ii

COMPARING SERUM PROCALCITONIN AND SERIAL

C-REACTIVE PROTEIN AS SCREENING MARKERS FOR

NEONATAL BACTERIAL SEPSIS AT THE LAGOS UNIVERSITY

TEACHING HOSPITAL, IDI-ARABA LAGOS, NIGERIA

THIS DISSERTATION IS SUBMITTED IN PART FULFILLMENT

OF THE REQUIREMENTS FOR THE AWARD FOR THE

FELLOWSHIP OF THE NATIONAL POSTGRADUATE MEDICAL

COLLEGE OF NIGERIA IN THE FACULTY OF PAEDIATRICS.

BY

DR. MATTHEW-OKORE AMALACHUKWU NNENNA

MBBS (NIG) 2004

NOVEMBER 2016

iii

DECLARATION

It is hereby declared that this work is original unless otherwise acknowledged. The work has not been

presented to any other college in part fulfilment of the requirements for the award of a fellowship, nor

has it been submitted for publication in any academic journal.

……………………………………….

DR. MATTHEW- OKORE AMALACHUKWU

DATE: JANUARY 26TH 2017.

iv

ATTESTATION

We hereby certify that this work was done by Dr Matthew-Okore Amalachukwu in the department of

paediatrics Lagos University Teaching Hospital, Idi- Araba Lagos state under our supervision. we

also supervised the writing of the dissertation.

Names and addresses of the supervisors:

FIRST SUPERVISOR:

V.C EZEAKA, FWACP( PAED),

Professor/Consultant Paediatrician,

Department of Paediatrics,

College of medicine, university of Lagos and Lagos University Teaching Hospital,

Idi- Araba, Lagos.

………………………………

Signature/Date

Second Supervisor:

DR O.F ADENIYI, FMC(PAED),

Consultant Paediatrician,

Department of Paediatrics,

College of medicine, university of Lagos and Lagos university teaching hospital,

IDI- ARABA, LAGOS.

………………………………

Signature/Date

Third Supervisor

DR R OLADELE,

Consultant Microbiologist,

Department of Microbiology,

College of Medicine, University of Lagos and Lagos University Teaching Hospital,

IDI- ARABA, LAGOS.

………………………………

SIGNATURE/DATE

v

TABLE OF CONTENTS

TOPIC PAGE

Title i

Declaration ii

Attestation iii

Table of contents iv

Dedication vi

Acknowledgements vii

List of abbreviations viii

Definition of terms ix

List of tables xi

List of figures xii

Summary 1

Introduction 3

Literature review 7

Aim and specific objectives 23

Subjects and methods 24

Results 43

Discussion 60

Conclusion 68

Recommendations 69

Limitation 70

Areas of future research 71

References 72

vi

Appendix Ia (ethical approval) 86

Appendix Ib (extended ethical approval) 87

Appendix ii (consent form) 88

Appendix iii (Study Proforma) 93

Appendix iv (Researcher at Work) 97

Appendix v (Positive Procalcitonin Cassettes) 98

Appendix vi (Negative PCT cassettes) 99

Appendix vii (PCT interpretation card) 100

Appendix viii (Positive CRP Strips) 101

Appendix ix (positive blood culture samples) 102

vii

DEDICATION

This work is dedicated to:

The almighty god, without whom I am nothing.

My parents, Barr sir and Lady Goddy C Agbasi.

My husband, mr matthew okore and my wonderful children, Amarachi, Chidera,

Chukwuebuka and Onyinyechi.

To the Nigerian newborn- surviving against all odds!

viii

ACKNOWLEDGEMENTS

This work would not have been successful without the contributions from different persons in diverse

ways. Their kind contributions without which this work would not have been is eternally appreciated.

I express my deepest gratitude to my supervisors: Prof V.C Ezeaka, Dr (Mrs) O.F Adeniyi and dr.

(mrs) r. Oladele for their guidance. They painstakingly reviewed my work and guided me through the

rudiments of research. I deeply appreciate their efforts.

I also want to appreciate all the consultants in the department of Paediatrics who at different times

read through and added useful suggestions to my work. Their investment into my training is highly

appreciated.

I also appreciate the residents in the department of Paediatrics, especially the 2010 set.

I cannot forget Dr Bode- Sojobi, Dr Alli, Dr Igwe and Mrs Makinwa of the microbiology department

for taking me through the process of blood culture and analysis. Dr akpa, dr abiola and dr adeniran of

the community medicine department helped me through the confusing time of statistical analysis. I

deeply appreciate their kindness.

My appreciation also goes to Mr Sunday Isife and Mr Peter of the Paediatrics department laboratory.

They made the laboratory conducive for me to analyse my CRP and PCT samples, and shared my

excitement each time i got results. Dr. Moses, my research assistant, i am grateful.

My very sincere appreciation goes to all the parents and the subjects in this study.

Finally, I celebrate my parents, Barr. (Sir) and lady Goddy C. Agbasi who have never stopped

encouraging me to achieve my dreams. I cannot say how blessed i am to be your child. My siblings

Chizoba , Ijeoma, Uju and Obinna whose loss in the course of this work set me back, I appreciate

your support. To my aunty, Mrs Anthonia Ekeh, ‘Daa”, and my nanny, peace Ekawo, God bless you

richly.

ix

To my husband, Matthew and my wonderful children- Amarachi, Chidera, Chukwuebuka and

Onyinyechi, I will say, we did it!

To god who kept me alive and made all things possible be all the glory for ever more.

x

LIST OF ABBREVIATIONS

CBC COMPLETE BLOOD COUNT

CHER CHILDREN’S EMERGENCY ROOM

CRP C REACTIVE PROTEIN

ELBW EXTREMELY LOW BIRTH WEIGHT

EOS EARLY ONSET SEPSIS

IL INTERLEUKINS

LBW LOW BIRTH WEIGHT

LOS LATE ONSET SEPSIS

NBW NORMAL BIRTH WEIGHT

NICU NEONATAL INTENSIVE CARE UNIT

NPV NEGATIVE PREDICTIVE VALUE

PCT PROCALCITONIN

PPV POSITIVE PREDICTIVE VALUE

PROM PROLONGED RUPTURE OF MEMBRANES

SIRS SYSTEMIC INFLAMMATORY RESPONSE SYNDROME

TBA TRADITIONAL BIRTH ATTENDANTS

TNF TUMOR NECROSIS FACTOR.

VLBW VERY LOW BIRTH WEIGHT

WBC WHITE BLOOD CELL COUNT

WHO WORLD HEALTH ORGANISATION

xi

DEFINITION OF TERMS

1. Accuracy- The Degree To Which The Result Of A Measurement, Calculation Or

Specification Conforms To A Correct Value Or Standard.

2. Neonatal Sepsis- The Presence Of Infection (Probable Or Documented) With Systemic

Manifestations Of Infection In Babies Less Than 28 Days Of Life.

3. Early Onset Sepsis (Eos) - Sepsis Occurring Within The First 72 Hours Of Life.

4. Late Onset Sepsis (Los) – Sepsis Occurring From 4th To The 28th Day Of Life.

5. C- Reactive Protein (Crp) Level- Level Of C – Reactive Protein In A Patient Which Is

Measured In Mg/L Or Mg/Dl. (10mg/L= 1mg/Dl)

6. Procalcitonin (Pct) Level – Levels Of Procalcitonin In Patients Plasma Which Is Measured In

Ng/Ml. Normal Values Are <0.5ng/Ml.

7. Preterm Baby- Baby Delivered At Less Than 37 Completed Weeks Of Gestation.

8. Term Baby- A Baby Delivered At 37 To 41 Completed Weeks Estational Age.

9. Post Term Baby- A Baby Delivered At 42 Weeks Gestational Age And Above.

10. Extremely Low Birth Weight (Elbw) - Birth Weight Of Less Than 1000grams.

11. Very Low Birth Weight (Vlbw) - Birth Weight Of Less Than 1500grams.

12. Low Birth Weight (Lbw) - Birth Weight Of Less Than 2500grams.

13. Normal Birth Weight (Nbw) - Birth Weight Of 2500g To 4000grams.

14. Inborn Neonates- Babies Born At The Lagos University Teaching Hospital

15. Outborn Neonates- Babies Born Outside The Lagos University Teaching Hospital.

16. Definite Infection- Any Neonate With A Maternal Risk Factor For Sepsis Or At Least One

Clinical Sign Of Sepsis, Or Had A Positive Crp Or Pct And A Positive Blood Culture.

xii

17. Probable Infection- Any Baby With A Maternal Risk Factor For Sepsis Or At Least One

Clinical Sign Of Sepsis Who Had At Least A Positive Crp Or Pct And A Negative Blood

Culture.

18. No Infection- Any Baby With Any One Of The Risk Factors Of Sepsis Nor Any Of The

Clinical Features Of Sepsis, And Negative Crp, Pct And Blood Culture.

19. Sensitivity – The Ability Of A Test To Correctly Identify Subjects With A Disease. (True

Positives)

20. Specificity- The Ability Of A Test To Correctly Identify Subjects Without A Disease.(True

Negatives)

21. Positive Predictive Value (Ppv) - The Proportions Of The Positive Results In Diagnostic Tests

That Are True Positive Results.

22. Negative Predictive Value (Npv) - The Proportions Of The Negative Results In Diagnostic

Tests That Are All True Negative Results.

xiii

LIST OF TABLES

PAGE

TABLE I- ASSESSMENT OF VALIDITY 42

TABLE II- SOCIO-DEMOGRAPHIC CHARACTERISTICS OF STUDY

POPULATION 44

TABLE III- DESCRIPTIVE TABLE ON THE DISTRIBUTION OF SEPSIS BY SEX

AND PLACE OF BIRTH 45

TABLE IV- RELATIONSHIP BETWEEN SOCIAL CLASS AND CULTURE PROVEN

SEPSIS 46

TABLE V- SERIAL C-REACTIVE PROTEIN VALUES DURING THE COURSE OF

THE ADMISSION 49

TABLE VI- PROCALCITONIN VALUES AT PRESENTATION 50

TABLE VII- RELATIONSHIP BETWEEN MARKERS AND GESTATIONAL AGE 51

TABLE VIII- RELATIONSHIP BETWEEN MARKERS AND ONSET TIME OF SEPSIS 52

TABLE IX- BLOOD CULTURE RESULTS 53

TABLE X- RELATIONSHIP BETWEEN SERIAL CRP, PCT AND BLOOD CULTURE

OF SUBJECTS 56

TABLE XI- ASSESSMENT OF VALIDITY OF THE MARKERS 58

TABLE XII- RELATIONSHIP BETWEEN CRP AND PCT WITH OUTCOME OF

CARE 59

xiv

LIST OF FIGURES

PAGE

FIG 1A- SCHEMATIC REPRESENTATION OF CRP ASSAY PROCEDURE 34

FIG 1B- SCHEMATIC REPRESENTATION OF CRP ASSAY PROCEDURE 35

FIG 2- SCHEMATIC REPRESENTATION OF PCT ASSAY PROCEDURE 38

FIG 3- BAR CHART SHOWING RISK FACTORS FOR SEPSIS IN THE SUBJECTS 47

FIG 4- BAR CHART SHOWING CLINICAL FEATURES OF SEPSIS IN THE

SUBJECTS 48

1

SUMMARY

Neonatal sepsis (NNS) is a leading cause of morbidity and mortality in the developing countries

requiring early diagnosis and prompt treatment. The diagnosis of neonatal sepsis is very

challenging to the physician as the symptoms are non-specific and blood culture which is the

gold standard in the diagnosis requires a lot of blood, has a low sensitivity, is expensive and has

a long result turn out time. Screening markers like C –Reactive Protein (CRP) and Procalcitonin

(PCT) can help to make an early diagnosis thereby reducing the cost of care of the newborn with

sepsis.

The aim of the present study was to compare serial C- reactive protein and Procalcitonin as

screening markers for neonatal bacterial sepsis against blood culture which is the accepted gold

standard.

The study population was made up of neonates who were managed at the Lagos University

Teaching Hospital (LUTH) over a 6 month period. One hundred and five (105) neonates were

recruited into the study and samples for CRP, PCT and blood culture were taken from each

newborn. The CRP and PCT were analysed using point of care kits while the blood culture

samples were collected in the BACTEC culture bottles and incubated in the BACTEC 9050

machine. All samples were taken on presentation, prior to commencement of antibiotics, and

additional CRP samples were collected at 24 hours and at 72 hours. Levels of CRP and PCT

obtained were compared with blood culture results. The relationship between CRP, PCT and

specified variables: gestational age, onset time of sepsis and outcome of the treatment was

determined. Data was analysed using the Epi InfoTM 2008 version 3.4.1 software.

2

The sensitivity of CRP at presentation (0 hour) was 100% while at 24 hours it was 82% and

70% at 72 hours. The specificity of CRP at 0, 24 and 72 hours were 65%, 66% and 75%

respectively while the negative predictive values of CRP at 0, 24 and 72 hours were 100%, 97%

and 96% respectively.

On the other hand, PCT done on presentation had 100% sensitivity and negative predictive

values of 100%.

Blood culture was found to be positive in 11 neonates giving an overall infection rate of of

10.5%.

Both CRP and PCT were found to have significantly higher positivity rates in the term babies

compared to the preterm neonates. Both markers were also found to be more significant in late

onset sepsis.

In conclusion, PCT on the day of presentation has a high sensitivity and NPV and is useful in

early diagnosis of neonatal sepsis. However, serial CRP with high sensitivity and NPV can also

be used.

3

INTRODUCTION

Sepsis is defined as the presence of infection (probable or documented) with systemic

manifestations of infection.1,2 Systemic manifestations of infection, which is the systemic

inflammatory response syndrome (SIRS), is described as the presence of at least 2 of the

following criteria ( one of which must be abnormal temperature or leucocyte count):2

Core temperature of >38.30C or <36oC.

Tachycardia - Heart rate >180 beats/minute or bradycardia (heart rate <100 beats/minute.

Tachypnoea- respiratory rate >60 cycles/minute

Leucocytosis or leucopenia.

Neonatal sepsis is a major cause of neonatal morbidity and mortality throughout the world and

especially in the developing countries.3 The World Health Organisation (WHO) has estimated

that 4 million babies die in the first 28 days of life, and neonatal sepsis accounts for 26% of those

deaths.4 Ninety eight percent(98%) of neonatal deaths occur in developing countries.5 In Nigeria,

sepsis contributes significantly to under 5 mortality6,7 and hospital based studies suggest it

accounts for 20-25% of neonatal mortality.8

The diagnosis of neonatal sepsis is challenging to the physician because the features are non-

specific, and overlap with other non-infectious causes of systemic inflammation.9 Despite

research over the past decades, there is still no single ideal test for early diagnosis of neonatal

sepsis.10

The gold standard in the diagnosis of sepsis is blood culture .11,12 Blood culture is expensive, and

there is a delay of 48 hours before preliminary results are obtained.13,14 The yield of blood

culture is between 30 -70% and some cases of neonatal sepsis go undetected.15 This is because

4

of difficulty in obtaining enough blood samples16 and increasing use of intra-partum antibiotics

in women during labour and delivery.17

Therapeutic antibiotic use is recommended for infants with clinical features or risk factors for

sepsis.18 However, this results in overtreatment, unnecessary painful intravenous cannulations,

and mother- infant separations. 19 Antibiotic therapy greater than 72 hours has been found to

increase colonization by gram negative organisms and emergence of drug resistant strains.18

There is also associated altered gut colonization and increased risk of Candida colonization20 and

death. Also, prophylactic antibiotic treatment may result in the treatment of as many as 30

uninfected neonates for every infected one.19 The dilemma therefore is, ‘which babies need

antibiotics and for how long?’

Several newer markers of sepsis have been studied including C- reactive protein (CRP)3,14,21,

Procalcitonin (PCT),22-24 Tumor necrosis factor α (TNF),25-27 Fibronectin28 and Interleukins.27,29

In Port Harcourt, Nigeria, West et al30 observed that the qualitative method of CRP estimation

could correctly identify three quarters of neonates with sepsis. Ayazi et al 31 in Iran found serial

C- reactive protein levels useful in the evaluation of neonates with suspected sepsis.

Benitz et al19 found that when 3 serial CRP tests were done, their sensitivities increased

progressively, and negative predictive value of 3 serial CRP was 99.7% for proven sepsis.

Ibeh et al 22 in Benin reported that procalcitonin was superior to CRP as a predictor of neonatal

sepsis.

Considering these findings, this study aims to compare serial CRP and PCT as markers of

neonatal sepsis. This research hypothesized that serial CRP measurements is as effective as one

PCT measurement which other researchers have found to be very sensitive in making an early

5

diagnosis of neonatal sepsis, but quite expensive. The research was aimed at answering the

following research questions:

(a) Considering the fact that neonatal sepsis is common in our environment and is a leading

cause of prolonged, and sometimes unnecessary hospital admission, can serial CRP or PCT

be useful in early decision making?

(b) Can serial CRP be utilized as an effective marker for neonatal sepsis in our resource poor

country?

(c) Can these markers be used to monitor response to treatment in neonates being treated for

neonatal sepsis?

JUSTIFICATION

The practice of commencing every neonate with suspected sepsis on antibiotics has increased the

emergence of drug resistant organisms. 32 The cost of antibiotic therapy is also challenging in a

resource poor country especially if unnecessary.

The use of a reliable marker with high sensitivity and negative predictive value to select neonates

who do not require prolonged antibiotic therapy is desirable. The kits used for this study were

point of care kits which can give results in as fast as 30 minutes thereby identifying babies who

may not require antibiotics early. Most of the studies on these markers were done in developed

countries. Additionally, the sensitivity and specificity of CRP improves with serial

measurements. In Nigeria, to the best of the authors’ knowledge, no study has compared serial

CRP and PCT as markers of neonatal sepsis. Ibeh et al 22 in Benin compared PCT with a single

CRP reading.

6

Additionally, CRP is more readily available than PCT, so it would be useful to compare the

sensitivity and specificity, positive and negative predictive values of serial CRP and PCT to

determine if the use of serial CRP measurements can help to promptly identify newborns with

sepsis. This would significantly reduce duration of hospital stay, prevent emergence of antibiotic

resistance and ultimately reduce cost of healthcare. Early detection of infected neonates and early

discharge of the uninfected will help improve morbidity and mortality among these vulnerable

newborns.

It is hoped that the findings from this study will answer the research questions, add to the body

of knowledge, and contribute to policy formulation in the management of neonatal sepsis in

Nigeria.

7

LITERATURE REVIEW

Neonatal sepsis is a source of diagnostic challenge to the physician and a killer of newborns all

over the world. Among the major causes of death in the first month of life in Nigeria, severe

infections account for 26% and prematurity 28%.33 This is worrisome because preterm/low birth

weight is the risk factor most closely associated with early onset neonatal sepsis.34

Neonatal sepsis encompasses various systemic infections in the newborn such as septicemia,

meningitis and pneumonia, but does not include superficial infections like conjunctivitis and oral

thrush.35

CLASSIFICATION

Neonatal sepsis is classified into 2 major categories:36

Early onset sepsis (EOS): This usually occurs within the first 72hours. Neonates with (EOS)

typically present with respiratory distress.

Late onset sepsis (LOS): Usually occurs from 4 – 30days of delivery and is acquired in the

newborn unit or community.

Late, Late onset sepsis occurring after 30days has been reported, especially in the preterm very

low birth weight infants.36

Risk factors for early onset sepsis include: prematurity, chorioamnionitis, febrile illness in the

mother (within 2 weeks prior to delivery) and prolonged rupture of membranes (>18hours).

8

Preterms have a 3 -10 fold risk of acquiring infections more than term, normal weight infants.36

This is because of immaturity of the immune system, and maternal genital tract infection is an

important cause of preterm delivery.

Chorioamnionitis is characterized by intrapartum fever(>38oC), leucocytosis (WBC>15,000)

and uterine tenderness.37

THE BURDEN OF NEONATAL SEPSIS

Neonatal sepsis is accepted worldwide as a devastating complication for the newborn. This is

because of the high mortality associated with it, alongside long term complications such as

neurodevelopmental impairment. In the United States, the estimated annual incidence of severe

sepsis in the newborn is 0.3/ 1000 live births.10 The estimated mortality is 10.3%, with most

deaths occurring in the first 48 hours of infection.10 Neonatal sepsis contributes between 13- 15%

of neonatal deaths globally,38 and in developing countries, 30-50% of neonatal deaths.6,38,39

In 2012, West et al in Port Harcourt reported a prevalence of 33.1%.40 This is similar to 31.7%

reported by Antia- Obong et al in Calabar 7and 34.4% reported by Bode- Thomas et al in Jos.41

Lowest prevalence documented so far has been 10.7%42 reported by Njokanma et al in Ogun

state about two decades ago. The higher prevalences in Jos, Calabar and Port Harcourt may have

been because the studies were prospective and infants who had prior antibiotic therapy or whose

mothers were on antibiotics were excluded from the study.40 The use of antibiotics prior to blood

culture may suppress growth of bacteria in cultures.

9

At the Lagos University Teaching Hospital, Iroha et al 43 showed a prevalence of 35 per 1000

live births and accounted for 36% of neonatal deaths during the study period. Adejuyigbe et al 44

in Ile- Ife in 1995 had an incidence of 22.9 per 1000 live births and a mortality rate of 33.3%.

These studies from Nigeria are in sharp contrast to findings from the United Kingdom where an

incidence of 8/1000 live births was recorded,45 and the United States where an incidence of

0.76/1000 live births was recorded.46 In Saudi Arabia, 4.9/1000 live births was reported.47

The socio-economic burden of long term complications of neonatal sepsis has also been studied.

Mwaniki et al 48 in a review article documented that amongst 2442 neonates with sepsis, 40%

had sequelae which ranged from cognitive and learning difficulties, cerebral palsy, hearing loss,

loss of vision and co-ordination. These impairments may constitute huge financial, emotional

and social challenges for affected families.

PATHOGENESIS OF NEONATAL INFECTIONS

Before birth, the fetus is maintained in a sterile environment. The chorioamniotic membranes,

placenta and antibacterial factors in the amniotic fluid protect the fetus from infections.49 Sepsis

can begin in utero if the fetus swallows infected amniotic fluid. Organisms causing early onset

sepsis can also ascend from the birth canal either at rupture of amniotic membranes or during

labour, especially if the membranes ruptured for longer than 18 hours. The neonate can also

develop sepsis in the hours or days after birth when the mucosal surfaces are compromised ( e.g

in babies with omphalocele) 37

Sepsis results from the complex interaction between the invading organism and the host immune,

inflammatory and coagulation response.50 After invasion of the microorganisms, an acute

10

localized inflammatory reaction is initiated. The contact with bacterial endo or exotoxins initiates

the release of prostaglandin, leukotriene and histamine which activate further inflammatory cells.

Activated fibroblasts and leucocytes produce pro inflammatory cytokines including Interleukins

1 and 6, TNFα. IL-6 in synergy with IL-1 induce the hepatocytes to produce CRP.32

The high levels of cytokines and bacterial endotoxin in sepsis cause elevation of PCT by

inhibiting the final step in conversion of PCT to calcitonin.51 It is also postulated that the

elevation of PCT due to endotoxin release occurs mainly in parenchymal tissues. Parenchymal

cells unlike neuroendocrine cells lack the ability to cleave PCT into its mature form calcitonin

leading to a circulation of PCT in large amounts.52

Other factors predisposing to sepsis in the neonate include :diminished concentrations of

immunoglobulin, and reduced function of neutrophils and other cells involved in the response to

infection in both term and preterm infants.36 Despite these alterations in immunologic factors,

newborn infections are uncommon in the absence of predisposing obstetric factors.36

CAUSATIVE ORGANISMS AND CHANGING PATTERNS

Organisms causing neonatal sepsis vary according to locations, and even in the same location,

studies have shown that causative organisms vary periodically.

Iroha et al,43 at LUTH reported the common causative organisms to be more of gram negatives,

with Klebsiella pneumonia being dominant in both early and late onset sepsis. This was at

variance with a study done 20 years earlier in the same centre where E.coli was dominant.53

Other significant pathogens isolated in the LUTH study were Staphylococcus aureus and

Coagulase negative staphylococcus.43

11

Awoniyi et al,54 in Ife, Nigeria in 2009 found Staphylococcus aureus ( Staph aureus) to be the

dominant pathogen, followed by Pseudomonas(13%) , Klebsiella (13%) and Proteus (10%).

Adejuyigbe et al39 eight years earlier in the same center reported a similar pattern except for the

isolation of Listeria monocytogenes which is not a common cause of neonatal sepsis in the

tropics. These studies did not clearly state the pattern of infection amongst inborns and outborns,

however Adejuyigbe et al 39 noted that 56.6% of the neonates with risk factors and/or clinical

features of sepsis were delivered outside the hospital. Mokuolu et al 55 in 2002 in Ilorin in a

study that screened 198 neonates, isolated gram negative bacilli in early onset sepsis and Staph.

aureus was the commonest pathogen (29.5%) in late onset sepsis. 52% of the newborns with

sepsis were out born.

In 2007 at the University of Port Harcourt Teaching Hospital, West et al,56 in a study of 406

neonates with 169 positive cultures, isolated Klebsiella pneumonia and Staph aureus

predominantly in early onset sepsis(EOS) and late onset sepsis (LOS) respectively. Both

organisms were also predominant in the preterm and term newborns. In Bauchi, Yusuf et al 57 in

2008 isolated Escherichia coli as the predominant pathogen in EOS while Staph. aureus was the

predominant pathogen in LOS.57

These patterns are in contrast with studies from the United States where Group B streptococcus

(GBS) is the predominant pathogen in early onset sepsis followed by E. coli.34 GBS remains the

most significant pathogen in term infants, and E. Coli the most significant pathogen in preterm

infants with EOS.34 Similarly, a study from the United Kingdom has reported GBS to be the

most frequent pathogen, followed by coagulase negative staphylococcus.58 Though it appears

that GBS is uncommon in developing countries , a study done by Dawodu et al 59 in Ibadan

showed that there is a high vaginal carriage rate of GBS but low incidence of disease. Suara

12

et al 60 in Gambia postulated that the rarity of GBS disease in developing countries may be due

to the prevalence of maternally derived GBS antibodies, and lower prevalence of the virulent

GBS serotype III. From the studies above, it is notable that causative organisms of neonatal

sepsis vary from center to center, hence the need for continuous research and re-evaluation of

antibiotic sensitivity patterns.

DIAGNOSIS OF NEONATAL SEPSIS

Diagnosis of sepsis in the newborn is difficult because of non-specific clinical features which can

also be observed in non-infectious conditions. Available diagnostic testing is not useful in

deciding which neonate requires empirical antibiotics treatment, ( due to poor positive predictive

values e.g. full blood count ) but can help with the decision to discontinue treatment.61 The

diagnostic tests for neonatal sepsis may be specific or non-specific.

SPECIFIC TESTS:

BLOOD CULTURE

Blood culture is still recognized as the “gold standard” in the diagnosis of neonatal sepsis.14

However, it requires 48- 72 hours to get a preliminary result, and with maternal antibiotics, as

few as 2.7% of neonates with clinical sepsis may have positive cultures.62 The yield of blood

culture is 30-70%,15 and data shows that even in neonates who were critically ill and died, only

80% of autopsy proven sepsis was diagnosed by pre-mortem blood cultures.63 A positive blood

culture with a pathogenic organism confirms the diagnosis of neonatal sepsis, but a negative

blood culture does not rule out the disease.64 The use of antibiotics prior to collection of blood

13

culture samples can also affect the results. However, the BACTEC® blood culture systems

contain resins which neutralizes antimicrobial activity of antibiotics.65 Microbial culture using

BACTEC® has multiple advantages over conventional blood culture including rapidity, cost

effectiveness, recovery of fastidious organisms and lower risk of contamination due to the closed

system of blood collection.66 With the BACTEC® Peds Plus bottle, as low as 1ml of blood can

be used which makes it very useful for neonates.65

BACTEC® also improves the yield of clinically significant isolates with reduced time to

detection.67

Other specific tests include: Cerebrospinal fluid culture, urine culture and tracheal aspirate

cultures.

NON SPECIFIC TESTS:

I COMPLETE BLOOD COUNT.

The complete blood count (CBC) is one of the earlier markers evaluated. However, in the

interpretation of CBC, timing is important.68 The Manroes’s criteria, using the total white cell

count, immature neutrophil count and bands to neutrophil ratio is widely used.69 Makkar et al 70

in India showed immature to total neutrophil ratio as the most reliable indicator of sepsis in

neonates.

In Nigeria, micro ESR ( Erythrocyte Sedimentation Rate) has been studied by Boma West et al,71

and found to have a sensitivity of 75.7% which increased to 95.9% when combined with band

forms. This is also similar to 71% reported by Akpede and Abiodun.72

14

II INFLAMMATORY CYTOKINES

Inflammatory cytokines, (eg interleukins, tumor necrosis factor) are released in response to

inflammation. Interleukins 6 and 8 have been extensively studied. Cytokines are promising

diagnostic markers and their levels are increased early in the infective processes.27,73

Tumor necrosis factor has been studied by Ako-Nai in Ile- Ife,26 Nigeria and found to be a useful

marker of neonatal sepsis.

Some other biomarkers studied include serum amyloid,74 haptoglobulins75 and fibronectin.28,75

It has been postulated however, that a combination of markers often produces a screening test

with better sensitivity and specificity than a single marker.17,76,77

III C – REACTIVE PROTEIN

CRP is one of the most studied laboratory tests for neonatal sepsis. It was first described in 1930

by Tillet and Francis at Rockefeller University.78 A precipitation reaction was observed between

serum from patients suffering from acute pneumococcal pneumonia and the extracted

polysaccharide fraction C from the pneumococcal cell wall. This reaction was not observed when

using serum of healthy controls, or the patients after they had recovered.78

CRP is an acute phase reactant protein, produced in reaction to acute tissue injury or infection.

The production of CRP in the hepatocytes are induced by interleukin 6.79 CRP is normally

present as a trace component of the plasma.80 However, in the acute phase response, hepatic

synthesis can reach 1000 fold levels.81` Levels remain high as long as the inflammation persists

and then decreases rapidly.

15

CRP is synthesized within 4-6 hours after exposure to an infective process or tissue damage,

peaks at 24-48 hours and then diminishes.82 In studies using CRP 1mg/dl as cut off value, the

range of reported statistical outcomes for culture proven sepsis is as follows: sensitivity 30-40%,

specificity 88-91% and negative predictive value of 97-99%.19,83 CRP elevations in non-infective

conditions such as meconium aspiration, recent vaccination, surgery, perinatal asphyxia and

intraventricular hemorrhage are also thought to affect the specificity of the test.84

The predictive value of CRP improves with time and is most predictive 24- 48 hours after a

newborn presents with suspected infection. Serial measurements (more than one) are therefore

recommended rather than a single value.19 Benitz et al 19 performed 3 CRP measurements- on

admission and next two mornings, while Nuntnarumit et al 85 performed 2 measurements at

initial assessment and 24-48hours later. In both studies, the predictive values of CRP increased

with serial testing.

Ayazi et al,31 in a study done in Iran determined CRP levels in neonates with suspected bacterial

infection. CRP levels were determined at initial evaluation and 24 hours later. Their results

showed that the sensitivity, specificity, positive and negative predictive values of CRP increased

with the second test. They concluded that normal CRP values obtained 24 hours after the initial

CRP indicate that infection is unlikely.

Lee et al,21 also applied a protocol of measuring CRP serially in the management of suspected

early neonatal sepsis. They measured CRP levels at initial evaluation, 8- 16 hours later and 24

hours later. Antibiotics were discontinued if two serial CRP were within normal range and blood

culture negative at the time of CRP report. They found that this protocol reduced the duration of

16

hospitalization and antibiotic use in the patients and none of the subjects required re-admission

within 1 week of discharge.

Benitz et al 19 in the study titled,’’ Serial serum C- reactive protein levels in the diagnosis of

neonatal infection”, evaluated 1002 newborns between 32weeks – 40 weeks gestational age.

CRP was done at initial evaluation and the next 2 mornings. The negative predictive values for 3

CRP tests were 99.7% and 98.7% for proven and probable sepsis. They concluded that two

normal CRP levels obtained 24 hours apart, show that a bacterial infection is unlikely. The

sensitivity of a normal CRP at the initial evaluation is not sufficient to justify withholding

antibiotic therapy.19 Furthermore Phillip et al 86 in their study guided the duration of antibiotic

therapy using serial monitoring of CRP. With this protocol, antibiotics were discontinued when

CRP levels became normal. The mean duration of treatment was thus reduced to 3.1 days and no

infant initially treated with antibiotics and discharged when the CRP returned to normal was re-

admitted.86

A normal repeat CRP 24-48 hours after the initiation of antibiotic therapy has been reported to

have a 99% negative predictive value in accurately identifying infants not infected in the

neonatal period.86

In summary, determination of serial CRP levels can be helpful in diagnosing neonatal sepsis,

monitoring the response to treatment in infected neonates and determining the duration of

antibiotic therapy .19,86

17

METHODS OF ASSAYING CRP

CRP can be assayed by several methods which may be qualitative, Semi quantitative or

quantitative. All methods are based on the ability of CRP to form antigen antibody complexes,

clump and precipitate, and then be visualized and measured. Examples of these methods are:

Latex agglutination method: This was the first laboratory method developed for CRP

assay. It is a qualitative method which measures the presence or absence of agglutination

or precipitation and indicates only whether CRP is present or absent in the sample.

Turbidimetric method: This quantitative method assesses agglutination of latex particles

coated with antibody against CRP by quantifying the absorbed light with a

spectrophotometer.87

Nephelometric method: This quantitative method measures the agglutination of particles

by quantifying the scattered light using a nephelometer.

Immunochromatography method: This method is based on the principle of flow through

immunochromatography and uses whole blood. It is a semi- quantitative method.

The quantitative methods are more widely used in developed countries. They provide highly

sensitive and specific results but require costly analysers.88 On the other hand, the qualitative and

semi quantitative methods are available for bedside testing and can provide results within 5-15

minutes. They also require less skill and may be more feasible in resource poor settings.

Furthermore, some researchers have found that the CRP values from semiquantitative methods

correlate well with the quantitative methods.88

18

IV PROCALCITONIN

Procalcitonin (PCT), an acute phase marker has been investigated for its diagnostic role in

neonatal sepsis since the mid 1990’s. Serum levels are reported to rise from 2 hours after

exposure to bacterial endotoxin, peak at 6-8 hours and reach a plateau after about 12 hours.89 It

reduces to its normal values after 2-3 days.82 It is the preliminary molecule of calcitonin. It is a

116 amino acid polypeptide with a molecular weight of 145kda.90

Procalcitonin is produced and secreted in the C cells of the thyroid gland in normal metabolic

conditions and levels are undetectable in the blood of healthy people.91 In cases of sepsis, PCT

has an extrathyroidal production in macrophages and monocytic cells of organs like the liver.89,92

In severe bacterial, fungal and parasitic infections with systemic manifestations, a significant rise

in procalcitonin levels are seen. Normal serum PCT level is <0.5ng/ml.93,94 Levels above this are

accepted as pathological. The unique feature that PCT levels increase in bacterial and fungal

infections , but remain unchanged even in severe viral infections makes it attractive as a potential

diagnostic variable for the diagnosis of bacterial infection.94

There are some challenges which have been noted with the use of PCT in the diagnosis of

neonatal infections. First is that infants with respiratory distress syndrome, perinatal asphyxia,

intracranial hemorrhage and pneumothorax may have raised serum PCT which may not differ

from those of septic neonates after onset of clinical signs of distress or infection.95 This raised

PCT is because the systemic response seen in severe infections could also occur in inflammatory

processes without infection such as ischaemia, multiple trauma and conditions listed above. 96

Available data also shows that PCT and CRP values are higher for neonates born to diabetic

mothers.97,98 Also, a physiological increase in PCT has been reported up to 36 hours

19

postpartum.99 Chiesa et al in Rome concluded that despite increase in PCT caused by these

perinatal events, PCT response to infection is much higher.97 They found that PCT levels in

infected neonates got as high as 500ng/ml depending on the severity of infection, while increases

caused by perinatal events such as respiratory distress rose to a mean of 2ng/ml.

METHODS OF PROCALCITONIN ASSAY.

Procalcitonin can be assayed quantitatively or semi- quantitatively. The semi- quantitative

method is an immunochromatographic assay. It utilizes the point of care kits which are faster to

use, inexpensive and do not require equipment or calibration. However, the colour intensity must

be evaluated by a technician and so is subject to individual observer variation.100

The quantitative assay is a luminometric immunoassay. Two antigen specific monoclonal

antibodies that bind PCT are added to serum or plasma. One of the antibodies is luminescence

labelled (the tracer). The antibodies react with the PCT and form “sandwich complexes”. The

luminescence labelled antibody then binds to the inner surface of the tube and excess tracer is

then removed. The amount of tracer on the test tube wall is then read with a luminometer. This

method is cumbersome, requires equipment and is more expensive.

The quantitative method is thought to be more reliable than the semi- quantitative method,

however, Manzano et al found the correlation between the two methods is moderate.100

20

COMPARING CRP AND PROCALCITONIN

CRP and PCT have been studied both independently and together. CRP is thought to be the most

extensively studied biomarker of sepsis and is the most frequent marker used in diagnosis and

monitoring of infections in the neonate.10

For a screening marker to be described as effective, ideally it should recognize all infected

neonates (highly sensitive) so that disease can be excluded with negative results (High negative

predictive value).101 Khashabi et al102 stated that the most important criteria of a reliable test are

its high sensitivity combined with a high negative predictive value. Kocabas et al27 also stated

that it is desirable for an ideal diagnostic marker to have about 100% sensitivity and NPV. It

should also have a reasonably high specificity and PPV of greater than 85%.

In Port Harcourt, Nigeria, West et al 30 studied 420 neonates and found that the qualitative

method of estimating CRP was cheap, had moderate sensitivity, specificity and negative

predictive value and could correctly identify close to three quarters of neonates with sepsis.

Similarly, Ibeh et al 22 in a study done among sixty neonates in University of Benin Teaching

Hospital, Nigeria found that PCT could be helpful for early detection of neonatal sepsis, and that

any increase in PCT in a critically ill neonate should warn of possible sepsis. Arowosegbe et al

103 in Abeokuta Ogun state studied 85 neonates with risk factors and/or clinical features of sepsis.

They found that at a cut off level 0.5ng/ml, the negative predictive value (NPV) of PCT was 80%

and the positive predictive value (PPV) was 39%. They also found that a higher percentage of

neonates who died (96%) had elevated PCT levels compared to those who survived (46%). They

concluded that any increase in PCT in an ill neonate should suggest the possibility of a

21

septicemic infection. They also stated that PCT is of great advantages where prediction of

severity and mortality is concerned.

Even though other critical illnesses in the newborn (Respiratory distress syndrome, severe

perinatal asphyxia) can elevate PCT, Chiesa et al97 found that PCT response in infection is much

higher, so a high index of suspicion of neonatal sepsis is required when PCT is elevated.

Naglaa et al 104in Egypt studied 60 neonates with suspected sepsis and found that PCT and CRP

are both reliable with the same diagnostic accuracy. In their study, both CRP an PCT had

positive predictive value (PPV) of 90%, while the negative predictive values ( NPV) were 87.5%

for PCT and 66.3% for CRP. The area under the curve (AUC) values were 0.92 and 0.88 for

PCT and CRP without any significant statistical difference. Their findings agree with

Franz et al 105 in Germany and Blommendahl et al106 in Finland.

However, some studies reported that PCT is a more reliable marker than CRP. This was found by

Sucilathangam et al in India93 who studied 50 neonates and found that PCT was superior to CRP

in diagnosing neonatal sepsis. In their study, an elevated PCT was detected in 22 of the 50

neonates, whereas an elevated CRP was detected in 18 cases. 14 of the 50 neonates were culture

positive, and PCT was elevated in 13 cases (92.85), while CRP was elevated in 7 cases (50%).

Based on their results, they recommended that commencement of antibiotics in newborn infants

should be based on the PCT results on the day of admission to the Neonatal Intensive Care Unit

(NICU).

Koksal et al107 in Turkey studied 67 neonates and found that PCT was superior to CRP in

diagnosing neonatal sepsis, detecting the severity of illness and evaluating response to

22

treatment. Furthermore, Mamdouh et al in Egypt108 and Adib et al109 also reported similar

findings.

Considering all the findings of previous researchers, this study seeks to compare serial CRP and

PCT as we seek to further improve newborn care. It is hoped that the results from this study will

significantly impact on policy formulations and management of neonatal sepsis in Nigeria.

23

AIM AND OBJECTIVES

AIM

1. This study aims to compare the efficacy of procalcitonin and serial C- reactive protein as

screening markers of neonatal bacterial sepsis at the Lagos University Teaching Hospital,

Idi Araba, Lagos.

SPECIFIC OBJECTIVES

1. To determine the levels of C-reactive protein and procalcitonin in the study subjects.

2. To compare C- reactive protein and Procalcitonin levels in neonatal sepsis by gestational

age, onset time of sepsis and socio-demographic factors.

3. To compare the sensitivity, specificity, positive and negative predictive values of serial

measurements of C- reactive protein with procalcitonin in neonatal sepsis using blood

culture as the gold standard.

24

SUBJECTS AND METHODS

STUDY LOCATION

The study was conducted at the inborn and out born wards of the neonatal unit at the Lagos

University Teaching Hospital (LUTH), Idi Araba, Lagos. Lagos is a metropolitan city in

Southwest Nigeria with an estimated population of 18 million, annual birth rate of 360,000 and

about 14,000 neonatal deaths annually, 20% of which are caused by infections.110

LUTH is a tertiary hospital with about 760 beds and provides healthcare services to the residents

of Lagos and its neighboring states. It is a training centre for undergraduates, post graduate

students and resident doctors.

The neonatal unit consists of 2 wards, the New Neonatal Unit (NNU) and Ward D1. The unit

averages 1400 admissions yearly and is run by 4 consultants, 8 resident doctors and 8 interns.

The NNU (in born ward) caters for inborn babies delivered in the labour ward of LUTH. It

shares the same complex with the delivery rooms and theatre. Babies are admitted here mainly

for prematurity, risks for sepsis, neonatal jaundice and congenital anomalies. This unit has the

capacity for 36 newborns.

Ward D1 (outborn unit) caters for referred babies admitted via the Children’s Emergency Room

(CHER). They are referred from other hospitals, maternity homes and traditional birth

attendants. Reasons for admission are similar to NNU except for neonatal tetanus, severe birth

asphyxia and severe hyperbilirubinaemia which are uncommon in inborn patients.

25

Babies born in LUTH without clinical features or risk factors for sepsis join their mothers in

postnatal wards C2 and C3. These babies are reviewed daily by the neonatal team. They are

discharged after 72hours except the baby develops jaundice or other problems.

For this study, subjects were recruited from NNU, D1 and Children Emergency Room after

obtaining parental consent. Both term and preterm neonates were recruited.

STUDY DESIGN

This was a cross sectional study carried out between September 2015 and February 2016.

STUDY POPULATION

The study population comprised of newborns, aged 0-28 days with risk factors and/ or clinical

features of sepsis. Neonates were recruited from the NNU for inborn babies, and CHER or ward

D1for outborn babies.

For the purpose of this study, the subjects were divided into 3 groups according to the criteria

suggested by White et al111:

Definite Infection: Any neonate with a maternal risk factor for sepsis or at least one clinical sign

of sepsis, or had a positive C-reactive protein or Procalcitonin, and a positive blood culture.

Probable Infection: Any baby with a maternal risk factor for sepsis or at least one clinical sign of

sepsis who had at least a positive C- reactive protein or Procalcitonin and a negative blood

culture.

26

No infection: Any baby with any one of the risk factors of sepsis or at least one of the clinical

features of sepsis, and negative CRP and PCT and blood culture.

INCLUSION CRITERIA.

Neonates who had risk factors for neonatal sepsis. These factors include

chorioamnionitis, maternal pyrexia > 38oC, foul smelling liquor, prematurity, prolonged

rupture of membranes > 18hours, history of maternal urinary tract infection.

Neonates who had clinical signs suggestive of sepsis. These factors include temperature

instability, feeding difficulties, abdominal distension, apnoea, abnormal glucose

homeostasis, non-physiologic jaundice, tachypnea, grunting respirations, irritability,

lethargy or seizures.

EXCLUSION CRITERIA

Babies who had already commenced antibiotics.

Infants of diabetic mothers.

Neonates with severe birth asphyxia.

No parental consent.

27

SAMPLE SIZE DETERMINATION.

Subjects were patients who met the inclusion criteria

The level of significance and power of the study was set at 5% and 80% respectively.

The formula for comparison of 2 proportions according to Varkevisser et al112 was used:

N= (u+ v)2{P1 (100- P 1) + P2 (100- P2) }

(P1 – P2)2

Where:

N = Sample size

u = One sided percentage point of the normal distribution, corresponding to 100% - the

power = 0.84.

v= Percentage point of the normal distribution, corresponding to the 2 sided 5%

significance level = 1.96

P1 = Proportion of proven septic neonates with positive CRP according to a previous

study93 = 50%

P2 = Proportion of non-septic neonates with positive CRP93 = 30.5%

N= (0.84 + 1.96)2 87

(50-30.5)2

7.84{2500 + 2119.75}

380.25

7.84 x 4619.75 = 36218.84

380.25 380.25

95.25.

28

The same calculation was done for PCT using:

P1 = Proportion of proven septic neonates with positive PCT according to a previous study93 =

92.85%

P2 = Proportion of non-septic neonates with positive PCT93 = 25%.

(0.84+ 1.96)2 (92.85(100-92.85) + 25(100-25)

(92.85-18)2

7.84(663.88 +1875)

4603.62

= 4.32 (Not statistically significant)

The higher value was taken113: 95.25

However, in order to increase precision and possible attrition (from early neonatal deaths,

discharge against medical advice) 10% of the sample size was added.

95.25 + 9.525 = 104.775

105 subjects were recruited for this study.

29

CLINICAL AND LABORATORY DATA COLLECTION

STUDY PROCEDURE:

ETHICAL CONSIDERATION:

Ethical approval was obtained from the Lagos University Teaching Hospital’s Ethics and

Research committee before commencing the study. (Appendix I)

INFORMED CONSENT

Parents/ caregivers of newborns with risk factors and or clinical features of sepsis admitted into

the neonatal ward were approached for their consent for their wards to be enrolled in the study.

The details of the study were explained to them in simple language and they were given

opportunity to ask questions. An informed consent was obtained after this. (Appendix 1I)

PATIENT HANDLING PROCEDURE

Patients were recruited from NNU, ward D1 and Children’s Emergency Centre. At recruitment,

the parents / caregivers were interviewed. Informed consent was obtained for each subject.

Relevant history which included socio-demographic data, weight, gestational age, ante natal,

pregnancy and delivery history was obtained. Gestational age was determined from the last

menstrual period (LMP), but if unknown, antenatal ultrasound reports or Ballards score was

utilized if baby presented within 72 hours of life.

Particular attention was paid to obtaining information on the risk factors for sepsis for each

newborn. A questionnaire and consent form was given to the parent of each subject after which

a physical examination was done. The questionnaires were filled by the parents with the research

30

assistant providing support to the illiterate and semi-literate parents. This included a general

examination for pallor, fever, jaundice, respiratory distress and lethargy and systemic

examination which included a central nervous system examination, respiratory and abdominal

examination was also done. The babies were weighed free of any clothing, using the My weigh®

(Ultrascale MBSC-55) weighing scale. The scale was adjusted to zero before each measurement.

The researcher was assisted by a trained medical officer. Three residents in the neonatal unit

were also trained on sample collection for blood culture and assisted the researcher in taking

some samples.

SAMPLE COLLECTION

On recruitment, blood samples were taken (following strict universal precaution protocol for

asepsis) from the subjects before the commencement of antibiotics. Samples were taken for

CRP, PCT and blood culture. Sample bottles for the research were labelled and made available in

all the neonatal wards.

On presentation, 1ml of blood was collected in an EDTA bottle for serum procalcitonin and

CRP. Samples were collected with the babies kept under a radiant warmer or in other areas

designated for sample collection. The samples were analysed immediately, but if collected after

work hours, was refrigerated at 2-8oC and analysed not later 48 hours after collection.

The BACTEC Peds Plus® (Becton Dickinson, New Jersey, USA) blood culture broth media

were used. In the subjects, a peripheral vein was located and the area was cleaned with

methylated spirit, then povidone iodine. To reduce the risk of contamination, closed blood

sample collection system (butterfly needle attached to the BACTEC® bottle) was used to collect

31

the sample (about 2 mls). At least 2mls of blood was collected to increase the likelihood of

recovering an organism, especially for fastidious organisms like Haemophilus species. The flip

off caps of the vials were removed and the septum swabbed with alcohol pads before introducing

the sample. The samples were transported immediately to the Department of Medical

Microbiology laboratory; overnight samples collected were kept outside the refrigerator and sent

to the laboratory within 10 hours of collection.

Samples for repeat CRP (1 ml each) were collected at 24 and 72 hours (total of 3 samples) after

the initial CRP.

LABORATORY ANALYSIS

For PCT and CRP, samples were analysed immediately, but for babies whose samples were

collected after 4pm, the samples were stored at 2-8oC temperature and analysed within 48 hours.

The CRP and PCT samples were analysed by the researcher, supervised by the medical

laboratory scientist with experience in hematology and use of point of care kits in the Paediatrics

department side laboratory.

CRP ASSAY

For CRP, the actim® CRP by Medix Biochemica ,Finland114 was used. It was visually interpreted

based on immunochromatography method for semi- quantitative determination of CRP in whole

blood samples. This kit detected elevated serum CRP levels of >10mg/l (1mg/dl) to >80mg/l

(8mg/dl).

Each CRP test pack comprised of:

One dipstick in a sealed aluminium pouch with a dessicant.

32

One tube of specimen dilution buffer (0.5ml). The buffer solution contains buffer salts,

bovine serum albumin and preservatives.

10ul end to end capillary tubes.

The 10ul capillary tubes were used to take blood from the EDTA bottles and dropped into the

specimen dilution buffer tube. Sample was mixed into the buffer by inverting the tube upside

down repeatedly until the sample has completely transferred into the buffer and there is no

visible whole blood in the capillary.

The aluminum foil with the dipstick was opened carefully without touching the yellow dip area

at the lower part of the dipstick. The dipstick was used shortly after its removal from the pouch.

Specimen, if stored in the refrigerator was allowed to reach room temperature before testing. The

yellow dip area (lower end of the dipstick) was placed into the diluted sample and held there

until the liquid front entered the result area. The dipstick was removed from the solution and was

placed in a horizontal position.

The result was available in 5 minutes and lines appearing later than 5 minutes were ignored. A

red line (control line) appeared which confirmed correct performance of the test. If the control

line did not appear, the test is considered invalid and is repeated with another dipstick.

The results of the CRP are read off as follows114:

Control line with NO blue line- CRP<10mg/l

Control line with one blue line- CRP >10-40mg/l

Control line with 2 blue lines- CRP >40-80mg/l

33

Control line with 3 blue lines- CRP >80mg/l.

For ease of analysis, the results were categorised as follows:

A- Negative

B- >10 -40mg/l

C- >40-80mg/l

D- >80mg/l.

Babies who had values within the range of 10mg/l to >80mg/l fell into groups B- D and were

considered CRP positive.

34

SCHEMATIC REPRESENTATION OF CRP ASSAY PROCEDURE

FIGURE 1A: SCHEMATIC REPRESENTATION OF CRP ASSAY PROCEDURE

35

FIGURE 1B: SCHEMATIC REPRESENTATION OF CRP ASSAY PROCEDURE.

36

PROCALCITONIN ASSAY

For Procalcitonin a rapid diagnostic kit (one step procalcitonin test by Artron Laboratories

Canada)115 was used. It is a rapid and convenient immune chromatographic assay for the semi

quantitative detection of procalcitonin in human serum or plasma. Monoclonal antibodies

specifically against PCT are conjugated with colloidal gold and deposited on the conjugate pad

and immobilized in the test zone of the cassette. When an adequate volume of the test sample is

added, the gold antibody conjugate is rehydrated and the PCT, if any in the sample will interact

with the colloidal gold conjugated antibodies. The antigen- antibody colloidal gold complex

migrates towards the test window until the test zone where they are captured by immobilized

antibodies, forming a visible pink test line indicating a positive result. The colour intensity of the

band is directly proportional to the PCT concentration in the sample and is interpreted with a

reference card. This cassette can be used for serum or plasma. For PCT, values >0.5ng/ml was

taken as positive.

After the sample for CRP was taken, the remaining sample in the EDTA was centrifuged for

about 5mins to separate the plasma.

The testing device was removed from the sealed pouch and placed on a level surface. A sample

dropper (included in the pouch) was used to add three full drops (120ul) of the specimen into the

sample well marked with an arrow on the cassette.

The result was read in 30minutes and the colour blocks were compared with the interpretation

card included. If there was no visible band in the control region, the test was repeated with a

new device. Results were reported as:

<0.5ng/ml No T band – Negative. Systemic infections are unlikely.116

37

>0.5-<2ng/ml Intensity of the T band is stronger than colour block 0.5ng/ml, but weaker than

that of colour block 2. – Moderate risk for progression to severe infection.116

>2 -<10 intensity of the T band is stronger than colour block 2 but weaker than colour block 10.

High risk for progression to severe systemic infection.116

>10ng/ml the intensity of the T band is stronger than colour block 10ng/ml. High likelihood of

severe sepsis or septic shock.116 (See interpretation card in appendix VII)

38

SCHEMATIC REPRESENTATION OF PROCALCITONIN ASSAY PROCEDURE

39

CRP and PCT levels were analyzed by the researcher in the Paediatrics department laboratory.(

see appendix IV) At intervals, samples were also analysed by the laboratory scientist with a

different test kit to ensure accuracy and quality control.

The Blood culture samples were analysed by the microbiologist assisted by the researcher. The

researcher was trained on blood culture processing by the consultant microbiologist for 4 weeks

prior to commencement of the study.

The BACTEC Peds Plus™ / F culture vials were used. The vials are enriched with soybean-

casein digest broth and carbon dioxide and are for aerobic cultures. They are principally used

with the BACTEC(R) fluorescent series instruments for the qualitative culture and recovery of

aerobic microorganisms. These bottles require 1-3 mls of blood only which makes it a good

choice for neonates.

After the blood samples were collected, they were taken to the microbiology laboratory and

incubated in the BACTEC 9050 machine for a maximum period of 5 days. The BACTEC 9050

machine is a controlled environment where the vials are maintained at 370C. It is able to incubate

50 vials at the same time, and each vial is identified by its unique barcode. It continuously

agitates the vials, leading to an earlier detection of positives. Each culture vial has a sensor which

detects increase in carbon dioxide produced by the growth of microorganisms as they metabolise

the substrates present in the vial. The sensor is monitored by the instrument every 10 minutes for

an increase in its fluorescence, which is proportional to the amount of carbon dioxide present. A

positive reading indicates the presumptive presence of microorganisms in the vial. Bottles will be

checked for microbial activity evidenced by flagging of the machine. (This identifies the bottle

growing an organism). This usually occurs within 48-72 hours.

40

Bottles with growth were sub cultured on blood agar, chocolate agar and MacConkey agar and

organisms were further identified by the Microbact 24E oxoid identification system (This is a

semi-automated identification system). MacConkey agar is a differential medium and is useful

for enteric pathogens e.g Klebsiella, E.coli Blood agar is useful for Streptococcus species while

chocolate agar is useful for fastidious organisms e.g Haemophilus influenza and Neiserria

species.

A direct gram stain was done alongside the subcultures in the plate. This helps to rapidly identify

any organisms and rule out a contamination.

Positive isolates had antimicrobial susceptibility testing using disc diffusion method to determine

the antibiotic sensitivity pattern.

SOCIOECONOMIC CLASS (SEC)

Socio-economic class classification was done using the method described by Oyedeji117. Scores

from 1 to 5 were given for each criteria assessed. The criteria were maternal educational

attainment, maternal occupation, paternal educational attainment and paternal occupation. The

score from the 4 criteria were added and an average found by dividing by 4. The result

approximated to the nearest whole number gave the socio-economic class 1 to 5. For analysis,

SEC 1 and 2 were lumped together as the upper socioeconomic class; SEC 3 was regarded as

middle class while SEC 4 and 5 were put together as lower class.

41

DATA MANAGEMENT

Data was initially entered into a Microsoft excel spreadsheet (2010 version) then analysis was

done using the Epi InfoTM 2008 version 3.4.1 software.

Quantitative variables were presented as mean ± standard deviation in a normal distribution.

However, if the distribution was skewed, median and interquartile range were used to present the

data. Qualitative variables were presented as frequency tables and charts.

Association between two categorical variables were assessed using chi square statistics or

Fisher’s exact test when expected values in any of the cells is less than 5, while the difference in

mean was assessed using student t test statistics in two groups and Analysis of variance

(ANOVA/ F test) in > 2 groups.

Assessment of validity was by calculating sensitivity, specificity, positive predictive value and

negative predictive value. Calculation of validity followed the 2x2 table shown in table 1.

42

Table I: Assessment of validity

Sensitivity = a/ a+c

Specificity = d/ b +d

Positive predictive value (PPV) = a/ a+b

Negative predictive value (NPV) = d/c+d

Level of statistical significance was put as p < 0.05.

43

RESULTS

DEMOGRAPHIC CHARACTERISTICS OF THE STUDY POPULATION

A total of 120 hospitalised neonates were screened for the study out of which 105 neonates were

recruited. They all had blood culture and procalcitonin tests assayed. All babies also had 3 CRP

tests except for the 9 mortalities who had at least 2 tests done. The study population consisted of

72 (68.6%) preterm and 33(31.4%) term neonates. 54 of the subjects were males (51.4%) while

51 (48.6%) were females with a male:female ratio of 1.1:1. The postnatal ages ranged from 1

hour to 624 hours with a mean age (SD) of 78.05(202.2) hours. The estimated gestational ages of

the babies ranged from 27 to 43 weeks with a mean (SD) of 33. 5(4.3) weeks. Majority of the

study participants were between 27-36 weeks (68.5%) while 1.9% were 43 weeks. (See Table II).

The weight of the subjects at the time of recruitment was between 500 grams and 4100 grams

with a mean weight (SD) of 1939 (842.6) grams. One third of the subjects (30.5%) were Low

birth weight (LBW) while 14.3% were extremely low birth weight (ELBW). (See table II)

Most of the study participants were recruited from the New Neonatal Unit (54.3%) which is for

inborn neonates. Sixty (57.1%) babies were delivered through caesarean section while 45

(42.9%) were spontaneous vaginal delivery.

Amongst the 11 babies with confirmed sepsis, 8(72.7%) were males and 8 (72.7%) of these

babies were outborn. (See table III)

44

Table 1I SOCIODEMOGRAPHIC CHARACTERISTICS OF STUDY POPULATION

Variable Number (%) of subjects

N=105(100%)

MEAN (SD)

SEX

Male

Female

54(51.43%)

51(58.57%)

M:F ratio= 1.1:1

GESTATIONAL AGE (WEEKS)

27-30

31-33

34-36

37-39

40-42

43

26(24.7%)

21(20%)

25(23.8%)

27(25.7%)

4 (3.8%)

2(1.9%)

33.5(4.3)

WEIGHT (GRAMS)

<1000g (ELBW)

>1000g - <1500g (VLBW)

>1500g -2499g ( LBW)

>2500-4000g (NBW)

15(14.3%)

28(26.7%)

32(30.5%)

30(28.6%)

1939.1g(842.6g)

ELBW-Extremely Low Birth Weight, VLBW- Very Low Birth Weight, LBW-Low Birth Weight, NBW- Normal

Birth Weight.

45

Table III: Descriptive table on the distribution of sepsis by sex and place of birth.

VARIABLE Definite

SEPSIS

N= 11

PROBABLE

SEPSIS

N= 48

No

SEPSIS

N= 46

SEX

Male

Female

8 (72.7%)

3 (27.3%)

26 (54.1%)

22 (45.8%)

20 (43.5%)

26(56.5%)

Place of birth

Inborn

LUTH

Outborn

PRIVATE HOSP

GENERAL HOSP

TBA

HOME DELIVERY

3 (27.3%)

6(54.5%)

1(9.0%)

1 (9%)

0(0%)

25 (52.0%)

17 (35.4%)

3 (6.3%)

1 (2.0%)

2 (4.2%)

27 (58.6%)

11 (23.9%)

5 (10.4%)

2 (4.3%)

1 (2.0%)

TBA-Traditional Birth Attendant. LUTH-Lagos University Teaching Hospital

46

RELATIONSHIP BETWEEN SOCIAL CLASS AND CULTURE PROVEN SEPSIS

Social classification was done according to Oyedeji’s117 classification. Forty two (40%) of the

mothers were in social class III (see table IV). It was also found that culture proven sepsis was

highest in the lower social class 5(45.4%).

Table IV: Relationship between socioeconomic classification and culture proven sepsis

Social class culture proven sepsis

N 105(%) N 11(%)

Upper 35 (33.3%) 2(18.1%)

Middle 42 (40.0%) 4(36.3%)

Lower 28 (26.7%) 5(45.4%)

47

RISK FACTORS AND CLINICAL FEATURES OF SEPSIS

Among the risk factors for sepsis, prematurity was the highest with 72 babies (68.6%), while

maternal UTI was the lowest in 8 babies (3.8%) (See Fig 3). Respiratory distress was present in

72.4% of babies. (See fig 4)

Fig 3: Risk factors for sepsis in the subjects

48

Fig 4: Clinical features of sepsis in the subjects.

49

VALUES OF MARKERS FOR SEPSIS

Values of C- reactive protein

On presentation, 61(58.1%) babies had a negative CRP while 44(41.9%) had positive CRP.

At 24 hours, 63(60%) babies had negative CRP while 41(39%) had positive CRP.

At 72 hours, 67(63.8%) of the babies were CRP negative which was a 3.8% increase from the

number at 24 hours. However, 9(8.6%) of the subjects had died. (See table V)

Table V: Serial C-reactive protein values during the course of the admission

CRP(mg/l) Negative 10-40 40-80 >80

Presentation 58.1 25.7 7.6 8.6

24hrs 60 12.4 15.2 11.4

72hrs 63.8 14.3 5.7 6.7

Values are in percentage(%)

- -

50

PROCALCITONIN VALUES OF SUBJECTS AT PRESENTATION

Sixty one babies (58.1%) were procalcitonin negative on presentation, while 16 (15.2%) had

values greater than 10ng/ml on presentation. (See table VI)

Table VI: Procalcitonin values at presentation

PCT (N = 105) %

A NEGATIVE(<0.5ng/ml) 61 58.1

B 0.5-<2ng/ml 13 12.4

C >2-<10 ng/ml 15 14.3

D >10ng/ml 16 15.2

Key: PCT-Procalcitonin, N- Number, %-Percentage. A-Negative, B-0.5-<2mg/ml, C->2-

<10ng/ml, D->10ng/ml.

51

RELATIONSHIP BETWEEN MARKERS AND GESTATIONAL AGE.

Table VII denotes that CRP was positive in 18(25%) of the preterm babies. For the term babies,

CRP was positive in 33(100%). Similarly, PCT was positive in 11(15.3%) of the preterms and

33(100%) of the term babies. (See table VII).

Table VII: Relationship between markers and gestational age.

Variables CRP 1 Total PCT Total

GEST(Weeks) Neg Pos Neg Pos

Preterm 61(84.7) 11(15.3) 72(100.0) 61(84.7) 11(15.3) 72(100.0)

Term

Post term

0(0.0)

0(0.0)

31(100.0)

2(100.0)

31(100.0)

2(100.0)

0(0.0)

0(0.0)

31(100.0)

2(100.0)

31(100.0)

2(100.0)

Total 54(51.4) 44(41.9) 105(100.0) 61(58.1) 44(41.9) 105(100.0)

χ2 = 50.9 P = 0.000* χ2 = 66.7 P = 0.000*

52

RELATIONSHIP BETWEEN MARKERSAND ONSET TIME OF SEPSIS

The positivity rates were higher in late onset sepsis than early onset sepsis for both CRP and

PCT. (See table VIII)

Table VIII: Relationship between markers and onset time of sepsis

CRP PCT

SEPSIS ONSET Negative Positive Total Negative Positive Total

Early 50(63.3) 29(36.7) 79(100.0) 58(73.4) 21(26.6) 79(100.0)

Late 4(15.4) 22(84.6) 26(100.0) 3(11.5) 23(88.5) 26(100.0)

Total 54(51.4) 51(48.6) 105(100.0) 61(58.1) 44(41.9) 105(100.0)

χ2 = 17.9 P = 0.000* χ2 = 30.8 P = 0.000*

Key: CRP- C-reactive protein, PCT-Procalcitonin. *- Statistically significant

53

BLOOD CULTURE RESULTS AND MICROBIOLOGICAL PROFILE OF SUBJECTS

Blood culture results are as shown in figure 6. Eleven (10.5%) samples had positive growth on

blood culture while 89.5% of the neonates had a negative growth on blood culture.

Acinetobacter Baumanii, Eschericia coli, Klebsiella, Streptococci and Methycillin resistant

staphylococcus aureus (MRSA) were isolated from two neonatal samples each while Coagulase

negative Staphylococcus(CONS) was isolated from one sample.

Table IX: BLOOD CULTURE RESULTS

BLD CULTURE ORGANISM N = 105 % = 100.0

A.BAUMANII 2 1.9

CONS 1 1.0

E.COLI 2 1.9

KLEBSIELLA 2 1.9

STREPTOCOCCI 2 1.9

MRSA 2 1.9

NEGATIVE 94 89.5

54

Relationship between CRP, PCT and positive blood cultures.

CRP was positive in 11(100.0%) neonates with positive cultures, and in 40 (42.6%) neonates

with negative cultures. (See table X). PCT was positive in 11(100.0%) neonates with positive

growth on blood culture, and also positive in 33(35.1%) neonates with a negative blood culture.

55

Table X: Relationship between serial CRP, PCT and blood culture of subjects

Blood Culture

Test Positive Negative χ2 P value

CRP 1

Positive 11(100.0) 33(35.1) 17.0 <0.001

Negative 0(0.0) 61(64.9)

Total 11(100.0) 94(100.0)

CRP2

Positive 9(81.8) 32(34.4) 9.3 0.003*

Negative 2(18.2) 61(65.6)

Total 11(100.0) 93(100.0)

CRP3

Positive 7(70.0) 21(24.7) 8.8 0.006*

Negative 3(30.0) 64(75.3)

Total 10(10.5) 85(89.5)

PCT

Positive 11(100.0) 33(35.1) 17.0 <0.001

Negative 0(0.0) 61(64.9)

Total 11(100.0) 94(100.0)

CRP- C-Reactive protein. CRP1- on admission. CRP2- 24hours of admission CRP 3- at 72hours.

PCT-Procalcitonin on admission.* Statistically significant

56

The sensitivity of CRP 1 was 100%, Specificity was 65%, Positive and negative predictive

values were 25% and 100% respectively. (See table XI).

The sensitivity, specificity, positive and negative predictive values of PCT were 100%, 65%,

22% and 100% respectively. (See table XI)

Based on the results of the CRP, PCT and blood culture, babies could be said to fall into 3

groups as stated earlier based on the classification used by White et al.111 Eleven (10.5%)

neonates were found to have definite sepsis, 48(45.7%) had probable sepsis while 46(43.8%) had

no sepsis.

57

Table XI: Assessment of validity of the markers.

Sensitivity Specificity PPV NPV

CRP1 100% 65% 25% 100%

CRP2 82% 66% 22% 97%

CRP3 70% 75% 22% 96%

PCT 100% 65% 25% 100%

Key: CRP- C reactive protein. PCT- Procalcitonin, PPV- Positive Predictive Value, NPV-

Negative Predictive Value.

58

Relationship of CRP and PCT to outcome of study participants

Of the 105 subjects, 1(1.0%) discharged against medical advice (DAMA) due to financial

constraints. 15(14.3%) babies died during the admission out of which 10 babies died within 72

hours of admission. Of the 15 mortalities, 2(13.3%) had CRP >80mg/l at presentation. Also

amongst the mortalities were 9 (60%) babies who had PCT >10ng/ml on presentation (See table

X)

59

Table XI : Relationship between CRP and PCT with outcome of care

VARIABLE NUMBER(%AFFECTED

CRP 1 DAMA DEAD ALIVE P-VALUE

A. 0(0.0) 5(33.3) 56(62.9) χ2 = 14.0 0.1222

B. 1(100.0) 4(26.7) 22(24.7)

C. 0(0.0) 4(26.7) 4(4.5)

D. 0(0.0) 2(13.3) 7(7.9)

Total 1(100.0) 15(100) 89(100.0)

CRP 2

A. 0(0.0) 1(6.7) 62(69.7) χ2 = 35.3 0.0001

B. 0(0.0) 2(13.3) 11(12.4)

C. 0(0.0) 7(46.7) 9(10.1)

D. 1(7.7) 5(33.3) 7(7.9)

Total 1(100.0) 15(100.0) 89(100.0)

CRP 3

A. 0(0.0) 1(14.2) 66(74.1) X2 = 48.5 0.0000

B. 0(0.0) 0(0.0) 15(16.9)

C. 0(0.0) 1(14.2) 5(5.6)

D. 0(0.0) 5(71.4) 2(2.2)

Total 0(0.0) 7(100.0) 89(100.0)

PCT

A. 0(0.0) 1(6.7) 60(67.4) X2 = 41.8 0.0000

B. 0(0.0) 1(6.7) 12(13.5)

C. 1(100.0) 4(26.7) 10(11.2)

D. 0(0.0) 9(60.0) 7(7.86)

Total 1(100.0) 15(100.0) 89(100)

Key: CRP 1-C reactive protein on presentation, CRP 2- at 24hours, CRP 3- at 72 hours.

CRP A- Negative, B-10-40mg/l, C- 40-80mg/l, D- >80mg/l.

PCT- PROCALCITONIN PCT- A- Negative, B-0.5-<2mg/ml, C>2-<10ng/ml, D>10ng/ml.

60

DISCUSSION

This study aimed to compare the efficacy of serial CRP and PCT as screening markers of

neonatal bacterial sepsis.

CRP and PCT were compared with blood culture which is widely reported as the gold standard

in the diagnosis of NNS. In the present study, the incidence of culture proven sepsis was low

(10.5%). This is similar to the findings of Iroha et al43 also at LUTH. The reason for this low

yield may be because 93% of the subjects in this study were born in a hospital environment with

better hygiene practices than the non- hospital environment. However, Boma West et al71 in

2012 in Port- Harcourt Nigeria had an incidence of 41.6% which may have been because of a

much larger neonatal sample size in addition to a high number of outborn subjects. Naher et al118

in Dhaka, Bangladesh reported a low incidence of 6% which they attributed to sample collection

after commencing antibiotics and faulty sample collection procedure. It has been generally

reported that the yield of blood culture is generally poor, and serial blood culture sampling

which can be used to improve yield is not practicable in the newborn due to low blood volumes

and difficulty in collecting adequate samples.119

In this study, CRP correctly identified 100% of the babies with culture proven infection and

42.6% of babies with probable infection. This is higher than the results of West et al30 in Port

Harcourt, Nigeria who found that CRP correctly identified 74% babies with culture proven

(definite) infection. This difference may be because the kits used in this study were semi-

quantitative and may be more sensitive than the qualitative assay used by West et al30. West et

al30 opined that that this finding would mean that one out of every four neonates with sepsis will

be missed which is unacceptably high considering the devastating effects of untreated neonatal

61

sepsis. This finding by West et al30 is one of the reasons why serial CRP sampling is preferred.

Ibeh et al22 in Benin found CRP levels in proven and probable sepsis to be as high as 130.1mg/l

and 96.7mg/l respectively. Sucilathangam et al93 in India also found high levels of CRP in

proven and probable sepsis. Similarly, Benitz et al19 found that proven or probable sepsis was

strongly correlated with elevated CRP levels.

PCT levels in this study were also found to be elevated in neonates with definite infection and

probable infection. This finding is similar to findings from studies done by Arowosegbe et al103

and Ibeh et al22 in Nigeria. Sucilathangam et al93 also found that PCT levels were remarkably

high in neonates with definite and suspected sepsis. In their study, in some cases of culture

positive sepsis, other sepsis screening tests (e.g White cell count) were negative but PCT was

elevated. Furthermore, this present study found that 45.7% of the subjects had probable sepsis

and their CRP and /or PCT were elevated, alongside risk factors and clinical features of sepsis,

however they had negative blood cultures. Arowosegbe et al also described a similar pattern

whereby 21.2% of babies with clinical features and positive PCT had negative blood cultures.

Other than sepsis, this pattern may also be seen in babies with clinical findings like respiratory

distress and intracranial haemorrhage which can also trigger off the inflammatory response.

These findings re –emphasize previous postulations that blood culture can remain negative

despite bacterial sepsis.27,107,118

CRP was found to be significantly positive in the term and post term babies, compared to the

preterm babies. The explanation for this may be the same as adduced by Hofer et al32 who also

reported lower CRP response to infection in preterms and suggested that reasons for this may

include the use of more frequent prophylactic antibiotics in the mothers of the preterm babies

and timing of blood sampling which is usually earlier in the preterm newborns.32 They also

62

stated that CRP is thought to play a role in innate immunity, as an early defense system against

infections and as far as the endogenous immune response depends on the gestational age, CRP

responses may be lower due to the preterms less mature immunological system.32

Similarly, Kawamura et al120 also reported a lower sensitivity of CRP in preterms compared to

term newborns.

For PCT, 100% of the term babies had positive PCT compared to 15.3% of the preterms. This

finding may be due to the fact that PCT is thought to rise slower in the preterm, possibly because

of their immature immune system. Liu et al in China studied physiological changes in

procalcitonin concentration in neonates and found that serum PCT of premature infants will

remain at relatively low levels within 36 hours after birth while term babies peak within 24

hours.121

Nevertheless, Arowosegbe et al103 in Nigeria found that PCT seemed equally accurate for the

diagnosis of neonatal sepsis in both preterm and term neonates. This is similar to the findings of

Savagner et al10 and Kocabas et al27 in Turkey. The conclusion from this study is that there is a

significant difference in the reaction of procalcitonin in term and pre term babies.

The relationship between CRP, PCT and onset time of sepsis denotes that CRP was found to be

significantly positive in 84.6% of newborns with late onset sepsis (LOS). This may be because

the babies have started building up some innate immunity which is important in CRP response.

In Ife, Nigeria Ako- Nai et al26 found no difference in the rate of CRP elevation in early and late

onset sepsis. However, they studied only 30 subjects and so their findings may not be very

representative of the general populace. Naglaa et al104 using categorization of EOS to be < 7

days and term babies only found CRP to be significantly higher in early onset sepsis. The

63

importance of stating their criteria for onset time of sepsis is that after 72 hours, other researchers

would have classified the babies as having late onset sepsis. Benitz et al19 however found CRP to

be more sensitive in late onset sepsis (<72 hours) and suggested that this may be because their

infections had been present for a longer time.

PCT in this study was also significantly more sensitive in late onset sepsis. PCT was positive in

26.6% of early onset sepsis and 88.5% of late onset sepsis. The reason for this is not immediately

known. However, Arowosegbe et al103 in their study found no difference in PCT performance in

early and late onset sepsis. In contrast, Naglaa et al104 found PCT to be more sensitive in early

onset sepsis, but as has been stated above , their definition of early onset sepsis differs from the

generally accepted 72 hours, and some of the babies may actually have late onset sepsis.36 The

conclusion from this study however, is that procalcitonin is more sensitive in late onset sepsis.

The relationship of social class with sepsis has also been well documented. In our study, 73.3%

of the neonates were in the upper and middle class while 26.7% were from the lower social class.

This may be attributed to the fact that in Lagos state, free healthcare for children under age 5 is

provided in state owned facilities so more people of lower socioeconomic status will likely

patronize those centres more than a fee paying centre like LUTH. However, amongst the

neonates with culture proven sepsis, 45.4% were from the low socio economic status. Other

workers in Nigeria found a significant relationship between culture proven sepsis and social

class.39,122 This is in consonance with the results of Javed et al123 in Pakistan who also found that

mothers belonging to lower social income groups and significantly, mothers with primary

education had more neonates with sepsis than mothers with secondary education. This

association of social class with proven sepsis may be because mothers of lower social income

64

groups are more likely to deliver in unsterile environments, and have poorer health seeking

behaviours than those of the upper class.

In terms of place of delivery and the incidence of culture proven sepsis, a high proportion of the

outborn babies (72.5%) had confirmed sepsis. Adejuyigbe et al103 documented a similar finding

with 72.7% of the subjects with culture proven sepsis being outborn. Mukuolu et al in their study

in Ilorin and West et al56 in Port Harcourt had similar findings. This pattern is not surprising as

most deliveries outside government owned hospitals, or well-equipped private hospitals are

usually done in very unsterile environments.

The overall sensitivity of CRP in this present study was found to be 100%, and NPV was 100%.

However, West et al30 in Port Harcourt, Nigeria got sensitivity of 74% in their study. The

difference in results may be due to the methods of assay used. In our study, a semi quantitative

assay method was used which may have a higher sensitivity than the qualitative assay used by

West et al. However, Nuntnarumit85 in Bangkok, using a quantitative assay got an overall

sensitivity, specificity, PPV and NPV of 100%,94%, 91.6% and 100% respectively. These values

were similar to the results of the present study.

C reactive protein levels are elevated in only 35% to 65% of neonates at the onset of the

illness.19 Recognising that there is a delay of at least several hours due to the cascade of events

which leads to the elevation of CRP (activation of neutrophils, production of interleukin-6 and

induction of hepatic synthesis of CRP) led to an initial criticism of this test as having insufficient

sensitivity to guide therapy.19 However, with further research, Phillip et al124 noted that CRP

levels are consistently elevated 24 to 48 hours after the onset of infection and suggested that

serial normal levels may indicative of absent bacterial infection.124 In this study the sensitivity of

65

CRP 1 was higher than CRP 2 and 3. This is in contrast to the findings of Benitz et al19 who

demonstrated increasing sensitivity (35%,78.9% and 88.9%) over 3 days in both preterm and

term babies. This pattern was also found by other researchers.31,85,120 The reason for this

difference in pattern may be that with response to antibiotic therapy, CRP levels which were

initially elevated will start returning to normal range. Hindocha et al125 in their study also

observed this. However, the negative predictive values (NPV) were 67%, 88% and 83%

respectively which is similar to the pattern observed by other researchers.19 Similar results were

also obtained by Lee et al21. Khashabi et al102 in Iran studied 110 patients out of which 91 had

negative CRP and blood culture. The overall NPV of serial CRP was used as a guide to

discontinue antibiotic treatment. They reported that only one patient of this cohort was re-

admitted after 21 days of discharge with symptoms unrelated to the initial admission. Mathai et

al 126 in India concluded that serial testing of CRP may not be of much use in diagnosis but may

help in identifying uninfected babies. Based on the findings from this study, it may also be

concluded that serial negative testing of CRP will be more useful in identifying uninfected

babies.

In this study, at a cut off value of 0.5ng/ml, the sensitivity and NPV of PCT were both 100%. In

their studies, Arowosegbe et al103 in Abeokuta and Ibeh et al22 in Benin, Nigeria also got high

values in these validity tests. Arowosegbe used a semi quantitative point of care test, similar to

what was used in this study while Ibeh et al used the quantitative Kryptor® immunoassay random

assess analyser. The PPV and NPV from Arowosegbe et al were 84% and 83%, while Ibeh et al

reported 91.6% and 80%. The moderate difference between both methods of assay further

buttresses the fact that in centres with minimal resources who cannot afford quantitative

analysers, point of care kits could be used. Similarly, Suciathangam et al93 in India using a

66

quantitative immune- luminometry method got 92.8%,75%,59% and 96% for sensitivity,

specificity, NPV and PPV respectively. They studied a similar cohort of subjects with this study

and concluded that PCT done on the day of admission can help in early diagnosis of neonatal

sepsis.93 Furthermore, Kocabas et al27 in Turkey using a quantitative assay got sensitivity and

NPV of PCT to be 100% . \

The results from the present study showed that 93.3% of the 15 mortalities showed progressively

rising CRP values (See table XI). Among the mortalities in this study, CRP levels progressively

rose to >80mg/l over 72 hours getting to such high levels in 71.4% of the mortalities at 72hours.

This is similar to Lai et al127 in their study at Taiwan who found that high CRP levels were

significantly associated with more severe clinical symptoms and worse outcomes. They also

found that more mortalities (13.6%) occurred in the groups with CRP >100mg/l compared to

4.9% in the low CRP group.127 Oberhoffer et al128 also postulated that high levels of CRP on

admission may predispose to a fatal outcome in patients with sepsis.

Similarly, PCT values in neonatal sepsis may be a good predictor of mortality. This study shows

that 93% of the mortalities had PCT levels of >2mg/ml to >10 ng/ml. Of this number, 60% had

PCT levels of >10ng/ml which is indicative of a high likelihood of severe sepsis or septic shock.

This is in agreement with Arowosegbe et al103 in Abeokuta, Nigeria as 96.3% of the mortalities

in their study had elevated PCT. Adib et al109 and Suciathangam et al93 also found that PCT

correlated well with the progression and severity of infection and is useful for prognostication.

The results from this study have shown that PCT on the day of presentation has a high

sensitivity and NPV and is useful in early diagnosis of neonatal sepsis and prognostication.

67

Similarly, CRP which also has equal sensitivity with PCT and a good NPV can also be used in

making early diagnosis of neonatal sepsis.

68

CONCLUSION

1. The levels of CRP and PCT rise in the presence of neonatal bacterial sepsis.

2. Both CRP and PCT perform better as markers in the term babies and in late onset sepsis.

3. Serial negative CRP in a neonate may confidently rule out the possibility of a septicaemic

illness.

4. PCT has a high sensitivity and NPV for sepsis if done within 24hours.

69

RECOMMENDATIONS

The present recommendations are made based on the results and conclusions from the present

study:

1. CRP and PCT should be routinely used in our newborn nurseries as an adjunct to blood

culture for the screening of newborns with risk factors and / or clinical features of sepsis.

2. Serial CRP measurements done over 72 hours in conjunction with clinical and other

laboratory parameters can be used to facilitate early decision on discharge for babies with

suspected sepsis.

70

LIMITATION

1. A quantitative assay using nephelometry method would have been a more objective

assessor of the values of CRP and PCT. However, the cost of doing this was out of the

researchers reach.

71

AREAS FOR FUTURE RESEARCH

1. A multi-center study on these markers should be carried out. This will help to obtain a

larger sample size which would be more reflective of the general population, and

subsequently nationally accepted protocols for the management of this group of newborns

can be formulated.

2. Research comparing the use of point of care kits in comparison with quantitative methods

of assay can be conducted.

3. A research can also be conducted on using serial CRP testing to discontinue antibiotic

therapy in the newborn unit. It would be interesting to find out how it impacts on cost of

care, service delivery and patient satisfaction.

72

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86

APPENDIX I

ETHICAL APPROVAL 1a

87

APPENDIX I

EXTENDED ETHICAL APPROVAL 1B

88

APPENDIX I1

INFORMED CONSENT AND CONSENT FORM (Subjects)

“To evaluate serum procalcitonin and serial C-reactive protein as screening markers for

neonatal bacterial sepsis at the Lagos University Teaching hospital.”

Name and affiliation of researcher.

This research is being conducted by Dr Matthew-Okore Amalachukwu, a senior registrar in the

Department of Paediatrics, Lagos University Teaching Hospital, Idi-Araba, Lagos State.

Sponsor of Research

This research is sponsored by Dr Matthew-Okore A

Purpose of Research

The purpose of this research is to find out if serial C reactive protein is comparable to serum

procalcitonin as markers of neonatal sepsis in LUTH.

Procedure of the Research “To compare serum procalcitonin and serial C-reactive protein

as screening markers for neonatal bacterial sepsis at the Lagos University Teaching

hospital Idi - Araba.”

On admission, blood samples will be taken under strict asepsis using sterile gloves and injection

packs. 1ml will be collected in an EDTA bottle for serum procalcitonin and CRP in subjects. A

repeat sample for CRP will be collected at 24 hours and 72 hours in the subjects.

The BACTEC blood culture systems will be used for blood culture. 2mls of blood will be

collected.

89

Expected duration of research and of participant(s)’ involvement

The research is expected to last four (4) months. You will be interviewed on admission and small

quantity of blood sample will be collected thrice.

Risks

The study will not cause any physical harm to your child. There will be slight pain when taking

the blood sample from the vein but this will resolve with pain reliever.

Cost Implication to the participants

Participation in this research will be free of any financial cost to the parents/guardian.

Benefits

Studies have shown that use of early markers for diagnosis of neonatal sepsis can reduce hospital

stay and reduce costs in management of neonatal sepsis. This study seeks to evaluate serial CRP

and procalcitonin to determine which of the markers that could be adapted to our environment.

Confidentiality

Any information you provide will be treated with utmost confidentiality.

Your child’s name or any identifier will not be used in any publication or report from this study.

Voluntariness

Your participation in this study is entirely voluntary.

Alternatives to Participation

90

Your refusal to participate in the study will not in any way affect the healthcare services you

would receive in this hospital.

Due Inducement

If you are requested to come after clinic hours, you will be compensated for cost of

transportation to and from research site but you will not be paid any dues for participating in this

research.

Consequences of participants’ decision to withdraw from research and procedure for

orderly termination of participation.

You are free to withdraw your participation at any time in the study even if you had earlier

agreed but changed your mind.

Your decision will not affect your ability to receive medical care or lose any benefit to which

you would, otherwise, be entitled.

Modality for providing treatment and action(s) to be taken in case of injury or adverse

event(s):

If you suffer any injury as a result of your participation in the research, you will be treated in the

Lagos University Teaching Hospital and the research will bear the cost of treatment.

What happens to the research participants when the research is over?

During the research, you will be given any information that may affect your continued

participation or your health. There is no expected negative effect on participant at the end of

91

research. If your child is identified with neonatal sepsis, he or she would be treated by the

neonatal unit.

Statement about sharing of benefits of this research:

The results of the tests will be communicated to you. And may be helpful in your treatment by

your doctors.

Statement of person obtaining informed consent:

I have fully explained this research to ………………………………………………………. and

have given sufficient information, including risks and benefits, to make an informed decision.

DATE:__________________ Signature_____________________

Name_________________________________

Statement of the person giving consent:

I have read the description of this research or have had it translated into language I understand

and I have also talked it over with the doctor to my satisfaction. I understand that my

participation is voluntary. I know enough about the purpose, methods, risks and benefits of the

research to judge that I want to take part in it. I understand that I may freely stop being part of

this study at any time. I have received a copy of this consent form and additional information

sheet to keep for myself.

Date_______________ Signature __________________

Name____________________________Name of parent/guardian _____________________

92

Signature_____________ Date_______________________

Researcher’s Contact:

Dr Matthew-Okore Amalachukwu;

Department of Paediatrics, Lagos University Teaching Hospital, Idi-Araba, Lagos.

Telephone mobile: 08033941972

Email: doctorminix@yahoo.com

Institutional Health Research Ethics Committee’s Contact:

LUTH Health Research Ethics Committee;

Room 107, Administrative Building, LUTH, Idi-Araba, Lagos.

93

APPENDIX III

STUDY PROFORMA

Neonatal data

Identification/ Personal data

Serial No …………………………… Interview date …………………………

Date of birth …………………….

Sex Male Female

Age ……………………………………..

Gestational age……………… APGAR Score ………………Birth weight ……………

Ballards score ………………………..Address ……………………………

Tribe Hausa Yoruba Igbo

Religion Christianity Islam Traditional Others.

Educational level of parents: Father Mother

Post graduate

University graduate/equivalent

SSCE/GCE+ Professional training

94

SSCE/GCE+ Grade teacher training

SSCE/GCE

JSS completed

Primary school

No formal education

Occupation of parents/ care giver

Senior public servant/Professional/manager

Large scale trader/Contractor

Intermediate public servant

JSS teachers/clerk/typist/artisan

Petty trader/laborer/messenger

Unemployed /full time house wife/student

MATERNAL DATA

Age

LUTH Patient Yes No

Mode of delivery SVD Caesarean section Others (Specify).........

Place of delivery Govt hosp Private hosp TBA Home delivery

95

RISK FACTORS FOR SEPSIS

Maternal factors

Chorioamnionitis Prolonged labour Maternal UTI

PROM > 18hours

Maternal pyrexia (>380C) Meconium stained liquor

NEWBORN FACTORS

Prematurity

Very Low birth weight

Sustained Fetal tachycardia (>180beats/min)

Multiple gestation

CLINICAL EVALUATION

Fever

Poor suck

Seizures

Hypothermia / Hyperthermia

Respiratory distress

Abdominal distension

Skin mottling/ sclerema

96

Lethargy

Others (please indicate)………………………………………..

Samples taken CRP PCT Blood culture Time of collection

97

APPENDIX IV

RESEARCHER AT WORK

98

APPENDIX V-

POSITIVE PROCALCITONIN CASSETTES.

99

APPENDIX VI

NEGATIVE PROCALCITONIN CASSETTES.

100

APPENDIX VII

Artron PCT Interpretation card

101

APPENDIX VIII

POSITIVE CRP STRIPS

102

APPENDIX IX

POSITIVE (CLOUDY) BLOOD CULTURE BOTTLES