

It was for these reasons that this study was conducted at the Special Care Baby

Unit of Ahmadu Bello University Teaching Hospital, Zaria, so that the current epidemiological and bacteriological patterns will be determined and documented since the last of such evaluations was done over 18 years ago. This may provide a basis for anticipatory care for babies at risk and better antibiotic choice for those with

42 provisional diagnosis of septicaemia, and a consequent reduction in the overall mortality due to neonatal septicaemia.

43 OBJECTIVES OF THE STUDY

General Objective:

To determine the current pattern of neonatal septicaemia in the Special Care

Baby Unit of Ahmadu Bello University Teaching Hospital, Zaria.

Specific Objectives:

1. To determine the incidence of neonatal septicaemia among babies delivered

in Ahmadu Bello University Teaching Hospital, Zaria.

2. To identify and compare the risk factors for neonatal septicaemia among

neonates admitted with working diagnosis of septicaemia.

3. To identify the bacteria in current pathogenesis of neonatal septicaemia and

their in-vitro antibiotic sensitivity.

4. To compare the current epidemiologic and bacteriologic data with those

previously reported from the unit.

44 PATIENTS AND METHODS

Study Location: Zaria is located on a Plateau in the centre of northern Nigeria at a height of about 660 metres (2200 feet) above sea level.133 Ahmadu Bello University

Teaching Hospital, Zaria, is primarily a tertiary centre but also provides primary and secondary health care services. The neonatal unit of the hospital admits about 697 babies annually13 into a 20-bed special care baby unit (SCBU) that has separate compartments of 6-bed space for the out-born babies coming through the out-patient department and 14-bed space compartment for in-born patients coming through the maternity ward of the hospital.

Neonatal Care Practices: To keep nosocomial infections to the barest minimum, the out-born patients are admitted into a separate section of the ward. Strict asepsis is observed for all procedures. All dressing materials are autoclaved in the hospital’s central sterilization department. The babies are managed by a team consisting of a consultant neonatologist, a senior registrar, registrars and paediatric nurses. Babies suspected of having septicaemia are screened and placed on empirical antibiotics while awaiting the results of bacteriological studies. Those considered to be of early- onset are treated with benzyl penicillin and gentamycin, while those considered to be of late-onset are treated with ampicillin-cloxacillin and gentamycin combination.

Patients with overwhelming infections are treated with ceftriaxone. Changes in the antibiotic regimen are made as dictated by the patients’ clinical response and in-vitro sensitivity studies. For proven infections, the duration of treatment is 10 - 14 days.

The study did not interfere with routine patient management protocols.

Study Design: The study was a hospital-based prospective study, conducted in the

SCBU of the hospital.

Ethical Considerations: The approval of the Medical Ethical Committee of ABUTH,

Zaria, was sought for and obtained before the commencement of the study (Appendix 45 II). Informed consent was obtained from the parent(s) or guardian of babies before they were recruited into the study. The possible harmful effect of the study on the patients such as pain and minimal bleeding at venipuncture site was explained to them. In addition, they were also informed of the possible benefit of the study to their patient such as better treatment outcome, since the results of the bacteriologic studies and hence the antibiotic sensitivity profile will influence the ultimate choice of antibiotics for treating their patient.

Patients: Consecutive newborns admitted into the SCBU, both in-born and out-born, that satisfied the inclusion criteria were enrolled in the study.

Inclusion criteria:

The presence of risk factors for and/or clinical features of neonatal septicaemia

(such as fever, jaundice, poor feeding, vomiting, diarrhoea, difficulty in breathing, excessive crying, abdominal distension, convulsion, et cetera) served as the inclusion criteria.

Exclusion criteria:

1. Babies who were on antibiotics prior to presentation.

2. Babies born to mothers who had been on antibiotics within 2 weeks prior to

delivery.

3. Babies with congenital malformations that can alter the normal mechanical

immunological barriers such as dysraphisms (spinal bifida cystica, ectopia

vesicae, omphalocoele and gastroschisis).

Sample size estimation: The minimum sample size needed was calculated using the formula:134

n = z2 (p)(1-p) d2

46 Where:

n = desired minimum sample size when the study site population is more than

10,000.

z = standard normal deviate which is 1.96 at 95% confidence interval.

p = previously reported prevalence of neonatal septicaemia in the SCBU,

ABUTH Zaria = 25.1%.15

d = level of precision = 5%.

n = 1.962 x 0.251x (1-0.251) = 288.9. 0.052

A previous study in SCBU ABUTH, Zaria, has reported an annual admission rate of 697 babies in 1996.13 Applying the correction for finite population, the

134 corrected sample size (nc) was calculated using the formula:

nc = n 1+ n N

Where:

nc = the desired minimum sample size when studying proportions with population

less than 10,000

n = the desired minimum sample size when studying proportions with population

more than 10,000 = 288.9.

N = the estimate of the population size = 697.

nc = 288.9 1 + 288.9 69

= 204.5 ≈ 205.

Since 2.5% of patients with septicaemia could be missed when a single blood culture is done5, a correction factor of 2.5% (5.1 patients ≈ 6 patients) was added to arrive at a

47 total sample size of 211 patients. Patients that fulfilled the inclusion criteria were enrolled into the study until the desired sample size of 211 was obtained.

Data Collection:

The relevant data were collected using a proforma specifically designed for this purpose and administered by the researcher. The information obtained and recorded in the proforma included: the demographic characteristics of the parents, maternal illnesses and symptoms that suggest maternal infection during pregnancy such as fever, diarrhoea, copious/offensive vaginal discharge and dysuria. Other data collected were: duration of membrane rupture, nature of liquor, duration of labour, place and mode of delivery.

Baby’s name, postnatal age, sex, gestational age calculated from the first day of the last regular menstrual period and / or Dubowitz gestational age score, birthweight and the 1-, 5- and 10- minutes Apgar scores were recorded. The presenting complaints such as fever, jaundice, poor feeding, vomiting, diarrhoea, difficulty in breathing, excessive crying and convulsions were also recorded.

A detailed physical examination was carried out on each neonate and the findings obtained were entered into the proforma. All specimens for infection screening were obtained before commencing antibiotic therapy. The results of the bacteriologic studies were communicated to the managing team as soon as they were known. The babies were monitored for possible outcome (but without interfering with their treatment protocols) until they were discharged.

LABORATORY METHODS

Blood Culture Specimens: The site of venipuncture was cleansed using 1% povidone iodine followed by 70% alcohol. The skin was swabbed thoroughly beginning at the point of venipuncture and moving outwardly in a concentric manner. The top of 48 each blood culture bottle was cleansed with a sterile swab pre-soaked with alcohol.

Using a sterile syringe and a sterile size 23-gauge needle, two millilitres of venous blood was withdrawn from a peripheral vein by the investigator or by an assistant

(another resident in paediatrics department) when the investigator was unavoidably absent. The sampled blood was inoculated aseptically into a blood culture bottle containing thioglycollate broth using a fresh sterile 21-gauge needle inserted through the rubber liner of the bottle cap (Tryptone soya diphasic medium and other media were not available at the time of study, hence only thioglycollate broth was used). The top of each culture bottle was re-wiped with sterile swab soaked with alcohol and the blood in the culture bottle was gentle mixed to prevent it from clotting. Each bottle was labelled with the name and number of the patient and the date and time of sample collection. Each specimen was then subjected to a standard microbiologic culture technique135 in the medical microbiology laboratory of the hospital with the assistance of a medical laboratory scientist. Daily macroscopic inspection of the inoculated medium was done after 24 hours of incubation at 370C, looking for evidence of bacterial presence such as cloudiness, gaseousness, haemolysis or obvious growth.135 For those that showed any of such evidence, subcultures were performed and Gram staining done at any point where bacterial growth was noticed. Inoculated media were discarded on the 8th day if there was no bacterial growth.

Gram-staining Procedure135

A single colony obtained from the subculture medium with bacterial growth was emulsified in a drop of normal saline on a clean grease-free glass slide. The slide was then fixed by passing it three times over a gentle flame. Crystal violet solution was poured on the slide, left for 60 seconds and then washed with water. The slide was then flooded with streams of Lugol’s iodine for another 60 seconds and subsequently

49 washed with water. Alcohol decolourisation process was carried out with acetone for

10 seconds and the slide was rinsed with clean water. Counter-staining was done with neutral red stain for 2 minutes after which the slide was rinsed with clean water and allowed to air dry. The slide was then examined under the oil immersion (X 100) microscope objective. The organisms that appeared purple-blue under the microscope were considered as Gram-positive agents while those appearing red to pink were considered to be Gram-negative organisms.

Biochemical and enzymatic tests:

Further identification of bacterial pathogens was done using various enzymatic and biochemical tests. Catalase test with 3% hydrogen peroxide was used to differentiate Staphylococci from Streptococci.135 Coagulase test was done to differentiate coagulase negative Staphylococci from the coagulase positive strains.135

Bacitracin and optochin sensitivity tests were done to identify some strains of

Streptococcus species. Gram-negative bacteria were also identified using various biochemical and enzymatic reactions, including, reactions on Simon citrate agar,

Kligler Iron agar, oxidase test, urease test and indole test.135

Antibiotic sensitivity testing:

Antibiotic sensitivity testing of the isolates was done by the Stoke’s disc- diffusion technique135 using antibiotic discs manufactured by Oxoid Ltd and Abtek

Biological Ltd. The antibiotics tested included: ampicillin 25g, amoxicillin 25g,

Augmentin® 30g, cloxacillin 5g, cotrimoxazole 25g, chloramphenicol 10g, cefuroxime 30g, ceftriaxone 30g, ceftazidime 30g, ciprofloxacin 5g, erythromycin

10g, gentamycin 10g, tetracycline 25g and ofloxacin 5g. Staphylococcus aureus

(ATCC 25923) was used as control for Gram-positive organisms while Escherichia coli

50 (ATCC 25922) was used as control for Gram-negative organisms. An isolate was reported sensitive to the antibiotic tested if the diameter of the zone of inhibition was wider than that of the control, equal to or not more than 3.0 millilitres smaller than the control. Those with a zone of inhibition of equal to or less than 2.0 millilitres were reported resistant to the antibiotics tested.135

Other Laboratory Samples:

Other samples obtained for bacteriological examinations, as part of the unit’s routine infection screen included suprapubic urine aspirates, cerebrospinal fluid, stool and swabs of skin eruptions, umbilical, eye or ear discharges. Urine was obtained by inserting a sterile 23- gauge hypodermic needle about 2.5cm above the pubic symphysis into the bladder after cleaning the skin with 1% povidone iodine and 70% alcohol. The urine samples were transferred to a sterile universal container and were subjected to microbiological examination within two hours of collection.135 The cerebrospinal fluid obtained from lumbar puncture under aseptic conditions, was collected in a sterile container and subjected to standard microbiological procedures immediately.135

Haematological indices such as haematocrit, blood film, total and differential white blood cell counts and platelet counts were also determined with the assistance of the laboratory scientist in the haematology department of the hospital.

DATA ANALYSIS:

The data obtained was collated, checked for errors and subjected to statistical analysis using the data management software (EPI Info version 6). Frequency Tables were generated for simple proportions. The association between categorical variables were tested using the Chi square (X2) test with Yates correction or Fisher’s exact test where the expected value in a cell or more is less than 5. The significance of the 51 difference between the means of the various haematological parameters were tested using the Z test or Student’s t test where applicable. A p-value < 0.05 was considered as statistically significant.

52 RESULTS

During the period of study (May 25, 2004 to May 31, 2005), 573 neonates were admitted into the Special Care Baby Unit of the hospital. Two hundred and eleven (36.8%) of them satisfied the inclusion criterion and were studied. Sixty-nine (32.7%) of the 211 patients studied were delivered in ABUTH delivery suite, while 142 (67.3%) were delivered elsewhere. One hundred and twenty-two (57.8%) of the study population were males while eighty-nine (42.2%) were females, giving a male : female (M:F) ratio of 1.37:1. The ages of the patients studied ranged from birth (day 0) to 28 days with a mean (1SD) of 7.5  6.7 days. Fifty-one (24.2%) of them presented in the first 48 hours of life, while 160 (75.8%) presented after 48 hours of life. Of those presenting later than 48 hours of life, 83 (51.9%) presented within seven days of delivery, while 77 (48.1%) presented between the 8th and 28th days of life. The mean gestational age (1SD) of the patients was 39.1  1.9 weeks with a range of 29 to 42 weeks. Twenty-five (11.8%) of them were preterm while 186 (88.2%) were full term. Forty-three (20.4%) of the newborn infants were low birthweight, 82 (38.9%) were of normal weight, 7(3.3%) were macrosomic, while in 79 (37.4%) of them, the birthweight was not known, because they were delivered at home. Their admission weight ranged between 1000 grams and 5300 grams, with a mean (1SD) of 2890  650 grams. Seventy-five (35.5%) of the 211 neonates had culture-proven septicaemia while 136 (64.5%) had sterile blood cultures. Twenty-one (28.0%) of the 75 septicaemic neonates were delivered at ABUTH delivery suite, while 54 (72.0%) were out-born, giving the prevalence of septicaemia among babies born in ABUTH and among those brought from outside as 30.4% (21/69) and 38.0% (54/142) respectively (2 = 0.86, df = 1, p = 0.3535). Incidence of neonatal septicaemia in ABUTH: A total of 1268 babies were delivered in the hospital during the period of study, 78 of them were stillbirths, while 1190 were live births. Twenty-one of the in-born live births had positive blood culture results; thus the incidence of neonatal septicaemia among in-born infants was 17.6 per 1000 live births.

53 FACTORS INFLUENCING THE INCIDENCE OF NEONATAL SEPTICAEMIA

Place of Delivery: Table I shows the places of delivery of the patients studied and the corresponding infection rates. On the whole, infection rate was higher among babies delivered at home than among those delivered in health facilities, even though this difference did not reach statistical significance (2 = 0.52, df = 1, p = 0.4721). Similarly, infection rates among neonates delivered in private hospitals and clinics were higher than those delivered in other health facilities, but again the difference was not statistically significant (2 =0.86, df =1, p =0.3535).

Table I: Frequency of septicaemia in relation to place of delivery in 211 neonates

S e p t i c a e m i a

Place of delivery Present (%) Absent (%) Total

Home 31 (39.2) 48 (60.8) 79

ABUTH 21 (30.4) 48 (69.6) 69

Private hospital/clinics 13 (41.9) 18 (58.1) 31

Primary health care centre 7 (29.2) 17 (70.8) 24

State hospital 2 (28.6) 5 (71.4) 7

University Health Centre 1 (100) - 1

Total 75 (35.5) 136 (64.5) 211

54 The relationship of gestational age, postnatal age and sex on infection rate is shown in Table II below. Gestational Age: Infection rate was higher among babies born at gestational ages of less than 37 weeks when compared with the infection rate in neonates born at/or after 37 weeks of gestation (2 =6.24, df=1,p =0.0125).

Table II: Infection rate in relation to gestational age, postnatal age and sex

S e p t i c a e m i a

Age and Sex Present (%) Absent (%) Total 2 df p-value

Gestational age(weeks)

<37 15 (60.0) 10 (40.0) 25 6.24 1 0.0125

≥37 60 (32.3) 126 (67.7) 186

Postnatal age (days)

0 – 2 24 (47.1) 27 (52.9) 51

3.92 2 0.1406 3 – 7 27 (32.5) 56 (67.5) 83

8 – 28 24 (31.2) 53 (68.8) 77

Sex

Male 42 (34.4) 80 (65.6) 122

0.06 1 0.8011

Female 33 (37.1) 56 (62.9) 89

55 Age at Presentation: Infection rate was highest among neonates that presented within the first 48 hours of life (47.1%) as against 32.5% and 31.2% among neonates that presented at ages 3-7 days and 8-28 days respectively. The higher infection rate among neonates that presented early did not reach statistically significant level when compared with the infection rates at the other age groups (2 =3.92, df=2, p =0.1406).

Sex: The proportion of females (37.1%) with proven septicaemia was higher than among males (34.4%), even though the difference did not reach statistical significance (2 =0.06, df=1, p =0.8011) (Table II). However, the male : female (M:F) ratio of the 75 septicaemic neonates was 1.21:1. With further regrouping of the neonates based on their ages at presentation and their sexes, septicaemia occurred more commonly among females that presented at ages 0 – 7 days as in Table III, but again the differences were not statistically significant.

Table III: Age by sex distribution of the 211 neonates

S e x Age(days) Male Female p-value

Had sep (%) No sep (%) Total Had sep (%) No sep (%) Total

0 – 2 14(42.4) 19(57.6) 33 10(55.6) 8(44.4) 18 0.5456

3 – 7 15(30.6) 34(69.4) 49 12(35.3) 22(64.7) 34 0.8341

8 – 28 13(32.5) 27(67.5) 40 11(29.7) 26(70.3) 37 0.9872

TOTAL 42(34.4) 80(65.6) 122 33(37.1) 56(62.9) 89 0.8011

56 Maternal Risk Factors: Analysis of individual maternal risk factors showed strong relationship between presence of maternal infections during pregnancy (p=0.0420), prolonged rupture of membranes (p=0.0085), prolonged labour (p=0.0032), lack of antenatal care (p =0.0234) and the occurrence of early-onset neonatal septicaemia (Table IV).

Table IV: Maternal risk factors and rate of neonatal septicaemia in 211 neonates

Age at presentation (days)

Maternal 0 – 2 3 – 7 8 – 28 factors Had No Total p-value Had No Total p-value Had No Total p-value sep sep sep sep sep sep

Had ANC 15 25 40 23 48 71 14 46 60 No ANC 9 2 11 0.0234 4 8 12 1.0000 10 7 17 0.0127

PROM Present 15 6 21 0.0085 6 4 10 0.0709 0 0 0 - Absent 9 21 30 21 52 73 24 53 77

Prolonged labour Present 10 1 11 0.0032 5 2 7 0.0342 0 0 0 - Absent 14 26 40 22 54 76 24 53 77

Maternal infections Present 6 1 7 0.0420 4 2 6 0.0844 0 0 0 - Absent 18 26 44 23 54 77 24 53 77

Maternal fever Present 4 4 8 1.0000 1 0 1 0.3253 3 2 5 0.1718 Absent 20 23 43 26 56 82 21 51 72

Delivery Supervised 15 21 36 0.3749 20 41 61 0.9671 8 27 35 0.2339 Unsupervised 9 6 15 7 15 22 16 26 42

Maternal infections = Copious/offensive vaginal discharge, diarrhoea, or dysuria.

57 Host Factors: Host factors for neonatal septicaemia are presented in Table V below.

Preterm delivery is significantly associated with prevalence of septicaemia (2 = 6.24, df =1, p = 0.0125). Infection rate among babies whose umbilical cord was not treated with antiseptic agents was higher than those with antiseptic agents treatment, even though the difference just fell short of statistical significance (2 = 3.79, df = 1, p = 0.0516). Birth asphyxia was assessable only among the 69 in-born infants.

Twenty-five (36.2%) of them had birth asphyxia out of which 13 (52.0%) had septicaemia while 12 (48.0%) did not. This difference was statistically significant

(2 = 7.09, df = 1, p = 0.0078).

Table V: Influence of host-related risk factors on infection rate

S e p t i c a e m i a

Host factors Present (%) Absent (%) Total 2 df p-value

Preterm 15 (60.0) 10 (40.0) 25 6.24 1 0.0125 Term 60 (32.3) 126 (67.7) 186

Birth asphyxia present 13 (52.0) 12 (48.0) 25 1 7.09 0.0078 Birth asphyxia absent 8 (18.2) 36 (81.8) 44

Multiple deliveries 2 (22.2) 7 (77.8) 9 Fisher’s Singleton delivery 73 (36.1) 129 (63.9) 202 exact - 0.4962

Uvulectomy present 11 (35.5) 20 (64.5) 31 0.04 Uvulectomy absent 64 (35.6) 116 (64.4) 180 1 0.8451

Umbilical cord care

Without antiseptic agents 41 (43.2) 54 (56.8) 95 3.79 1 0.0516 With antiseptic agents 34 (29.3) 82 (70.7) 116

58

CLINICAL FEATURES Symptoms: The symptoms commonly encountered among septicaemic neonates were fever, yellowness of the eyes /skin, refusal of feeds, excessive crying, abdominal distension and vomiting. There was no statistically significant difference between the frequency of these symptoms in septicaemic and non-septicaemic neonates as shown in Table VI.

Table VI: Presenting symptoms in 211 neonates studied

Septicaemia

Symptoms Present Absent Total 2 df p-value

(n = 75) (n =136)

Fever 59 (78.7) 113 (83.1) 172 0.37 1 0.5440

Yellowish 26 (34.7) 53 (39.0) 79 0.22 1 0.6386 eyes/skin

Refusal of feeds 22 (29.3) 47(34.6) 69 0.39 1 0.5345

Excessive crying 18 (24.0) 37(27.2) 55 0.12 1 0.7309

Abdominal 12 (16.0) 15 (11.0) 27 0.69 1 0.5819 distension

Vomiting 11(14.7) 14 (10.3) 25 0.52 1 0.4726

Convulsion 7(9.3) 15 (11.0) 22 0.02 1 0.8803

Breathing difficulty 5 (6.7) 15 (11.0) 20 0.62 1 0.4295

Pustular skin 7(9.3) 8 (5.9) 15 0.43 1 0.5132 rashes

Umbilical discharge 5 (6.7) 9 (6.6) 14 Fisher’s - 1.0000 exact

Eye discharge Fisher’s 5(6.7) 7(5.2) 12 exact - 0.7580

59 Physical signs: Pyrexia was the commonest sign in septicaemic neonates.

The frequency of pyrexia, respiratory distress, tachycardia, hepatomegally, impetigo, lethargy and hypothermia were higher in neonates with septicaemia than in those without septicaemia (Table VII).

Table VII: Distribution of signs of septicaemia among the study population

Septicaemia

Total df Physical signs 2 p-value Present Absent  (n = 75) (n = 136) Pyrexia (RT ≥38.0oC) 57(76.0) 86 (63.2) 143 3.05 1 0.0809

Respiratory distress 23 (30.7) 33 (24.3) 55 0.71 1 0.3979

Jaundice 18 (24.0) 35 (25.7) 53 0.01 1 0.9105

Hepatomegally (>3cm) 18 (24.0) 23 (16.9) 41 1.13 1 0.2874

17 (22.7) 20 (14.7) 37 1.60 1 0.2054 Tachycardia( HR>160/min)

Lethargy 17 (22.7) 15 (11.0) 32 4.22 1 0.0399

Pallor 9 (12.0) 19 (14.0) 28 0.04 1 0.8478

Septic umbilical cord 5 (6.7) 10 (7.4) 15 0.01 1 0.9249

0.5449 Abdominal distension 3 (4.0) 9 (6.6) 12 Fisher’s -

exact

0.1201 7 (9.3) 5 (3.7) 12 Fisher’s - Impetigo exact

Hypothermia (RT≤ 36.40C) 12 (16.0) 7 (5.1) 19 5.09 1 0.0179

60 HAEMATOLOGICAL PROFILE

The mean of haematocrit value (1SD) among neonates with septicaemia (0.35  0.10 l/l) was lower than those of neonates without septicaemia (0.37  0.08 l/l) (z = 1.492, p>0.05). The haematocrit values (1SD) ranged from 0.15 l/l to 0.54 l/l among neonates with positive blood cultures while it ranged from 0.20 l/l to 0.54 l/l among their non-septicaemic counterparts.

The mean total white blood cell count in patients with septicaemia (10.3  6.4 ×

109/l) was higher than those of neonates without septicaemia (9.3  5.8 ×109/l)

(z = 1.159, p>0.05). The white blood cell counts ranged from 2.8 to 31.6 × 109/l in septicaemic neonates while it was between 3.1 and 31.0 × 109/l in non-septicaemic neonates. Neutrophil values of greater than 8.0 × 109/l occurred in 16 (21.3%) of the

75 septicaemic neonates while 16 (11.8%) of the 136 non-septicaemic neonates had such level of neutrophil counts. This difference was not statistically significant

(2 = 2.74, df = 1, p = 0.0980).

Neutrophil toxic granulation occurred in 41 (54.7%) of the 75 septicaemic neonates and in 19 (14.0%) of the 136 non-septicaemic neonates. Neutrophil toxic granulation had a strong association with septicaemia (2=37.37, df=1, p< 0.0001)

When the haematologic parameters for term and preterm were compared, the mean haematocrit values in septicaemic term babies were significantly (p<0.05) lower than the values in non-septicaemic term babies (Table VIII). The mean of total leucocytes count, absolute neutrophil counts and absolute lymphocyte counts were higher in term neonates with septicaemia than in neonates without septicaemia, though the differences were not statistically significant. The mean of absolute neutrophil counts in term neonates with septicaemia (6.2  4.8 ×109/l) was higher than the mean of absolute neutrophil counts in preterm septicaemic neonates (4.9  0.7 ×

109/l). This difference was statistically significant (z=2.1197, p<0.05). 61 The mean of total leucocytes count and mean of absolute neutrophils count were higher in preterm septicaemic babies compared to their non-septicaemic counterparts, though only the difference in the mean of absolute neutrophil counts reached statistical significance (p < 0.05) (Table VIII).

Table VIII: Mean haematological indices of 211 infants studied

Term Preterm Parameter Had sep No sep z- p- Had sep No sep t- p- (n = 60) (n = 126) score value (n =15) (n =10) value value

Haem 0.350.10 0.38 0.10 1.9608 <0.05 0.36 0.12 0.350.10 0.2176 NS ( l/l ) TWBC 10.4 6.5 9.3  5.8 1.1162 NS 8.6 3.7 8.5  6.9 0.0472 NS (×109/l) ANC 6.2  4.8 5.0 3.2 1.9059 NS 4.9  0.7 4.1 0.4 3.2626 <0.05 (×109/l) ALC 4.4  3.5 3.8  2.6 1.1818 NS 3.6  0.5 3.9  0. 4 1.5856 NS (×109/l) Platelet 228.450.6 222.178.6 0.6230 NS 202.365.0 218.4 0.0 0.5441 NS 9 (×10 /l) 82.8

NS = Not significant.

Haem = Haematocrit.

TWBC = Total white blood cell count.

ANC = Absolute neutrophil count.

ALC = Absolute lymphocyte count.

62 BACTERIOLOGY

There were 77 isolates in 75 neonates with septicaemia; two of the neonates had double isolates consisting of Staphylococcus aureus and Escherichia coli. Forty (51.9%) of the isolates were Gram-positive while 37(48.1%) were Gram-negative. The commonest organism was Staphylococcus aureus, which accounted for 42.9% of all the isolates followed by Escherichia coli (19.5%). Proportions of isolates are shown in Table IX

Table IX: Bacterial isolates in 75 septicaemic neonates

Isolates Number Percentage of total Percentage of GM+ve / GM-ve Gram-positive bacteria 40 51.9 100.0

Staphylococcus aureus 33 42.9 82.5

Streptococcus pyogenes 4 5.2 10.0

Untyped Streptococcus 2 2.6 5.0

Streptococcus pneumoniae 1 1.3 2.5

Gram-negative bacteria 37 48.1 100.0

Escherichia coli 15 19.5 40.5

Klebsiella pneumoniae 6 7.8 16.2

Proteus mirabilis 5 6.5 13.5

Citrobacter species 3 3.9 8.1

8.1 Acinetobacter species 3 3.9 5.4 Enterobacter species 2 2.6

Pseudomonas aeruginosa 1 1.3 2.7

Aeromonas specie 1 1.3 2.7

Alcaligenes specie 1 1.3 2.7 Total 77 100

GM+ve = Gram-positive GM-ve = Gram-negative 63 When the isolates were regrouped according to the age of patients at presentation

(Table X), 60.0% of the isolates from blood of infants that presented within the first 48 hours of life were Gram-negative bacteria, while Gram-positive bacteria predominated in neonates that presented after 48 hours (2 days) of life. However, as high as 58.3% of isolates in neonates aged 8 - 28 days were Gram-negative agents.

Table X: Distribution of bacterial isolates according to age at presentation

Age groups (days)

Agents 0 – 2 3 - 7 8 - 28 n (%) n (%) n (%) Total Gram-positive 10 (40.0) 20 (71.4) 10 (41.7) 40

Staphylococcus aureus 7(28.0) 18 (64.3) 8 (33.3) 33

Streptococcus pyogenes 2 (8.0) 2 (7.1) - 4

Untyped streptococcus 1 (4.0) - 1 (4.2) 2

Streptococcus pneumoniae - - 1 (4.2) 1

Gram-negative 15 (60.0) 8 (28.6) 14 (58.3) 37 Escherichia coli 6 (24.0) 5 (17.6) 4 (16.7) 15

Klebsiella pneumoniae 1 (4.0) 1 (3.6) 4 (16.7) 6

Proteus mirabilis 2 (8.0) 1 (3.6) 2 (8.3) 5

Citrobacter species 1 (4.0) - 2 (8.3) 3

Acinetobacter species 2 (8.0) - 1 (4.2) 3

Enterobacter species 2 (8.0) - - 2

Alcaligenes specie 1 (4.0) - - 1

Pseudomonas aeruginosa - - 1 (4.2) 1

Aeromonas specie - 1 (3.6) - 1

Total 25** 28** 24 77

** = One of the patients in each of the asterisk groups had double isolates. n = Number of bacteria isolated from each age group. % = Percentage of total bacteria isolated from each age group.

64 Staphylococcus aureus septicaemia was more common among the out-born than the in-born (Table XI), (2 = 4.57, df = 1, p = 0.0325). In contrast, streptococcal septicaemia occurred more commonly among in-born than out-born babies (Fisher’s exact, 2-tailed p-value = 0.0054). The frequency of isolation of Escherichia coli,

Citrobacter species and Acinetobacter species were higher among in-born than out-born infants, though the differences were not statistically significant (p>0.05).

Table XI: Distribution of the common organisms in relation to place of delivery

Place of delivery

Organisms In-born (%) Out-born (%) Total 2 p-value (n=21) (n=54)

Staphylococcus aureus 5 (23.8) 28 (51.9) 33 4.57 0.0325

Streptococcus species 6 (28.6) 1 (1.9 ) 7 Fisher’s exact 0.0054

Escherichia coli 6 (28.6) 9 (16.7) 15 Fisher’s exact 0.5729

Klebsiella pneumoniae - 6 (11.1) 6 Fisher’s exact 0.1806

Proteus mirabilis 1 (4.8) 4 (7.4) 5 Fisher’s exact 1.0000

Citrobacter species 2 (9.5) 1 (1.9) 3 Fisher’s exact 0.2498

Acinetobacter species 2 (9.5) 1 (1.9) 3 Fisher’s exact 0.2498

65 Analysis of results of clinical specimens obtained from neonates with septicaemia showed that 8 (10.7%) of them had associated meningitis. In six of the eight cases of meningitis, the same organisms were isolated from the CSF and the blood, and they included Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli and

Proteus mirabilis. The predominant bacterial isolate from the swabs of eye, umbilical and skin discharges of the 75 neonates with septicaemia was Staphylococcus aureus.

Five (6.7%) of these septicaemic neonates had confirmed urinary tract infection, all of which were due to Klebsiella pneumoniae (Table XII). In four of these babies with urinary tract infection, the same organism was isolated from the blood and urine, while in one of the cases; the organism was isolated from the blood, urine and swabs of eye discharges.

Table XII: Distribution of bacterial isolates from clinical specimens of the 75 neonates with culture-proven septicaemia

Bacterial CSF Skin Umbilical Eye Ear SPA Isolates swabs swabs swabs swabs urine (n =75) (n = 7) (n = 5) (n = 5) (n = 4) (n = 75) Gram-positive 4 6 5 4 - -

Staphylococcus aureus 2 6 4 4 - -

Streptococcus 2 - - - - - Pneumoniae

Streptococcus faecalis - - 1 - - -

Gram-negative 4 1 - 1 2 5 Escherichia coli 2 - - - - -

Klebsiella pneumoniae - - - 1 - 5

Proteus mirabilis 1 1 - - - -

Pseudomonas - - - - 2 - aeruginosa

1 - - - - - Enterobacter species Total 8 7 5 5 2 5

66 Antibiotics Sensitivity Pattern: As depicted in Figure 1, the Gram-positive bacteria were generally more sensitive to the commonly used antibiotics than the Gram- negative bacteria. All isolates were sensitive to ofloxacin, while only 30.0% of the

Gram-positive and 10.8% of the Gram-negative bacteria were sensitive to ampicillin.

Apart from ofloxacin and ciprofloxacin, the other drugs to which most of the Gram- positive bacteria showed high in-vitro sensitivity included chloramphenicol (92.5%), ceftriaxone (90.0%), Augmentin® (90.0%), cefuroxime (85.0%), erythromycin (80.0%) and gentamycin (70.5%). While the Gram-negative bacteria showed high sensitivity to ceftriaxone (75.7%), ceftazidime (78.4%), gentamycin (78.4%) and ciprofloxacin

(97.3%).

10 0 10 0 10 0 9 7 .3 100 Gram -positive 9 2 .5 90 90 Gram -negative 90 85

80 7 8 .4 7 8 .4 80 7 5 .7

7 0 .5

70 6 4 .9 6 4 .9

60 5 7 .5 60

5 1.4 50

4 0 .5 40

% Sensitivity 3 2 .5 3 2 .4 30 30 2 3 .4

20

20 16 .2

10 .8 10

0 C o t A m p C e ft A m x T c n G e n E r y C e fu A u g C r o C h l C ip r o O flo

Antibiotics

Figure 1: Bar chart showing the antibotic sensitivity pattern of the organisms isolated

Amp – Ampicillin Chl – Chloramphenicol Ceft – Ceftazidime Amx – Amoxycillin Gen – Gentamycin Cefu – Cefuroxime Cot – Cotrimoxazole Tcn – Tetracycline Cro – Ceftriaxone Aug – Augmentin® Cipro – Ciprofloxacin Ery – Erythromycin 67 Oflo – Ofloxacin The antibiotic sensitivity profile of the Gram-positive bacteria showed that ofloxacin

and ciprofloxacin had the highest in-vitro activity against Staphylococcus aureus, while

cotrimoxazole (15.2%), ampicillin (33.3%) and cloxacillin (45.5%) were among the

drugs with the lowest in-vitro activity. As shown in Table XIII, the other drugs shown to

be active against Staphylococcus aureus are chloramphenicol (93.9%), ceftriaxone

(90.9%), cefuroxime (87.9%), Augmentin® (87.9%), erythromycin (84.8%) and

gentamycin (81.8%).

Table XIII: Gram-positive bacteria and their in-vitro antibiotic sensitivity rates

A n t I b i o t i c s (%)

ISOLATES AMP CLOX AMX AUG COT CHL ERY TCN GENT CEFU CRO CEFTA OFLO CIPRO

Staphylococcus 33.3 45.5 54.5 87.9 15.2 93.9 84.8 54.5 81.8 87.9 90.9 30.3 100 100 aureus

Streptococcus 0 75.0 75 100 25 100 25 75 75 75 75 50 100 100 pyogenes

Streptococcus 0 100 100 100 10 100 100 100 0 100 100 0 100 100 pneumoniae

Untyped 50 0 50 100 50 50 100 100 0 50 100 50 100 100

Streptococcus

Overall 30.0 47.5 57.5 90.0 20.0 92.5 80.0 60.0 70.5 85.0 90.0 32.5 100 100

Susceptibility

AMP – Ampicillin CHL – Chloramphenicol CEFT – Ceftazidime AMX – Amoxycillin GEN – Gentamycin CEFU – Cefuroxime

COT – Cotrimoxazole TCN – Tetracycline CRO – Ceftriaxone CLOX - Cloxacillin CIPR – Ciprofloxacin AUG – Augmentin® OFLO – Ofloxacin ERY – Erythromycin

Cloxacillin

68 Of the commonly used antibiotics, gentamycin had the best in-vitro activity against

Gram-negative bacteria. The sensitivities of Gram-negative bacteria to ceftriaxone and ceftazidime were generally high (75.7% and 78.4% respectively), but still in the range of that recorded for gentamycin. Sensitivity to ampicillin was disappointingly low

(Table XIV). Apart from Escherichia coli and Citrobacter species that showed a sensitivity of 20.0% and 33.3% respectively to the drug, all the other Gram-negative bacteria were resistant to ampicillin.

Table XIV: Gram-negative bacteria and their in-vitro antibiotic sensitivity rates

A n t i b i o t i c s (%)

ISOLATES AMP AMX AUG COT CHL ERY TCN GENT CEFU CRO CEFT OFLO CIPRO

Escherichia 20 46.6 73.3 13.3 100 60 53.8 93.3 53.3 80 80 100 100 coli

Klebsiella 0 0 33.3 0 66.6 33.3 83.3 83.3 33.3 66.6 83.3 100 100 pneumoniae

Proteus 0 20 40 40 40 20 80 60 20 100 100 100 100 mirabilis

Citrobacter 33.3 33.3 66.6 33.3 33.3 0 66.6 33.3 33.3 66.6 66.6 100 100 species

Acinetobacter 0 0 33.3 0 0 6.6 66.6 66.6 0 33.3 66.6 100 100 species

Others 0 0 20 20 40 20 60 80 0 80 80 100 80

Overall 10.8 24.3 51.4 16.2 64.9 40.5 64.9 78.4 32.4 75.7 78.4 100 97.3 susceptibility

69

CEFT – Ceftazidime AMP – Ampicillin CHL – Chloramphenicol CEFU – Cefuroxime AMX – Amoxycillin GEN – Gentamycin CRO – Ceftriaxone COT – Cotrimoxazole TCN – Tetracycline CIPRO – Ciprofloxacin AUG – Augmentin® OFLO – Ofloxacin ERY – Erythromycin

70 The antibiotic sensitivity pattern of isolated organisms in the present study was compared with those of previous studies in Table XV below. The sensitivity of pathogenic bacteria to cephalosporins is on the decline. Whereas the Gram-positive bacteria were becoming less sensitive to ampicillin and cloxacillin, gentamycin has maintained good in-vitro activity against Gram-negative bacteria.

Table XV: Comparison of antibiotic sensitivity patterns in previous studies and

present study

Agents Antibiotics Author and Date

Amiebenomo Ogala et al 16 Present et al15 1996 study 1984 - 1985 2004 – 2005

Gram-positive Ampicillin 41.4% 40.0% 30.0%

Cloxacillin 82.6% 43.0% 47.5%

Chloramphenicol 44.6% 83.0% 92.5%

Cephalosporins NT 100.0% 87.5% (Cefuroxime, Ceftriaxone)

Gram-negative Ampicillin 19.2% 10.0% 10.8%

Cotrimoxazole 50.0% 50.0% 16.2%

Gentamycin 69.2% 75.0% 78.4%

Cephalosporins NT 100.0% 77.1% (Ceftriaxone, Ceftazidime)

NT = Not tested.

71 OUTCOME

Patient’s outcome was known in 208 (98.6%) of the 211 neonates. As in Table XVI below, the proportion of septicaemic babies with fatal outcome was higher than that in non-septicaemic babies, though the difference just fell short of statistical significance (2 =3.56, p =0.0592). Of the 51 septicaemic neonates that presented within the first 7 days of life, 14 (27.5%) of them had fatal outcome, while only 3 (12.5%) of the 24 septicaemic neonates that presented at ages 8 – 28 days of life had fatal outcome. This difference was however, not statistically significant (2 = 1.32, p = 0.2513). Mortality among the 54 septicaemic out-born neonates was 12 (22.2%), while it was 5 (23.8%) among the 21 septicaemic in-born infants, again, this difference was not statistically significant (Fisher’s exact, 2-tailed, p-value =0.3452).

Table XVI: Outcome of patients’ management

Outcome Had septicaemia No septicaemia Total 2 p-value

Known 74 134 208 3.56 0.0592+ Well 57 118 175 Died 17 16 33 Unknown 1 2 3 Absconded - 1 1 DAMA - 1 1 - - Referred 1 - 1 DAMA = Discharged against medical advice.

Ten (58.8%) of the 17 fatal cases occurred within 48 hours of presentation. Five (50.0%) of these were due to Gram-positive agents, while Gram-negative bacteria contributed the other half. Of the 7(41.2%) deaths that occurred after 48 hours of admission, 4 (57.1%) were due to Gram-negative septicaemia. On the whole, mortality was higher in neonates with Gram-negative septicaemia (24.3%) than in those with Gram-positive septicaemia (20.0%), though the difference was not statistically significant (p>0.05).

72 PATTERN OF NEONATAL SEPTICAEMIA OVER THE YEARS

Table XVII shows the epidemiological and aetiological patterns of septicaemia in the unit in the last three decades. The sex ratios showed the continued predominance of males over females among neonates with septicaemia.

Aetiologically, Gram-positive agents (Staphylococcus aureus) were the predominant organisms isolated in neonates with septicaemia until 1996 when there was a switch over to Gram-negative predominance. In the present study, Gram-positive bacteria predominated. A downward trend in mortality is obvious, from 41.4% in 1976 – 1979 to

22.7% in 2004 – 2005.

Table XVII: Comparison of patterns of neonatal septicaemia in previous studies

and present study

Parameter Authors and Time

Winfred Amiebenomo Ogala et al16 Present study et al14 et al15 1976-1979 1984-1985 1996 2004-2005

Study population 445 187 41 211

Septicaemic neonates 116(26.1%) 47 (25.1%) 24 (58.5%) 75(35.5%)

Sex ratio (M:F) - 1.14:1 - 1.21:1 Total bacterial isolates 116 56** 24 77** Commonest isolates Staphylococcus aureus 38 (32.8%) 23 (41.1%) 3 (12.5%) 33 (42.9%) Escherichia coli 26 (22.4%) 5 (8.9%) - 15 (19.5%) Klebsiella species 12 (10.3%) 16 (28.6%) 7(29.2%) 6 (7.8%)

Mortality 48(41.4%) 16(34.0%) - 17(22.7%)

** Nine of the patients in Amiebenomo et al series and two of the patients in the

present study had double isolates.

73 DISCUSSION

In the present study, the incidence of neonatal septicaemia was 17.6 per 1000 live births. The incidence compares favourably with recent incidence rates across

Nigeria,17,18,29 but much higher than the 5 to 9 per 1000 live births recorded in the

1970’s and 1980’s.9,15,19 The current incidence rate in Zaria is about three and a half times higher than the 5 per 1000 live births reported by Amiebenomo et al15 from the same centre 18 years ago. The high incidence of neonatal septicaemia in the present study shows that the disease is still a serious problem in Zaria and this may be a reflection of the extent of the disease in Nigeria.

The high incidence could be due to increasing awareness of the disease in the populace and/or improvement in the laboratory facilities. It is possible that the high prevalence of maternal risk factors seen in this study such as failure to attend antenatal clinic during pregnancy, prolonged rupture of membranes and failure to come to hospital early enough in labour might have contributed to the relatively high infection rate. In addition, most of the infected babies were babies of mothers with symptoms suggestive of infections during pregnancy such as diarrhoea, purulent vaginal discharge and dysuria had septicaemia. Similarly, as high as 57.5% of babies whose mothers did not attend antenatal clinic had septicaemia. Failure to attend antenatal clinic has been associated with development of neonatal septicaemia.7,9,20

The role of maternal risk factors in the development of neonatal septicaemia has been documented by workers in Nigeria17-20 and parts of the world.7,31 But the

26.7% (20 out of 75) prevalence of prolonged rupture of membranes in this study is much higher than that reported in most of these series.7,17-20,31 This difference could be related to differences in obstetric care and rates of prolonged rupture of membranes in the respective regions.

74 In the present study, 72.0% of neonates with proven septicaemia were out- born, this compares favourably with the 74.0% reported in Ile-Ife20 but higher than the

61.7% earlier reported in the unit15 and the 59.0% in the series of Airede.18 The high prevalence of septicaemia among out-born infants could be related to the high prevalence of home deliveries also observed in the present study. Home delivery has been associated with development of infection most likely due to use of unsterilized instruments and the unhygienic environment in which the deliveries were conducted.17

Egri-Okwaji et al56 also recorded similar high prevalence of infection among out-born neonates in Lagos. Although, the infection rate in out-born infants (38.0%) did not reach statistical significant when compared to the rate in in-born infants (30.0%), the relatively higher infection rate among out-born babies in the present study underscores the need for aggressive campaign towards greater use of health care facilities in this locality. However, that an infection rate of 41.9% occurred among infants delivered in private hospitals also emphasizes the need for continuing education and enforcement of the rule of asepsis in the conduct of deliveries in these health care facilities.

Beside maternal risk factors, neonatal host factors were also found to contribute significantly to the increased prevalence of septicaemia in the unit. Notable among the neonatal factors were preterm delivery and perinatal asphyxia. The role of perinatal asphyxia and preterm delivery on infection rate has been documented by other researchers in Nigeria.17-19 The high infection rate (52.0%) among asphyxiated babies in the present study may be attributed to the depressive effect of hypoxia on the immune function as was earlier demonstrated by Ogala et al62 among 10 severely asphyxiated infants in the unit. Hypoxia coupled with high inoculum of bacteria that could follow some of the invasive procedures utilized during resuscitation could predispose severely asphyxiated babies to infection.

75 The gestational age at birth has variously been shown to have a negative correlation with prevalence of septicaemia.17,21,31 This was confirmed in the present study in which 60.0% of the 25 preterm babies studied had septicaemia. However the prevalence rate of septicaemia was the same for babies born before 33 weeks of gestation (early preterm) and those born between 33 and 36 weeks of gestation (late preterm). This may be related to the fact that both early preterm and late preterm have developmental immunologic deficiencies; in addition, the conditions precipitating delivery before term may be alike in both groups and as such all preterm babies should be offered services that reduce the rate of infection irrespective of their gestational ages.

The other host factor explored was the gender of the infants. Although there was male preponderance of 1.21:1 among the septicaemic neonates, the observed sex ratio was similar but lower than 1.37:1 in the entire cohort of the 211 neonates. It is a popular belief that males are more prone to infections because of the possession of a single X- chromosome as against the females that are doubly endowed.32,71 In the present study, however, the prevalence of infection was higher in females, though not to a statistically significant level.

The present study also revealed that neonates with septicaemia could present with a variety of signs and symptoms such as fever, refusal of feeds, jaundice, impetigo, tachycardia, hepatomegally, lethargy and respiratory distress. The multiplicity of symptoms and signs in these septicaemic neonates supports the general observation that the features of neonatal septicaemia are vague and non-specific.3,9,21

Although none of these features was pathognomonic of infection, their appearance in a high-risk infant should raise a strong suspicion of septicaemia. Various temperature patterns were observed in the neonates with septicaemia but pyrexia occurred more frequently than hypothermia and normal temperature. This is in keeping with the

76 reports of other authors in Nigeria9,15,17,20 and parts of Africa.7,8 The point re-emphasized by the present study was that various temperature patterns could be found in neonates with septicaemia and as such absence or presence of fever alone should not be used as indication for withholding or initiating antibiotic therapy.

A blood culture positivity rate of 35.5% was observed in the present study. This is comparable to the 30.8% and 36.0% reported in Ilorin and Lagos by Mokuolu et al29 and Egri-Okwaji et al56 respectively, but differs from the 25.0% and 58.5% earlier reported by Amiebenomo et al15 and Ogala et al16 respectively, in the same unit and the 59.8% reported in Calabar.17 This wide variation in blood culture positivity rates may be attributed to differences in the selection criteria, volume of blood culture samples, local laboratory and personnel factors. The level of sincerity of the respondent of the various studies might also be contributory since some mothers may not voluntarily divulge the history of antibiotic self-medications, with consequent inclusion of neonates of such mothers in the studies.

Most of the positive cultures in this study were monomicrobial except in two patients that had polymicrobial infection. This is similar to the reports from other regions.9,18,136 Polymicrobial infection has been reported as mostly being hospital- acquired.137 Continuous surveillance of infection in hospitals might help to clarify the polymicrobial nature of nosocomial infections.

Gram-positive bacteria were the commonest aetiologic agents of septicaemia in the present study, in contrast to the high prevalence of Gram-negative septicaemia recorded in the unit about 10 years earlier.16 The result was however in keeping with the reports of Winfred14 and Amiebenomo et al15 who respectively recorded 55.2% and

51.8% frequency of Gram-positive septicaemia. The 52.0% prevalence of Gram- positive infection in the present study compares favourably with results from other parts of Nigeria.17,19,29

77 The high prevalence of Gram-positive septicaemia in the present study is closely related to the predominance of Staphylococcus aureus septicaemia among the out-born patients. As was revealed by this study, 81.8% of the Staphylococci were isolated from out-born patients, majority of which were home deliveries (55.6%), thus emphasizing the role of the community in the acquisition of Gram-positive septicaemia.

Apart from handler of babies in the community, deliveries outside the hospital are usually conducted by traditional birth attendants who may be harbouring these organisms in their hands and nostrils and whose unhygienic methods contribute to infections in these babies.17 The high prevalence of Staphylococcus aureus among out-born babies in the present study demonstrate the continued predominance of

Staphylococcus aureus septicaemia in this environment and this supports the unit’s practice of including anti-staphylococcal agent in the empirical treatment protocol for all infected out-born babies. In contrast, Streptococcus species predominated among the in-born infants with Gram-positive septicaemia, a finding that justifies the inclusion of penicillin in empirical treatment protocol for early-onset infection in the unit.

The frequency of Gram-negative septicaemia in the present study was 48.0%.

This is similar to the 48.2% and 44.8% in the series of Winfred14 and Amiebenomo et al15 respectively, both from this unit. The prevalence of Gram-negative septicaemia was even higher (60.0%) among neonates with early-onset infection. This is similar to the reports of Nathoo et al7 who recorded 62.3% Gram-negative septicaemia among neonates with early-onset disease and to the series of Dawodu and Alausa9 with over

75.6% of the Gram-negative septicaemia occurring in neonates less than 48 hours of life. The reasons for the predominance of Gram-negative bacteria in early neonatal septicaemia could be related to the colonization of maternal genitalia by Gram- negative enteric bacilli which could gain access to the fetus by ascending infection following prolonged rupture of the fetal membranes or during passage of the baby

78 through the birth canal at delivery. In addition, newborn babies are deficient in immune factors (such as complement, immunoglobulin M and fibronectin activities) that are central to the host handling of Gram-negative bacteria,21 hence, increasing the chances of newborn babies acquiring Gram-negative septicaemia when exposed to them.

The spectrum of organisms isolated in the present study is similar to earlier ones.14-16 Important pathogens isolated were Staphylococcus aureus, Escherichia coli,

Klebsiella pneumoniae, Proteus mirabilis and Streptococcus species, the most common agents being Staphylococcus aureus and Escherichia coli. This is in agreement with the findings of workers in Nigeria and parts of tropical Africa.7,15,17,136

In contrast, recent reports of studies on neonates with septicaemia in non-tropical countries28,37-39 showed the predominance of Coagulase-negative staphylococci and group B streptococci septicaemia. The use of total parental nutrition and invasive diagnostic and therapeutic devices in highly unstable neonates as well as regional differences in rates of maternal vaginal GBS colonization might have contributed to this difference in aetiologic agents of septicaemia.

Among the Gram-negative organisms isolated in the present study, a specific organism of note is Klebsiella species, which ranked fourth commonest bacterial isolates. Barely 10 years earlier, the organism was rated first among all the bacterial isolates in the unit.16 The relegation of Klebsiella species to the present position demonstrates the continued change in the predominant aetiological agent of septicaemia in this locality, and under-scores the need for continual surveillance of the neonatal unit. The 7.8% prevalence of Klebsiella septicaemia observed in the present study is similar to reports from Calabar,17 Benin-City19 and Ile-Ife;20 but much lower than the prevalence reported in Ibadan,9 Maiduguri43 and Lagos.56

79 The overall mortality rate of 22.7% in the present study is similar to the rate found in recent studies in Nigeria,18,20,29,56 but much lower than earlier mortality rates in the unit.14,15 Mortality rates from neonatal septicaemia depend on variable factors including the characteristics of the infant population, aetiologic agents, appropriateness and promptness of antimicrobial therapy and the level of neonatal care. The lower mortality in the present study compared to the earlier studies is most likely due to a combination of these factors. The appropriateness of the initial antibiotic therapy, especially the lower incidence of gentamycin resistant organisms, probably played a significant role. It is also relevant to mention that there were fewer paediatric staffs with special training in neonatal care in the hospital during the period of the previous studies as compared with the present study where such members of staff were readily available to offer standard, highly specialized patient care, training and supervision of other staff members. This factor was contributory to a decreasing mortality pattern in Ibadan about two to three decades ago.9 It will therefore be reasonable to suggest that the level of neonatal care during the present study was better than those of the previous studies, thus contributing to the lower mortality currently observed in the unit.

One of the striking features of the present study was the high rate of in-vitro resistance to the commonly used antibiotics. This was also demonstrated in a report from Lagos56 and is similar to the previous reports from the unit.15,16 Apart from chloramphenicol and gentamycin to which there was a marginal improvement in sensitivity, there was a steadily increasing resistance to all antibiotics when compared to the pattern earlier documented in the unit.15,16 On the whole, the highest resistant rate was with cotrimoxazole (80.0% for Gram-positive, 83.8% for Gram-negative), followed by ampicillin (70.0% for Gram-positive, 80.2% for Gram-negative). These findings have far reaching consequences in therapy since ampicillin (in combination

80 with cloxacillin) is one of the drugs for empirical treatment of neonates with septicaemia in the unit and this could partly account for the relatively high mortality rate seen among neonates with early-onset Gram-positive septicaemia. Even the cloxacillin that could have offered protection against -lactamase enzyme showed similar degree of resistance. The relatively free, uncontrolled use of antibiotics, especially the penicillins, might have contributed to this increasing resistant pattern.

However, the fact that the overall mortality rate in the present study was similar to those in other studies18,29,56 and even lower than those in previous studies14,15 in the same unit, suggests that in-vitro antibiotic sensitivity may not reflect the in-vivo situation. Synergism between ampicillin and gentamycin137 might have contributed to the cure rate observed in the present study

In spite of the general increase in the prevalence of antibiotic resistant pathogens, some drugs, like the quinolones and cephalosporins, have retained good sensitivity rates. Of the commonly used antibiotics, ceftriaxone appeared to be the most highly effective drug having sensitivity rates of 90.0% for Gram-positive and

75.7% for Gram-negative bacteria. However, the routine use of third-generation cephalosporins for suspected neonatal septicaemia is inappropriate because of the potential for rapid emergence of resistant organisms and the high cost of the drug. The rational use of antibiotics in neonates involves use of highly effective, non-toxic and readily affordable drugs when possible.21 Since completion of therapy is highly dependent on the ability of the caregiver to sustain drug supply, compliance may not be guaranteed when expensive drugs, such as ceftriaxone, are prescribed and as such ceftriaxone may not be recommended as drug of choice for empirical treatment of neonatal septicaemia.

The study also showed an increasing sensitivity of the pathogens to chloramphenicol when compared to previous studies.15,16 But the use of

81 chloramphenicol in neonates is restricted in view of the potential danger of gray baby syndrome associated with high doses and other side effects of the drug.109,137 The sensitivity of the most predominant isolate (Staphylococcus aureus) was 84.8% to erythromycin and 87.9% to cefuroxime but disappointingly low for cloxacillin (51.4%).

Unlike cloxacillin, gentamycin had high in-vitro activity against Staphylococcus aureus

(81.8%). This may be responsible for the high cure rate associated with the use of ampicillin-cloxacillin-gentamycin combinations in spite of the high level of resistance against ampicillin and cloxacillin in the study. The higher sensitivity of Staphylococcus aureus to gentamycin than to cloxacillin in the present study has been previously observed by Airede18 in Jos and Adejuyigbe et al20 in Ile-Ife.

The Gram-negative organisms also showed high degrees of resistance to all drugs tested except the quinolones. Even the third generation cephalosporins, which were particularly useful as anti-Gram-negative drugs, showed a resistance of 21.6% to

24.3%. On the whole, of the most commonly used antibiotics, gentamycin had the best coverage against Gram-negative bacteria with a sensitivity of 78.4%. The lesson of note in the present study is that, despite heavy usage, gentamycin has retained good sensitivity rates, thus the continued role of gentamycin in combination therapy for neonatal septicaemia appeared to be justified. It was for this high degree of sensitivity that Airede18 in 1992 and Antia-Obong et al17 in the same year recommended the use of gentamycin alone as the drug of empirical treatment of neonatal septicaemia. The problem with such monotherapy is rapid emergence of gentamycin-resistant mutant strains of Staphylococci.137 Apart from this observed phenomenon, the clinical efficacy of aminoglycosides alone in the treatment of serious staphylococcal infection has not been documented41,137 and as such use of gentamycin alone in neonates with serious staphylococcal septicaemia may be attended with higher morbidity and mortality.

82 Based on the information obtained from this study, empirical therapy of neonates with suspected septicaemia should include cefuroxime and gentamycin.

Although this regimen appears highly effective, the main problem is the cost of cefuroxime, a factor that could be down played when compared with the cost of prolonged hospital stay, treatment failure and mortality.

83 CONCLUSIONS

It is concluded from this study that:

1. The current incidence of neonatal septicaemia at the Ahmadu Bello

University Teaching Hospital is 17.6 per 1000 live births, which is relatively

high for a tertiary health institution.

2. The highest infection rate was found in babies delivered in private

hospitals/clinics and at home.

3. The risk of infection was found to be significantly higher in babies whose

mothers had infections during pregnancy, unsupervised antenatal period,

prolonged rupture of membranes and prolonged labour as well as in those

with perinatal asphyxia and in those delivered before 37 weeks of gestation.

4. The isolated aetiologic agents of neonatal septicaemia in A.B.U. Teaching

Hospital, Zaria were Staphylococcus aureus, Escherichia coli, Klebsiella

pneumoniae, Proteus mirabilis, Streptococcus pyogenes, Streptococcus

pneumoniae, untyped Streptococcus species, Citrobacter species,

Enterobacter species, Pseudomonas aeruginosa, Aeromonas species and

Alcaligenes species.

5. These organisms had developed high levels of resistance to ampicillin and

cloxacillin as well as other commonly used antibiotics.

6. The epidemiology and bacteriology of neonatal septicaemia in the present

study were generally similar to those of earlier reports from our newborn

unit, except for the higher incidence of the disease and the increasing

prevalence of antibiotic resistant pathogens.

84 RECOMMENDATIONS

From the results of this study, the following recommendations are made:

1. Since pregnant women still deliver at home, health education should be given to

people in the community on the ways of preventing infection of the newborn at

birth and the subsequent care of the baby.

2. Meticulous attention to infection control should be mandatory for all private

hospitals and clinics that wish to offer antenatal and delivery services.

3. The combination of cefuroxime and gentamycin is recommended as empirical

treatment protocol for neonatal septicaemia in A. B. U. Teaching Hospital,

Zaria.

4. Future trends in aetiology and antibiotic sensitivities of infecting organisms

should be monitored so that prompt and effective action can be taken should

any change occur.

5. In order to combat the problem of multi-drug resistant organisms, the hospital

should have an effective infection surveillance system and definite guidelines

on prudent use of antibiotics to which every physician must adhere.

85 THE LIMITATIONS OF THE STUDY

The study was limited by

1. The strike action of members of staff that lasted for 12 weeks during the study

period which resulted in low patients turnover and hence possible alteration of

the actual pattern of septicaemia in the unit.

2. Blood Culture was done only once in each patient due to financial constraints,

this might have caused some of those with septicaemia to be regarded as

negative. Up to 2.5% of neonates with septicaemia could be missed where only

one blood culture sample is taken.5

3. Anaerobic cultures were not done as the facilities for the test were not available

in the hospital at the time of study and they could not be purchased due to

financial constraints, since the project was totally self-financed.

86 FUTURE RESEARCH

1. There is need for detailed community-based prospective study to determine the

peculiar social, prenatal, perinatal and neonatal factors contributing to the high

neonatal infection rate in Zaria.

2. Studies to review the relationship between the identified risk factors and

outcomes of neonatal septicaemia are highly desired.

3. Studies on the increasing on the increasing antibiotic resistance in our

environment are desirable.

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99 APPENDIX I PROFORMA FOR THE STUDY OF NEONATAL SEPTICAEMIA IN A.B.U.T.H. ZARIA.

A BACKGROUND INFORMATION Name……………………………. Hospital Number ………………… Age at presentation …………… Date of Admission……………….. Sex………………………………. Date of discharge ……………….. Address: Total hospital stay……….…days Street:………………… Birthweight (if known)------grams. Location:……………… Gestational age------weeks.

B: PARENT’S DEMOGRAPHIC DATA: Age: Father………………………..Mother……………………… Education: Father………………………..Mother..……………………. Occupation: Father………………………..Mother………………………

C: PREGNANCY, LABOUR AND DELIVERY. Place of antenatal care……………………………………………………. Length of rupture of membranes before delivery……………………Hours Duration of Labour..…………………………………………………Hours. Nature of liquor: 1- Clear 2-Yellowish 3- Greenish 4- foul smelling Maternal fever during delivery 1- Yes 2- No Maternal infection within 4 weeks of delivery 1- Yes 2- No, if yes state type: Maternal antibiotics use within 2 weeks prior to delivery. 1- Yes 2- No Types of gestation. 1- Singleton 2- multiple Mode of delivery. 1- Spontaneous vaginal delivery. 2- Breech extraction 3-Caesarian section. 4- Forceps/vacuum. Place of delivery 1- Delivery suit (ABUTH). 2- Private Hospital/ Clinics 3- General Hospital 4-Primary Health Care centre 5- Home. 6- Others Recorded Apgar score 1min……… 5min……………10min…………… Duration of stay in the hospital after delivery…………….Hour Instrumentation: Intravenous infusion 1- Yes 2- No Umbilical catheterization 1- Yes 2- No 100 Venipuncture 1- Yes 2- No Uvulectomy 1- Yes 2- No Breast feeding practice: (1) Exclusive. (2) Predominant. (3) Breast milk substitute. (4) None Method of cord care: 1-Methylated spirit 2-Warm water compress 3-Others------Other risk factors for infection (specify)…………………………………………………..

D. PRESENTING COMPLAINTS: Symptoms (1) (2) Duration (if yes) Fever Yes No ……………………….. Refusal of food Yes No ……………………….. Irritability Yes No ……………………….. Vomiting Yes No ……………………….. Diarrhoea Yes No ……………………….. Abdominal distension Yes No ……………………….. Yellowish discoloration of the skin Yes No ………………………..

Difficulty in breathing Yes No ……………………….. Convulsion Yes No ……………………….. Others (specify) Yes No ………………………..

E. PHYSICAL SIGNS General Examination (1) (2) (3) (4) (5) Activity Active Diminished activity Lethargy Floppy Moribound Colour Pink Pale Dusky Cyanosis Jaundice Temp. Normal High (To= o c) Low (To= o c) (Rectal)

Focal signs None Impetigo Petechiae Sclerema Others

Anthropometry weight….kg Length……cm OFC…….cm

101 Systems: (1) (2) (3) (4) (5) Chest Normal Tachypnoea Respiratory Crepitation Others (RR >60/min) distress

CVS Normal Tachycardia Bradycardia Displaced Apex Others (HR>160/min) (HR>60/min)

Abdomen Normal Distended Hepatomegaly Splenomegaly Others CNS Normal Bulging Hypotonia Hypertonia Others Fontanelle

MSS Normal Swollen Tender Hyperaemia Others Limbs joint of the limb

F. LABORATORY RESULTS Haematology Differential Counts Blood film Haemoglobin mg/dl Neutrophils (%) Anisocytes Yes/No PCV % Lymphocytes (%) Poikilocytes Yes/No WBC Total (x 109/l) Eosinophils (%) Hypochromia Yes/No Platelet (x 109/l) Monocytes (%) Toxic granulation Yes/No

Blood culture Other cultures Other cultures Specify: Specify:

Cultured organism(s) Cultured organism(s) Cultured organism(s)

- Sensitive to - Sensitive to - Sensitive to

- Resistant to - Resistant to - Resistant to

G OUTCOME (1) (2) Alive and normal Yes No Alive with sequalae Yes No If yes, specify……………… Dead Yes No

102