Assessment of Immunity to Diphtheria in Mothers And

ASSESSMENT OF IMMUNITY TO DIPHTHERIA IN MOTHERS AND

THEIR NEWBORNS AT THE UNIVERSITY OF BENIN TEACHING

HOSPITAL, BENIN CITY.

A DISSERTATION SUBMITTED TO THE NATIONAL POST-GRADUATE

MEDICAL COLLEGE OF NIGERIA IN PART FULFILMENT OF THE

REQUIREMENTS FOR THE AWARD OF THE FELLOWSHIP OF THE

COLLEGE IN PAEDIATRICS.

DR CUMMINGS HENRY

MB, BS (ILORIN) 2001.

MAY, 2012

Declaration

I hereby declare that this dissertation is original unless otherwise acknowledged. It has not been submitted to any college for the purpose of a fellowship examination.

Signature: ……………………………………………………

Date:………………………………………………………….

Name of Candidate: Dr CUMMINGS Henry

Certification

We certify that Dr CUMMINGS Henry of the Department of Child Health,

University of Benin Teaching Hospital, Benin City, prepared this dissertation under our supervision.

FIRST SUPERVISOR: DR (MRS) A. E. SADOH

STATUS: CONSULTANT PAEDIATRICIAN/ SENIOR RESEARCH

FELLOW

ADDRESS: DEPARMENT OF CHILD HEALTH, UBTH, BENIN CITY

SIGNATURE: ……………………………………………………….

DATE:……………………………………………………….

SECOND SUPERVISOR: DR (PROF.) O. OVIAWE

STATUS: CONSULTANT PAEDIATRICIAN

ADDRESS: DEPARTMENT OF CHILD HEALTH, UBTH, BENIN CITY

SIGNATURE: ……………………………………………………….

DATE:………………………………………………………………..

iii

DEDICATION

This book is dedicated to all children who have suffered from diphtheria.

Then to my wife, Mrs. Omonigho Cummings and daughter, Efemena for their unwavering support.

ACKNOWLEDGEMENT

To God be the glory for the journey so far. I am heartily thankful to my supervisors- Professor O. Oviawe and Dr (Mrs) A.E. Sadoh for their guidance and support. My appreciation also goes to my teachers – Professors Ibadin M.O, Okolo A.I, Omoigberale A.I, Eregie C.O, Drs Sadoh W.E, Ofovwe G.E, Onyiriuka A.N, Odunvbun M, Iduoriyekemwen N, and to all my consultants; words cannot express the values you have invested in me.

To Dr A. Ande of the feto-maternal unit, Obstetrics and Gynaecology department, UBTH, thank you for your valuable support and contribution. To all mothers and their babies who made this work possible, I am eternally grateful. My gratitude also goes to Mr A.O. Oladipo and Dr E.B. Etenobo who assisted in the laboratory aspects of the study.

Finally, I wish to express my loving thanks to my wife and daughter for their understanding and encouragement.

Table of Contents

Title page……………………………………………………………. i

Declaration………………………………………………………….. ii

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

Dedication…………………………………………………………... iv

Acknowledgment…………………………………………………… v

Table of contents…………………………………………………… vi

List of Tables……………………………………………………….. vii

List of Figures………………………………………………………. viii

List of Abbreviations……………………………………………….. ix

Definition of terms…………………………………………………. .x

Summary……………………………………………………………. xii

Introduction ………………………………………………………… 1

Literature review ………………………………………………….... 4

Justification of study ………………………………………………. 49

Aims and Objectives ………………………………………………. 51

Subjects and Methods………………………………………………. 52

Data analysis ……………………………………………………….. 65

Results……………………………………………………………… 67

Discussion…………………………………………………………... 79

Conclusion………………………………………………………….. 85

Recommendation…………………………………………………… 86

Limitation of study…………………………………………………. 87

Future line of study………………………………………………… 88

References ………………………………………………………….. 89

Appendices ………………………………………………………… 102

vii

LIST OF TABLES Page

Table I: Socio-demographic characteristics of the subjects (mothers) 68

Table II: Characteristics of babies 69

Table III: Immunity status for diphtheria in mother-baby pairs 70

Table IV: Association between babies’ characteristics and their anti-

diphtheria immunity 71

Table V: Association between maternal age groups and

anti-diphtheria immunity in mother- baby pairs 74

Table VI: Association between maternal vaccination status and

anti-diphtheria immunity in mother- baby pairs 75

Table VII: Association between maternal ethnicity and anti-diphtheria

immunity in mother-baby pairs 76

Table VIII: Association between maternal place of abode and anti-

diphtheria immunity in mother-baby pairs 76

Table IX: Association between household size and anti-diphtheria

immunity in mother-baby pairs 77

Table X: Association between family socio-economic status

and anti-diphtheria immunity in mother-baby pairs 78

viii

LIST OF FIGURES Page

Figure 1: Age distribution of mothers 67

Figure 2: Prevalence of protective immunity in mother-baby pairs 70

Figure 3: Relationship between antibody titres in mother-baby pairs 72

List of Abbreviations

ANC - Antenatal care ANOVA - Analysis of variance CI - Confidence interval df - degree of freedom DT - Diphtheria-tetanus vaccine (paediatric type) dT - Diphtheria-tetanus vaccine (adult type) DPT - Diphtheria-pertusis-tetanus vaccine D & D - Dubowitz and Dubowitz gestational age estimation chart EIA - Enzyme immunoassay ELISA - Enzyme-linked immunosorbent assay EPI - Expanded Programme on Immunization IgG - Immunoglobulin G IM - Intramuscular IU - International unit IV - Intravenous Kg - kilogram LMP - Last menstrual period ml - Millilitres

NIS - Newly Independent States NPI - National Programme on Immunization OD - Optic density “r” - Pearson correlation coefficient r2 - Regression Tdap - Tetanus-diphtheria-accellular pertusis vaccine UBTH - University of Benin Teaching Hospital UK - United Kingdom USA - United States of America WHO - World Health Organization

Definition of Terms

Diphtheria: An infectious disease due to the bacterium Corynebacterium diphtheriae and its highly potent toxin; characterized by the formation of a highly adherent pseudomembrane on the mucous membrane of the nose, pharynx and sometimes the tracheobronchial tree.1

Infectious disease: A disease capable of being transmitted from person to person, with or without actual contact.1

Infection: Invasion of the body with organisms that have the potential to cause disease.1

Newborn: Synonymous with neonate; refers to an infant aged 28 days or younger.1, 2

Antibody: An immunoglobulin molecule produced by B-lymphoid cells that combines specifically with an antigen.1

Immune: Free from the possibility of acquiring a given infectious disease.1

Immunity: The status or quality of being immune.1

Active immunity: Resistance to disease resulting from previous exposure of an individual to an infectious agent or antigen.1

Passive immunity: Immunity acquired through transfer of antibodies from another person or animal, either naturally, as from mother to fetus, or by intentional inoculation.1

Toxin: A noxious or poisonous substance that is formed or elaborated either as integral part of the cell or tissue (endotoxin), as an extracellular product (exotoxin),

xii or as a combination of the two, during the metabolism and growth of certain microorganisms and some higher plants and animal species.1

Toxoid: A toxin that has been treated (commonly with formaldehyde) so as to destroy its toxic property but retain its antigenicity, that is, its capability of stimulating the production of antitoxin antibodies and thus of producing an active immunity.1

Vaccine: Any preparation intended for active immunologic prophylaxis.1

Vaccination: The act of administering a vaccine.1

Immunization: Protection of susceptible individuals from communicable diseases by administration of a vaccine.1

Booster dose: Additional dose given at some time after an initial dose to enhance the effect of the initial dose.1

Epidemic: The occurrence in a community or region of cases of an illness, specific health-related behaviour, or other health-related events clearly in excess of normal expectancy.1

Endemic: Denoting a temporal pattern of disease occurrence in a population in which the disease occurs with predictable regularity with only relatively minor fluctuations in its frequency over time.1

xiii

SUMMARY

Immunity to diphtheria has been noted to wane with age such that a significant proportion of women of reproductive age have been reported to have inadequate levels of immunity to diphtheria. Thus, it is envisaged that a significant proportion of newborns may inherit inadequate levels of immunity to diphtheria from their mothers. To evaluate this hypothesis, a cross-sectional analytical study was carried out to determine the prevalence of the different levels of anti-diphtheria immunity in mothers and their newborns at the Labour ward of the University of Benin Teaching Hospital (UBTH), Benin City. The study further evaluated the relationship between anti-diphtheria antibody titres in mother-baby pairs as well as associations between some socio-demographic factors and the levels of anti- diphtheria immunity in mothers and their babies.

Antibody titres of two hundred and thirty one mother-baby paired sera were assayed using Enzyme-linked immunosorbent assay (ELISA) technique. Data was analyzed using Statistical package for Social sciences (SPSS) version 18.0 with level of significance set at 0.05 or less at 95% confidence interval.

As much as 29.9% of both mothers and their babies had no protection (antibody titre < 0.01IU/ml) from diphtheria. Ninety (39.0%) mothers and 107 (46.3% ) babies were inadequately protected (antibody titre < 0.1IU/ml) from diphtheria. The difference in the mean antibody titres of mothers and babies was not found to be statistically significant (p= 0.12). There was a strong positive linear correlation between maternal and newborn antibody titres (“r” = 0.982, p= < 0.0001), such that, as mothers antibody titres increased, those of babies also increased in a linear pattern. Mothers who reside in urban settings had

xiv significantly lower mean anti-diphtheria antibody titre 0.21±0.27IU/ml than the mean 0.42±0.36IU/ml of those who reside in rural settings (p = 0.01). Similarly, babies of mothers from rural settings had a significantly higher mean titre 0.35±0.30IU/ml than the mean titre 0.17±0.23IU/ml of babies born to mothers from urban settings (p = 0.01). However, other socio-demographic factors such as maternal age, maternal childhood vaccination history, maternal ethnicity and the household size and socio-economic class did not significantly affect the levels of protection to diphtheria in mothers and their babies.

This study has shown that a significant proportion of mothers and their newborns are inadequately protected from diphtheria. Thus, vaccination of parturient women with booster doses of diphtheria toxoid vaccine may be indicated.

INTRODUCTION

Diphtheria, a Greek word for leather, refers to a specific infectious disease due to the bacterium Corynebacterium diphtheriae and its highly potent toxin.3 The disease is marked by severe inflammation that results in the formation of a membranous coating, due to the release of thick fibrinous exudates on the mucous membrane of the pharynx, the nose, and sometimes the tracheobronchial tree.3 Furthermore, the toxin produced by the bacterium leads to degeneration of peripheral nerves, heart muscle and other tissues such as the conjunctiva, external auditory canal and the genital tract.1,3

Diphtheria was one of the earliest infectious diseases that were controlled on the basis of principles of microbiology, immunology and public health.3 As a result of effective vaccination campaigns, diphtheria was reduced from a major cause of childhood death in the western hemisphere in the early 20th century to a medical rarity (less than 2 cases per annum).3, 5 However, this dramatic change did not occur in many parts of developing countries6, as diphtheria is still thought to be endemic in these regions. For example, Nigeria reported more than 2000 cases per annum within the last decade.7 More recently, re-emergence of diphtheria has been noted in several countries in Eastern Europe as well as Russia and other parts of the former Soviet Union.8-10 Outbreaks have also been recorded in Afghanistan, Lesotho, Sudan, India and Algeria.11-15 There has not been any recent report of diphtheria epidemic in most parts of sub-Saharan Africa. However, recent reports of clusters of diphtheria cases in Katsina and Edo states, led to speculations of possible resurgence of diphtheria in Nigeria.16, 17

In some of the outbreaks, more females than males were affected.18 Cases of diphtheria have been reported during pregnancy and post-partum period,19-22 as well as in the neonatal and early infancy periods.23-26 Reports on diphtheria outbreaks suggest that the disease affects mainly persons with inadequate anti-diphtheria immunity.27 Immunity to diphtheria appears largely to depend on the level of antibodies to diphtheria toxin in the serum at the time of onset of illness. Antibodies to diphtheria toxin (antitoxin) are able to neutralize the effect of the toxin and protect against disease.28 Stimulation of antitoxin production occurs with sub-clinical infection (commonly cutaneous infections), clinical disease or immunization with diphtheria toxoid. These antibodies are also transferred transplacentally (from mother to fetus), providing protection to infants during the first 3-6 months of life.28 Immunity to diphtheria derived from infection, vaccination and that transferred via the placental to fetus, diminishes with time.29, 30-34 Some studies35-37 have reported inadequate levels of anti-diphtheria antibodies not only in older children and adults but also in newborns whose immunity is solely dependent on that passively acquired from their mothers (who are members of the older/adult population with possibly low anti-diphtheria immunity). In some of these studies, both mothers and their newborns had inadequate immunity to diphtheria.36, 37 A serological survey conducted in Nigeria in 1967 indicated that about 90% of older children and adults had protective levels of anti-diphtheria immunity.38 Newborns were not included in that study.38 The high level of immunity was attributed to high frequency of natural infections (especially cutaneous diphtheria) which maintained immunity to diphtheria. The findings of this survey may no longer be applicable to present day Nigeria as there have been reported improvement in the level of personal and environmental hygiene in the country.39 It

2 has been shown that improvement in the level of personal and environmental hygiene results in reduced frequency of cutaneous diphtheria infection.28, 29 Furthermore, the Nigerian serological survey was carried out before formal immunization programme was initiated in the country.38 In Nigeria, the Expanded Programme on Immunization (EPI) was launched in 1979.40 It has been reported that, increasing immunization coverage with the third dose of the diphtheria- pertusis-tetanus vaccine (DPT3), results in reduction of the frequency of diphtheria infection in the populace and hence reduction in the immunity boosting effects derived from natural infections.29 According to the WHO,41 DPT3 coverage has progressively increased in Nigeria, from 5% in 1984 to 74% in the year 2010. It is thus possible that the older population in Nigeria may have inadequate immunity to diphtheria due to a combined effect of waning of immunity over time and the lack of boosting due to lack of continuous exposure to natural infections. Therefore, there is a need to assess the current status of immunity to diphtheria in the older population and more importantly, that of a vulnerable group of children - newborns, whose immunity is solely dependent on that of an older population – their mothers (whose immunity may be suboptimal). This study therefore sets out to assess the current status of immunity to diphtheria in mothers and their newborns delivered at University of Benin Teaching Hospital, Benin City. It is envisaged that the research findings will not only provide information on the current level/adequacy of anti-diphtheria immunity being passively transferred to this very vulnerable group of children (newborns), but also, provide insight on the level of immunity against diphtheria in a section of (mainly) adult population – their mothers. This information may also serve as a basis for reviewing the age at commencement of the immunization series, and/or the inclusion of diphtheria immunization for mothers.

LITERATURE REVIEW Historical Perspective

Diphtheria was first described in the 5th century BC by Hippocrates.3 He had recognized the unfavourable implication of a sort of spider web (or membrane) in patients with tonsillar ulcers.42 The name ‘diphtheria’ was coined from the Greek word diphthera meaning leather and was named in 1826 by French physician Pierre Bretonneau.42 This signifies the leather-like membrane (pseudomembrane) that grows on the tonsil and pharynx of a patient with diphtheria.3

In 1884, Friedrich Loeffler discovered the causative organism (Corynebacterium diphtheriae).42 Emile Roux and Yesin purified the diphtheria toxin in 1889.3 One of the first effective treatments of diphtheria was discovered in the 1880s by American physician Joseph O’Dwyer.42 O’Dwyer developed tubes that were inserted into the throat, and prevented victims from suffocating due to the membrane sheath that grows over and obstructs the airways. In the 1890s, the German physician Emil von Behring developed an antitoxin that did not kill the bacterium, but neutralized the toxic poisons that the bacterium releases into the body.42 He was awarded the first Nobel Prize in Medicine for his role in the discovery and development of a serum therapy for diphtheria.42 The first successful vaccine for diphtheria was developed in 1913 by Behring.42

The Pathogen The pathogen is of the kingdom: Bacteria, Phylum: Actinobacteria, Order: Actinomycetales, Suborder: Corynebacterineae, Family: Corynebacteriaceae, Genus: Corynebacterium, Specie: C. diphtheriae and C. ulcerans.43 Other species of the genus Corynebacterium (C.pseudodiphthericum and C.xerosis ) are ubiquitous in nature and are part of the normal commensal flora of human beings.4

Corynebacterium diphtheriae is an aerobic, non-encapsulated, non-spore- forming, mostly non motile, pleomorphic, gram-positive bacillus (rod).28 Corynebacteria measure 0.5-1µm in diameter and 2-4 µm long.44 Characteristically, they possess irregular swellings at one end that give them the “club-shaped” appearance. Irregularly distributed within the bacillus (often near the poles) are granules staining deeply with aniline dyes (metachromatic granules) that give the rod a beaded appearance.44 The organisms tend to lie parallel or at acute angles to one another such that when these patterns are superimposed on one another, they form the so called “Chinese lettering” appearance.44 C. diphtheriae is an exclusive inhabitant of human mucous membrane and skin.4 There are 4 biotypes of C. diphtheriae (mitis, intermedius, belfanti and gravis) and each is capable of causing diphtheria.28 They are differentiated by colony patterns, morphology, haemolysis, fermentation reactions and severity of disease produced by the organism. 44 C. diphtheriae is by far the most commonly isolated causative agent of diphtheria, however, C. ulcerans which is more commonly isolated from cattle can cause similar disease in humans.4 Differentiation of C. diphtheriae from C. ulcerans is based on urease activity, as C. ulcerans is urease-positive while C. diphtheriae is not.4 Isolation of C. diphtheriae is enhanced by use of a selective medium, such as cystine-tellurite blood agar, that inhibits growth of competing organisms and, when reduced by the C. diphtheriae, renders colonies gray-black.4 The pathogenicity of the organisms is dependent on their ability to produce an exotoxin.44 The ability to produce diphtheritic toxin results from acquisition of a lysogenic Corynebacteriophage virus by either C. diphtheriae or C. ulcerans, which encodes the diphtheritic toxin gene and confers diphtheria-producing potential to these strains.44 This process is called lysogenic conversion.29, 44 The virus provides no essential protein to the bacterium.44 There are three types of

5 lysogenic Corynebacteriophages; beta-tox +, gamma-tox +, and omega-tox+. 3 Highly toxic strains have 2 or 3 genes inserted into their genome.3 Expression of the gene is regulated by the bacterial host and is iron dependent. In the presence of low concentration of iron, the gene regulator is inhibited, resulting in increased toxin production.44 Other factors influencing the yield of toxin in vitro are osmotic pressure, amino acid concentration, pH, and availability of suitable carbon and nitrogen sources.44 The factors that control toxin production in vivo are not well understood. The diphtheria toxin is excreted from the bacterial cell and undergoes cleavage to form 2 chains, A and B, which are held together by an interchain disulfide bond between cysteine residues at positions 186 and 201.44 Demonstration of diphtheria toxin production or potential for toxin production by an isolate is necessary to confirm disease.29 The former is performed in vitro by the agar immunoprecipitin technique (Elek test) or by the in vivo toxin neutralization test in guinea pigs.4 The potential for toxin production can be detected by polymerase chain reaction which tests for carriage of the toxin gene. Toxigenic and nontoxigenic strains are indistinguishable by colony type, microscopy, or biochemical tests.44 Non-toxigenic C. diphtheriae can be rendered toxigenic and hence pathogenic after importation of toxigenic strains.29 In fact, the introduction of a toxigenic strain into a community may initiate an ourbreak of diphtheria by transfer of the bacteriophage to non-toxigenic strains carried in the respiratory tracts of the inhabitants of the community.29 Both toxigenic and non-toxigenic strains of C. diphtheriae may be isolated during such an outbreak.45 Whereas toxigenicity is under control of the phage gene, invasiveness is under control of bacterial genes.44

Epidemiology of Diphtheria Transmission Transmission of Corynebacterium diphtheriae primarily occurs via contact with airborne respiratory droplets, direct contact with respiratory secretions of symptomatic individuals, or contact with exudates from infected skin lesions. 3, 4 Asymptomatic respiratory tract carriers and toxigenic strains in skin ulcers are important in transmission. In diphtheria endemic areas, 3-5% of healthy individuals may harbour toxigenic strains in the upper respiratory tract.4 Carrier rates in Africa have been estimated to be as high as 9.3% in the general population.15 Prevalence In the 1920s, more than 125,000 cases of diphtheria were reported annually in the United States, with highest fatality rates among the very young(infants) and elderly patients.4, 46 In this pre-vaccine era, diphtheria was a dreaded, highly endemic childhood disease reported widely in the temperate climate.3 Despite a gradual decline in deaths in most industrialized countries in the early 20th century (associated with improving living standards), diphtheria remained one of the leading causes of deaths in children until widespread vaccination was implemented.3 Between 1937 and 1938 in England and Wales, diphtheria was second only to pneumonia among all causes of deaths in children, with an annual death rate of 32 per 100,000 in children younger than 15 years.3 Specific data on the prevalence of diphtheria during pregnancy and in the newborn period are however, not available. Superimposed on the high rates of endemic disease was an incidence periodicity that demonstrated peaks every several years.3 Epidemic waves were characterized by extremely high incidence in Spain in the early 1600s, New England in the 1730s, and Western Europe from 1850-1890.3 The factors governing the periodicity of diphtheria outbreaks are not understood.

Data from some developing countries (Afghanistan, Burma and Nigeria,38 India,47, 48 Sri Lanka,49 and Zaire.50) suggest that the pattern of infection in the 1960s resembled the pattern seen in Europe and the U.S. in the pre-vaccine era. Widespread vaccination with diphtheria toxoid was commenced in most developed countries in the 1950s. 4 Following the successful implementation and wide coverage of vaccination against diphtheria in the developed countries, the incidence of the disease steadily declined. In this vaccine era, the incidence steadily declined throughout the United States and Western Europe (after the immediate postwar period) so much that the disease became a clinical rarity.3 In these regions, the last outbreaks were reported between 1972-1982.3 At the beginning of the 1980s, many countries of the developed world were progressing toward the elimination of diphtheria. Diphtheria incidence rates reached their lowest levels, and there was optimism that elimination of indigenous respiratory diphtheria could be achieved in the European region by 1990 by maintaining and strengthening immunization services.51 However, a striking resurgence of epidemic diphtheria in the Newly Independent States (NIS) of the former Soviet Union has drawn attention to the lack of a full understanding of the epidemiology of the disease.14 The epidemic began in the Russian Federation at the end of the 1980s and had affected all 15 NIS countries by the end of 1994.14 More than 150,000 cases were reported from this region.4 Subsequently, a progressively increasing number of cases were reported in the rest of Europe: 1,778 in 1990 to 47,671 in 1994.5 Some of these reported cases were linked to transmission from the NIS epidemic.52 For example, in Poland, 19 of 25 persons diagnosed with diphtheria in 1992-1995 had travelled to Russia, Ukraine, or Belarus or had had contact with visitors from these countries.53 Importation of diphtheria to European countries and Mongolia, as well as diphtheria cases among US citizens travelling to or residing in the NIS54 gave rise

8 to the fear that the NIS epidemic might spread over a wider area. Not surprisingly, subsequent outbreaks were reported across the world; China, Ecuador, Jordan, Lesotho and Sudan.14,15, 55 In Algeria, a diphtheria epidemic broke out in 1993 with 163 confirmed cases and 31 deaths.5 In Afghanistan, an outbreak was noted in the summer of 2003.11 Between 2003 and 2006, resurgence of diphtheria was reported in Delhi, India.12 In Nigeria, resurgence of diphtheria has be reported in Edo and Kastina States between 2008 and 2010.16, 17 Generally, diphtheria is still thought to be endemic in many parts of the developing world, including countries of the Caribbean, Latin America and Africa.3 Estimates of the burden of diphtheria in Africa are unreliable given the low number of diagnosed (positive culture) or reported cases.56 Incidence data in Africa is limited to case series and hospital-based surveillance studies, where underreporting is likely, given that diphtheria is frequently misdiagnosed as non- specific upper respiratory infections.57 Also children may die at home from diphtheria as some families do not have access to or do not utilize orthodox health services.57 These cases would not be documented in official records. In 1973, Heyworth et al58 reported an annual incidence rate of six cases per 1,000 persons under the age of five years in the Gambia. Incidence report from Nigeria showed 5,039 confirmed cases in 1989, 3,995 in 2000, 2,468 cases in 2001, 790 cases in 2002, 312 cases in 2006, and no officially reported cases in 2008 and 2009.7 However, 5 cases were reported from Benin city over the period 2008-201017 while another 10 cases were reported from three contiguous local government areas of Katsina State over the period 2009-2010.16 The apparent progressive reduction in the number of cases from Nigeria may be attributed to improvements in the level of personal and environmental hygiene and in the immunization (DPT) coverage in the country. However, this reduction in incidence may be an ominous sign, as similar reductions were noted in some

9 countries that later suffered massive outbreaks of diphtheria.14, 51 Furthermore, the fall in incidence may be indicative of a decreasing rate of exposure of the Nigerian populace to natural diphtheria infections. Thus, the benefits – enhancement and sustenance of immunity to diphtheria, being derived from these natural infections may be on the decline (particularly in the older population). The aforementioned recent reports of diphtheria from Nigeria16, 17 may suggest the validity of these observations. There is a need therefore, for an evaluation of the level of immunity to diphtheria in newborns, as this vulnerable group of children may no longer be receiving adequate/protective amounts of anti-diphtheria antibodies from their mothers, who are members of the older population. Morbidity/Mortality The severity of the disease depends on the virulence of the organism (with the gravis strain usually accounting for the most severe disease),3 the age (it is said to be worse in infants and the elderly),3 the immunization status of the patient, and the site of involvement (worse in respiratory than in cutaneous infections).3, 4 For example, in the diphtheria epidemic that occurred in the Sudan between August and December 1988, all six children who died had not been vaccinated.55 Three of these children died from myocarditis and one from laryngeal obstruction.55 The causes of death of the other two children were not known. Other factors include the dose of the diphtheria bacilli and the general immune status of the person infected.59 In a study of three urban populations in Massachusetts, it was noted that a subgroup of patients in immunocompromised clinical states had low levels of immunity against diphtheria and were at risk of developing severe disease,60 while reports from Lesotho, suggest that certain population groups, such as alcoholics, may be more vulnerable to diphtheria.15 The prognosis also depends on the speed with which antitoxin is administered.3 For patients in whom the disease is recognized on the day of the onset of symptoms

10 and therapy promptly initiated, the mortality rate is approximately 1%.3 If appropriate treatment is withheld until the fourth day following the onset of symptoms, the mortality rate rises to 20%.3 Worldwide case fatality rates range from 3-23%.4 Death due to mechanical obstruction of the airway or cardiac involvement with circulatory collapse occurs in at least 10% of patients with respiratory tract diphtheria.3 Through the decades, the mortality rate has not improved and was approximately 20% in the outbreak that occurred in the NIS of the Soviet Union during the early 1990s.5, 9 While the case fatality rate was 23%, in the epidemic reported in Lesotho in 1989,15 the outbreak recorded in Algeria in 1993, resulted in 31 deaths (19%) from the 163 confirmed cases.5 Ngowu et al61 noted that childhood morbidity and mortality due to diphtheria ( and the other EPI targeted vaccine preventable diseases) in Nigeria, has remained high during the years 1970-2003. A study in Benin City, Nigeria, showed that as much as two out of five cases (40%) of diphtheria seen at a health facility over a one year period, had severe airway obstruction necessitating tracheostomy.17 One of them had had no dose of DPT vaccine. The case fatality in this report was 40% as two of the cases died,17 one of whom was an infant. Race Generally, no racial predilection is observed.3 However, in the U.S. during the period 1959-1970, the incidence rate among Native Americans was three times higher than in persons of black race and 20 times higher than in persons of white race.62 A strikingly higher incidence of diphtheria, particularly of the skin and ear, among American Indian populations has also been noted in Canada.63 These racial differences likely represent mostly differences in living conditions (such as increased contact rates and reduced hygiene) that promote transmission of diphtheria, particularly in its cutaneous form.

Sex In the diphtheria outbreak that occurred in the NIS of the former Soviet Union, there was no significant difference in the number of male and female patients.9 In contrast; Jones et al64 reported a higher attack rate in male than in female children aged less than five years in the epidemic that occurred in Yemen. In those over five years of age, the attack rate for males and females was similar. Pantavee et al 18 on the other hand, reported that more females than males were affected in the diphtheria outbreak that occurred in Thailand. A similar finding was noted in a report from Edo State, Nigeria where 80% of reported cases of diphtheria occurred in female children.17 However, Oyeyemi and other workers reported a male: female ratio of 1:1 in cases studied in Katsina State.16 Some studies have shown no gender based difference in the level of immunity to diphtheria.35 While others have noted significantly higher levels of immunity in males compared to females.59, 65 In Denmark and Sweden the immunity among men older than 20 years of age was higher than among women older than 20 years of age.59, 65 This may be explained by the fact that many men in these countries served in the military (in Denmark, 26% to 83% in various age groups65) and received booster injections of diphtheria-tetanus toxoid.59, 66 A survey conducted in some African countries including Nigeria,38 showed that immunity to diphtheria was higher in boys than in girls. Because boys tend to be more adventurous than girls, they may sustain more injuries and therefore suffer from more cutaneous diphtheria infections (with resultant immune boosting) than girls. If females (especially women of child-bearing age) have lower levels of immunity to diphtheria, newborns born to them, may receive amounts of passive immunity that are inadequate to protect them until the time they acquire active immunity from DPT vaccination. This further re-enforces the need to assess the immunity to diphtheria in mothers and their newborns.

Age When diphtheria was endemic in developed countries, it primarily affected children younger than 15 years.4, 29 Recently, the epidemiology has shifted to adolescents and adults who lack natural exposure to toxigenic C. diphtheria in the vaccine era and those who have low rates of receiving booster injections.3 Also, there have been reported cases in neonates and very young infants.23-26 Diphtheria outbreaks in developing countries in the last 3 decades document a shift in age distribution similar to the shift witnessed in developed countries 30- 40 years ago.29 For example, in the outbreaks that occurred in Jordan (1982-1983), China (1988-1989), Indonesia (1986), Sudan (1982-1985), Lesotho (1989) and Algeria (1993-1996), mainly adolescents and young adults were affected.67-69 However, in the recent suspected outbreaks in Nigeria, the mean age of children affected was 5.8 years with a range of 11months to 10 years in Edo State and 7.7 years with a range of 3-13 years in Katsina State.16, 17 It is possible that the older population in Nigeria are still exposed to recurrent natural subclinical infections that boost and maintain their anti-diphtheria immunity. However, under-diagnosis and under-reporting of diphtheria remain limiting factors in interpreting incidence reports from Nigeria and most parts of Africa.56, 57 What is presumptive from these reports29, 67-69 is that, there are increasing numbers of older children and adults with low and inadequate immunity to diphtheria. If women of child bearing age have low/no immunity, newborns will be born unprotected and become possible victims with resultant poor outcome. It is thus important to evaluate the anti-diphtheria immunity of mothers and their newborns.

Pathogenesis The principal human pathogen, C. diphtheriae, is acquired from airborne droplets or by contact with infected individuals.4 The bacilli then grow on mucous membrane or in skin abrasions, and those that are toxigenic start producing toxin. The pathogenesis of diphtheria is based upon two primary determinants70: (1) the ability of a given strain of C. diphtheriae to colonize the nasopharyngeal cavity and/or the skin, and (2) its ability to produce diphtheria toxin.

Colonization The emergence and subsequent predominance of a strain of C. diphtheriae in the population is due to its ability to colonize and effectively compete in their segment of the nasopharyngeal ecologic niche.70 Corynebacterium diphtheriae usually remains in the superficial layers of skin lesions or respiratory tract mucosa, inducing mild local inflammatory reaction.4 C. diphtheriae does not actively invade deep tissues and practically never enters the bloodstream.44 In rare occasions however, it may cause a bacteraemia with focal seeding of distant organs resulting in atypical illness like endocarditis and septic arthritis.4 However, little is known about differences between strains in their ability to invade or colonize epithelial cell surfaces. The exotoxin is considered as the primary virulence factor.70 Toxigenic strains seem to have a selective advantage over non-toxigenic strains in unimmunized population because diphtheria toxin causes local tissue destruction at the site of membrane formation, which may promote multiplication and transmission of the bacterium.4

Diphtheria Toxin All toxigenic strains are capable of elaborating the same disease-producing exotoxin.70 Diphtheria toxin is very potent.70 In sensitive species (for example,

14 humans, monkeys, rabbits, guinea pigs) as little as 100 to 150ng/kg of body weight is lethal.70 Diphtheria toxin is composed of a single polypeptide chain of 535 amino acids.70 The toxin binds to the target cell surface receptor and is internalized by receptor-mediated endocytosis.44 Upon acidification of the endosome, the transmembrane domain inserts into the vesicle membrane and the catalytic domain is delivered to the cytosol, resulting in inhibition of protein synthesis. The resultant abrupt arrest of protein synthesis is assumed to be responsible for the necrotizing and neurotoxic effects of diphtheria toxin.44 Pathology Diphtheria toxin is absorbed into the mucous membranes and causes destruction of epithelium and a superficial inflammatory response.70 The necrotic epithelium becomes embedded in exuding fibrin and red and white cells, resulting in a dense necrotic coagulum of organisms, epithelial cells, fibrin, leukocytes, and erythrocytes.44 This advances – commonly over the tonsils, pharynx, or larynx, and becomes a gray-brown, leather-like adherent pseudomembrane (Diphtheria is Greek for leather). Removal is difficult and any attempt to remove the pseudomembrane exposes and tears the underlying capillaries and thus reveals a bleeding edematous submucosa.44 In respiratory diphtheria, paralysis of the palate and hypopharynx is an early local effect of diphtheritic toxin.70 Also, the regional lymph nodes in the neck become enlarged, and there may be marked edaema of the entire neck.17 The diphtheria bacilli within the membrane continue to produce toxin actively.44 This is absorbed and results in distant toxic damage, particularly parenchymatous degeneration, fatty infiltration, and necrosis in heart, muscle, liver, kidneys, and adrenals.44 These manifest as renal tubular necrosis, thrombocytopenia, cardiomyopathy, and/or demyelination of nerves.44 Because the latter two

15 complications can occur 2-10 weeks after mucocutaneous infection, the pathophysiology in some cases is thought to be immunologically mediated.4

Clinical Features of Diphtheria The manifestations of C. diphtheriae infection are influenced by the anatomic site of infection,3 the immune status of the host,60 and the production and systemic distribution of toxin.4 Generally, infection may result in an asymptomatic carrier or disease.4 Carrier State Asymptomatic nasopharyngeal infection with C. diphtheria occurs more frequently than clinical disease.28 The duration of carriage was studied during the pre-antibiotic era, by examining the rate of disappearance of C. diphtheriae from the nasopharynx of either convalescent cases or identified carriers.71 Weaver71 studied the rate of disappearance of C. diphtheria among 500 cases. After the first week, approximately half of the cases that began the week culture-positive became negative during the following 7 days. By 3 weeks after onset, 71% had become culture negative, by 4 weeks, 83%, and by 8 weeks, 99%. Weaver71 described the association of chronic carriage with a large, irregularly shaped tonsil, and the value of tonsillectomy in eradicating carriage. However, this should not be necessary with the present availability of effective antibiotics.

Respiratory Diphtheria Infection limited to the anterior nares, which is more common in infants, manifests as chronic serosanguinous, seropurulent discharge or erosive rhinitis.4 A whitish membrane may be observed on the septum while shallow ulceration of the

16 external nares and upper lip is characteristic. Such patients may readily spread diphtheria.3, 4 Tonsilo-pharyngeal diphtheria is the most common form of respiratory diphtheria.4 In the state of Assam in India, a retrospective analysis of data elicited from clinical records of patients admitted in the state teaching hospital over a five year period (1997-2002), showed that, 90% of patients with diphtheria had pharyngeal involvement only.72 Both laryngeal and nasal diphtheria were each recorded in five per cent of cases.72 A report from Yemen showed that, the anatomical locations involved with diphtheria were as follows: pharyngeal only, 80.5%; laryngeal and pharyngeal combined, 14.1%; pharyngeal and either nasal or ocular, 2.0%; laryngeal only, 1.3%; site not recorded, 2%.64 No lesions consistent with cutaneous diphtheria were noted. In a report from Nigeria,17 all the diphtheria cases that were seen over a one year period had tonsilo-pharyngeal involvement. Eighty per cent of these patients also had nasal involvement.17 The absence of cutaneous diphtheria in these reports may be attributed to the fact that they were hospital based studies.17, 64, 72 Toxin-induced complications are said to be uncommon in cutaneous diphtheria,28 hence patients with cutaneous diphtheria may not present at health care facilities. Respiratory diphtheria begins insidiously with a sore throat (the universal early symptom),3 and mild to moderate fever (in only about half of the patients).4 In the Nigerian report,17 all the children presented with fever, dysphagia or drooling of saliva or inability to suck (in an infant case). Eighty per cent had nasal discharge while sixty per cent also presented with cough.17 Another Nigerian report16 noted that 70% of affected children presented with dysphagia and 60% with fever and neck swelling. There is an initial mild pharyngeal erythema, usually followed by progressive formation of whitish tonsillar exudates (which may be unilateral or bilateral),

17 which over 24-48hr changes into a gray adherent membrane, that is tightly adherent and bleeds upon attempted removal.3, 4 In more severe cases, the patient appears toxic and the membrane is more extensive involving the uvula and the glottis area.4 There may be toxin-mediated paralysis of soft palate, posterior oropharynx and hypopharynx.17 Underlying soft tissue edema and enlarged cervical lymph nodes can cause a bull-neck appearance, 17 In the report from Benin City, Nigeria, 40% of the children with diphtheria, had the typical bull neck appearance.17 Laryngeal involvement, which may occur on its own or as a result of membrane extension from the nasopharynx, presents as hoarseness, stridor, croupy cough and dyspnea.4 These patients are at significant risk for suffocation because of local soft tissue edema and airway obstruction by the diphtheritic membrane. 70

Cutaneous Diphtheria Classical cutaneous diphtheria is an indolent, non-progressive infection characterized by a superficial, ecthymic, punched out, non-healing ulcer with a gray-brown membrane.4 More commonly, however, C. diphtheria may be a secondary infecting agent of a variety of primary skin lesions like laceration, burns, bite, impetigo or chronic dermatitis.73 Extremities are more often affected than the trunk or head.4 Pain, tenderness, erythema, and exudate are typical.4 Respiratory tract colonization or symptomatic infection occurs in a minority of patients with cutaneous diphtheria.28 Recognized toxin-induced diphtheria complications are usually said to be uncommon in patients with cutaneous diphtheria, because toxin absorption from the skin is much less than from the nasopharynx.28 Among infected Seattle adults, 3% with cutaneous infections and 21% with nasopharyngeal infection, with or without skin involvement, developed

18 toxic myocarditis, neuropathy, or obstructive respiratory tract complications.73 All had received at least 20,000U of equine antitoxin at the time of hospitalization. Infection at Other Sites C. diphtheriae occasionally causes mucocutaneous infections at other sites, such as the ear (otitis externa), the eye (purulent and ulcerative conjunctivitis), and the genital tract (purulent and ulcerative vulvovaginitis).4 The clinical presentation of ulceration, membrane formation, and submucosal bleeding helps differentiate diphtheria from other bacterial and viral causes.4 Invasive Disease Uncommonly, C. diphtheria, both toxigenic and nontoxigenic, may cause invasive disease, including endocarditis, osteomyelitis, septic arthritis, and meningitis.3 Rare cases of septicaemia are described and are universally fatal.4 Frequently, but not always, these patients have predisposing factors such as prosthetic cardiac valve or underlying immunosuppression.74 Hence, isolation of the organism from sterile body sites should not be routinely dismissed as a contaminant without careful consideration of the clinical setting.

Maternal and Neonatal Diphtheria Respiratory diphtheria19-21 or vulvovaginal infection22 can occur during any trimester of pregnancy, at term, or in the postpartum period. The mortality rate of obstetric respiratory diphtheria is high (estimated at 50%) without infusion of diphtheria antitoxin, even with tracheostomy or intubation and is accompanied by

75 fetal loss or premature birth in approximately one third of survivors. Early treatment with serum diphtheria antitoxin improves survival and pregnancy outcomes, although complications of the disease might require prolonged supportive care.19-21

Parturient women with respiratory diphtheria can transmit C. diphtheriae to their newborns.19 Neonatal diphtheria may present as respiratory or cutaneous (post-circumcision wound infection) forms.24 Nasal form of respiratory diphtheria is particularly commoner in neonates and young infants.4 Neonates and young infants are more prone to the complications of diphtheria.25

Differential Diagnosis of Diphtheria The differential diagnoses to be considered may include; epiglottitis, exudative pharyngitis due to Streptococcus pyogenes and Epstein-Barr virus, viral laryngotracheobronchitis, bacteria tracheitis, Herpes Simplex virus infection, impetigo, mucositis, and Vincent’s angina. 3, 4

The characteristic adherent membrane, extension beyond the faucial area, dysphagia, and relative lack of fever helps differentiate diphtheria from exudative pharyngitis caused by Streptococcus pyogenes or Epstein-Barr virus.4 Vincent’s angina, infective phlebitis with thrombosis of the jugular veins, mucositis in patients undergoing cancer chemotherapy is usually differentiated by the clinical setting. Differentiation from bacterial epiglottitis, severe laryngotracheobronchitis, and staphylococcal or streptococcal tracheitis is hinged partially on the visualization of the adherent pseudomembrane at the time of laryngoscopy and intubation and thereafter on the isolation of the causative organism.4

Complications of Diphtheria Respiratory tract obstruction by pseudomembranes may occur suddenly. 17 Two other tissues usually remote from sites of C. diphtheriae infection can be significantly affected by diphtheritic toxin: those of the heart and the nervous system.4

Toxic cardiomyopathy occurs in 10–25% of patients with respiratory diphtheria and is responsible for 50–60% of deaths.4 Subtle signs of myocarditis can be detected in most patients, especially the elderly, but the risk for significant complications correlates directly with the extent and severity of exudative local oropharyngeal disease and delay in administration of antitoxin. 3, 4

Neurologic complications parallel the severity of primary infection and are multiphasic in onset.4 Acutely or 2–3 weeks after onset of oropharyngeal inflammation, it is common for hypoesthesia and local paralysis of the soft palate to occur.4 Weakness of the posterior pharyngeal, laryngeal, and facial nerves may follow, causing a nasal quality in the voice, difficulty in swallowing, and risk for aspiration.17 Cranial neuropathies characteristically occur in the 5th week, leading to oculomotor and ciliary paralysis, which can cause strabismus, blurred vision, or difficulty with accommodation.4 Symmetric polyneuropathy has its onset 10 days to 3 months after oropharyngeal infection and causes principally motor deficits with diminished deep tendon reflexes. Distal muscle weakness in the extremities that progresses proximally is more commonly described than proximal muscle weakness with distal progression.4

Immunity to Diphtheria Immunity against diphtheria is anti-body mediated.29 Because the lethality of diphtheria is almost entirely due to diphtheria toxin, immunity to diphtheria depends primarily on antibody against the toxin.29 This antibody, called antitoxin is primarily of the Immunoglobulin G (IgG) type.29 The antitoxin is distributed throughout the body and can pass easily through the placenta, providing passive immunity to the newborn during the first few months of life.29 The placental

21 transfer of the antitoxin is so efficient that breast milk does not significantly contribute to immunity against diphtheria in infants.76

Production and enhancement of anti-diphtheria antibodies Production of anti-diphtheria antibody (antitoxin) may be induced by diphtheria toxin produced by C. diphtheriae during the disease or the carrier state, or by diphtheria toxoid following immunization.29 These antibodies are identical and cannot be distinguished by any existing techniques.29 Murphy et al 75 proposed that following exposure to the diphtheria toxin, some immunological memory is induced in addition to production of antibodies. This immunological memory may lead to a more vigorous antibody response in subsequent contact with the antigen (diphtheria toxin).75 However, it is unclear if this memory plays any significant role in protection against diphtheria. Other workers have noted that although a boost in antitoxin levels may occur following the onset of disease in individuals who have been previously immunologically primed but whose antitoxin concentration has fallen below the protective level, in many cases the production of diphtheria toxin by the infecting organism is so overwhelming that the antitoxin response is not swift enough to prevent complications or even death.28 This emphasizes the need for individuals to have adequate antitoxin levels at all times. Universal immunization is the only effective control measure for diphtheria.29 Although immunization does not preclude subsequent respiratory or cutaneous carriage of toxigenic C. diphtheriae, it decreases local tissue spread, prevents toxic complications, diminishes transmission of the organism, and provides herd immunity when at least 70–80% of a population is immunized.29

Diphtheria toxoid is prepared by formaldehyde treatment of diphtheria toxin, standardized for potency, and adsorbed to aluminum salts, which enhance immunogenicity.4 Two preparations of diphtheria toxoids are formulated according to the limit of flocculation (Lf) content, a measure of the quantity of toxoid.4 The paediatric preparations ( DPT, DT) contain 6.7–25.0 Lf units of diphtheria toxoid per 0.5 mL dose.4 They are used for primary immunization series and booster doses for children through 6 years of age because of superior immunogenicity and minimal reactogenicity.3, 4 The adult preparation (dT) contains no more than 2 Lf units of toxoid per 0.5 mL dose.4 The lower concentration of diphtheria toxoid is adequately immunogenic and prevents the heightened reactogenicity that occurs with higher content of diphtheria toxoid in older persons. It is recommended for persons who are aged 7 years or older.4

Serum Concentrations of Anti-Diphtheria Antibodies and Immunity to Diphtheria The activity of the anti-diphtheria antibodies (antitoxin) is represented in international units (IU) /ml of serum.29, 35 This is the WHO standard for diphtheria antitoxin calibration.35

There is a good correlation between protection from diphtheria and the presence of serum antitoxin, whether this antitoxin results from disease or immunization.29 In the 1984 diphtheria epidemic in Sweden, all seven patients who had neurological complications or died, had antitoxin titres < 0.014IU/ml whereas, 92% of symptom-free diphtheria carriers showed antitoxin titres above 0.16IU/ml.77

However, it has also been shown that there is no sharply defined level of antitoxin that gives complete protection from diphtheria.78 A certain range of

23 variation must be accepted as the same level of antitoxin may give an unequal degree of protection in different persons.29 Other factors may influence vulnerability to diphtheria including the dose and the virulence of the diphtheria bacilli and the general immune status of the person infected.60, 79

In view of the above, the degrees of protection against diphtheria have been classified based on accepted range of serum concentrations of anti-diphtheria antibodies as follows;80

 No protection: This corresponds to a serum antitoxin level of less than 0.01IU/ml.  Minimal protection: This corresponds to a serum antitoxin level of 0.01to < 0.1IU/ml. These concentrations of antitoxin provide minimum but incomplete protection. In the diphtheria outbreak in Sweden, some of the patients who were symptom-free had serum antitoxin concentrations between 0.01 and 0.1IU/ml. Some others with similar range of antitoxin concentration suffered severe disease and neurological complications.77 It has been recommended that persons with serum antitoxin of < 0.1IU/ml should receive three doses of age appropriate diphtheria toxoid vaccine (DPT/dT) with each dose given at one month interval.81  Safe protection: This corresponds to a serum antitoxin level of 0.1 to less than 1.0IU/ml. These antitoxin concentrations provide safe and adequate protection from diphtheria for up to a period of one year. A booster diphtheria toxoid vaccine is recommended to be given within this period.81  Long term protection: This corresponds to a serum antitoxin level of ≥ 1.0IU/ml. Antitoxin levels of 1.0 – 1.9IU/ml provides protection from diphtheria for up to a period of 5 years. Diphtheria toxoid booster vaccine is thus recommended after 5 years.81 Antitoxin levels ≥ 2.0IU/ml provide

protection for up to a period of 10 years and booster vaccination is not recommended until after 10 years.81

While some early serological surveys of immunity to diphtheria used a serum antitoxin level of 0.01IU/ml as the cut off level for estimating percentage of individuals with protective immunity to diphtheria,38, 82 more recent studies used a more appropriate level of 0.1IU/ml as cut off for determining percentage of individuals with protective immunity to diphtheria.35, 37 Thus, the degree of protection may also be classified as;35

 Inadequate protection: This corresponds to a serum antitoxin level of less than 0.1IU/ml. It provides in complete protection from different.  Adequate protection: This corresponds to a serum antitoxin level of 0.1IU/ml or more. This provides complete protection from diphtheria.

Immune Response to Vaccination In scenarios where mothers have high anti-diphtheria antibody titres, a level of maternally acquired antibody higher than 0.1IU/ml may interfere with the immune response of infants to the first and to a lesser extent the second dose of DPT vaccine.29 However, following the second and third doses of vaccine, most infants develop adequate immune response.29 In the absence of high transplacental anti-diphtheria antibodies (< 0.1IU/ml), most infants would develop adequate immune response to the first and subsequent doses of the diphtheria toxoid vaccine.29 A study83 in Iran assessed the influence of pre-vaccination passive immunity on infants’ immune response to three doses of DPT vaccination. The study showed that diphtheria-tetanus toxoid components of DPT vaccine were highly immunogenic and maternal passive immunity did not affect the infants’ immune

25 response to DPT vaccination.83 The sample size used for this study was however small, as only 69 infants were assessed for their anti-diphtheria antibody titres following DPT vaccination. Studies show that primary immunization of infants with three doses of diphtheria toxoid stimulates antibody levels that considerably exceed the minimum protective level (0.01IU/ml).84 Generally, the antibody levels increase after the second dose of diphtheria toxoid and it is much higher after the third dose.84 Following the primary series, 94-100% of infants have antibody levels higher than 0.01IU/ml, with the mean level between 0.1 and 1IU/ml or higher.84 Following primary series of immunization in the Gambia, the mean titre of diphtheria antibody increased from 0.07IU/ml before the third DPT dose to 0.84IU/ml after the third dose of DPT.85 In Ghana, Hori et al86 reported that the mean antibody titre following the third dose of DPT was 1.18IU/ml. The difference in the reported mean titres between the Gambian and Ghanaian studies may be as a result of difference in the immunization schedule used. In the Gambian study, DPT was given at 8, 12 and 16 weeks of age whereas in the Ghanaian study DPT was given at 6, 10 and 14 weeks of age. Secondly, the difference in the dose of diphtheria toxoid in the vaccine used in the Gambian and Ghanaian studies may be another explanation for the differences in their results. The vaccine used in the Gambia contained 6.7Lf unit of diphtheria toxoid per 0.5ml while that used in Ghana contained 25Lf units/0.5ml.

In some countries where a booster dose of diphtheria toxoid is part of the immunization schedule, giving such booster doses 12-18months after completion of the primary series stimulates abundant production of diphtheria antibody, with the mean levels above 1IU/ml.84 In one study,87 the percentage of children with

26 protective antibody levels following a booster dose exceeded 95% for at least 6 years.

It has been shown that parturient women develop adequate immune response to diphtheria toxoid booster doses given during pregnancy. In a study83 that assessed the level of immunity to diphtheria in two groups of parturient women, one group had 2 booster doses of diphtheria-tetanus (dT) vaccine at the 4th and 6- 7th month of pregnancy. The other group did not receive any diphtheria booster dose. The mean concentrations of anti-diphtheria antibody titres were lower in unvaccinated than in vaccinated mothers: 0.31IU/ml vs. 0.78IU/ml. While all vaccinated mothers had adequately protective antibody titres, up to 27% of the unvaccinated mothers were susceptible to diphtheria.83

Fate of Immunity to Diphtheria

Studies indicate that the immunity to diphtheria passively received via the placenta or that actively developed following clinical or sub-clinical infections or immunization, wanes with time.29, 30, 35, 82, 83

There is a steady logarithmic rate of loss of antitoxin in newborns which averages 13.9% per week.29 The average antitoxin content of the serum of babies 10 days old is half that of the cord blood.29, 30 Subsequently, the babies lose half their antitoxin every four and a half (4½) weeks.30 This implies that an infant born to a mother with low anti-diphtheria antibody titre may rapidly become susceptible since the little antitoxin received from its mother is rapidly lost.

Most studies suggest limited persistence of diphtheria antibody following the primary series of three doses of DPT vaccine.84, 85 During the first year after the primary series, the mean level of diphtheria antibody declines threefold to fifty- fold; however, most children sustain levels of antibodies above the protective

27 threshold.84 One year after the primary series of DPT or DT vaccination, 75-97% of children studied in Russia87 and the USA88 had protective titres of diphtheria antibody. During the next two years the mean titre of anti-diphtheria antibody declined by 40% per year.87 In one study in the USA,89 children who received primary DPT immunization during infancy had diphtheria antibody below the protective level at 16-20 months of age.

Waning Diphtheria Immunity in Adults Following Introduction of Routine Childhood Immunization

In the pre-vaccine era, children were susceptible to diphtheria but adults were immune because of frequent exposure to C. diphtheriae and repeated natural boosting.29 Large-scale immunization of children with diphtheria toxoid has resulted in high levels of immunity in children and considerable decline in diphtheria incidence.4, 29 As the number of clinical cases of diphtheria declines, asymptomatic carriage of C. diphtheriae becomes rare.84

Pappenheimer90 noted in his work that toxigenic strains seem to have a selective advantage over non-toxigenic strains in unimmunized populations because diphtheria toxin causes local tissue destruction at the site of membrane formation, which may promote multiplication and transmission of the bacterium. He further noted that, in immunized individuals, toxigenic strains cannot benefit from this, and the metabolic cost of production of toxin may become significant and consequently a disadvantage for toxigenic strains. Pappenheimer90 concluded that this could explain why wide use of the diphtheria toxoid vaccine leads to diminished circulation of toxigenic strains of C. diphtheriae and that reduction in circulating toxigenic C. diphtheriae means that opportunities to build or maintain immunity amongst adults are limited.

Sustenance of Immunity to Diphtheria

In view of the progressive loss of immunity to diphtheria acquired from the primary series of immunization, there is need to ensure that older children and adults remain immune to diphtheria.29 This is particularly relevant to newborns as the adequacy of their immunity is dependent on their mothers maintaining high titres of diphtheria antibody through the child-bearing ages.

Two mechanisms have been reported to be involved in the sustenance of immunity to diphtheria through childhood and adulthood.29 The first results from recurrent subclinical infections by toxigenic strains of C. diphtheriae.29 These subclinical infections are mainly cutaneous diphtheria.28, 73 As the child grows up he/she is subjected to recurrent cutaneous infections that sustain immunity to diphtheria at high levels into adulthood. The likelihood of being exposed to cutaneous diphtheria is increased in settings of low socio-economic status, poor personal and environmental hygiene.29 It has also been suggested that improving immunization coverage with diphtheria toxoid results in reduced exposure to toxigenic strains in both respiratory and cutaneous infections.84, 90

In countries where the level of hygiene and DPT coverage (primary series) have improved, immunity to diphtheria in the older population have been reported to be waning and other means of sustaining this immunity in these older age groups are being sought.29 In developing countries like Nigeria, it is still being thought that the sustenance of immunity to diphtheria in the older population operates via recurrent cutaneous infections.3 However, the level of hygiene and DPT coverage have significantly improved over the years in Nigeria.39, 41

The second mechanism for sustaining immunity to diphtheria in the older population is through the administration of booster doses of diphtheria toxoid after

29 the completion of the primary series of immunization.4, 29 In countries where this is being practiced, two booster DPT doses are given at the ages 18 months and 4-6 years.84 In some other countries like the USA, booster diphtheria toxoid are also given to women of child-bearing ages and pregnant women in the form of tetanus- diphtheria-acellular pertussis combined vaccine (Tdap) or dT vaccine.75 This is meant to boost their immunity to diphtheria (as well as tetanus and pertussis) and consequently prevent these diseases in their infants. Ingestion of colostrum from an immune mother however does not result in an increase in or sustenance of the concentration of diphtheria antitoxin in infant sera.75

Generally, priority should be given to efforts to reach at least 90% coverage with three doses of DPT vaccine in children below one year of age.29 In addition, WHO29 recommends that in developing countries where diphtheria has been successfully controlled, the immunity level acquired through immunization in infancy should be maintained through properly-timed booster doses of DT or dT vaccines. dT vaccine should be used for older children (≥7 years) or adolescents leaving primary or secondary schools. 3, 4 Periodic booster doses are required to sustain protection throughout adulthood.29 Furthermore, routine serological studies have been recommended, as data from these studies may identify age groups at risk of diphtheria and serve as a valuable guide in deciding when booster doses are warranted.29

Factors that Affect Immunity to Diphtheria

The level of immunity to diphtheria in an individual (including parturient women) may be affected by some factors. These factors include;

 Immunization status: Vaccination of an individual with diphtheria toxoid is a major determinant of his/her status of immunity to diphtheria.29 In the childhood vaccination programme, three diphtheria toxoid doses are administered monthly commencing at the second month of life. WHO EPI, to which Nigeria subscribes recommends DPT vaccine containing 7-25Lf of diphtheria toxoid per 0.5ml of vaccine to be given at 6, 10 and 14 weeks of age.84 It has been shown that children who receive this vaccine develop adequate immunity to diphtheria.86 However, using other schedules such as giving the vaccine at 8, 12 and 16 weeks of life or not completing the required regimen may result in lower immune response.29, 85 Furthermore, not using the WHO recommended dose of diphtheria toxoid (7-25Lf/0.5ml) for vaccination may affect the immune response.84 In a Gambian series, vaccine containing diphtheria toxoid at a concentration of 6.7Lf/0.5ml was used for infant vaccination. Peak anti-diphtheria antibody titre following the third dose of vaccine was 0.84IU/ml .85 In a similar study86 conducted in Ghana, using 25Lf/0.5ml of diphtheria toxoid, peak antibody titre following the third dose of vaccine was 1.18IU/ml.86

Receipt of booster doses of diphtheria toxoid vaccine at later ages following the primary childhood vaccination, has also been shown to help sustain the immunity to diphtheria at adequate levels in older children and adults.84, 87 Studies have shown that pregnant women who received booster doses of diphtheria toxoid during pregnancy had higher levels of immunity to diphtheria compared to those who did not receive any booster dose.83

 Age: In the absence of regular booster doses of diphtheria toxoid, the immunity to diphtheria progressively wanes with age.29 Studies have shown that following the primary series of vaccination, children within the first

decade of life were the most protected against diphtheria compared to older persons.29 Some African studies have shown that as much as 30% of females of reproductive ages had no protection (antibody < 0.01IU/ml) to diphtheria.82, 91 Amongst the child population, newborns are peculiar since their immunity to diphtheria is solely dependent on that of their mothers (who are member of the older population). Not surprisingly, a study showed that as high as 70% of newborn had inadequate immunity to diphtheria.35

 Hygiene and Socio-economic Class: In settings with low vaccination coverage or no diphtheria vaccination at all, recurrent subclinical cutaneous diphtheria is an important means for maintaining and enhancing immunity to diphtheria.3 The likelihood of being exposed to cutaneous diphtheria is increased in settings of low socio-economic status, poor personal and environmental hygiene.29 In a 1967 Nigerian study,38 as high as 90% of older children and adults who had never been vaccinated with diphtheria toxoid, had protective titres of anti-diphtheria antibodies. These high antibody titres were attributed to recurrent cutaneous diphtheria. However, following improvements in socio-economic conditions and hygiene, the exposure to these natural infections diminishes and thus sustenance of immunity via these means is progressively removed.29

 Immuno-compromised states: Sargent et al 60 noted that a subgroup of patients suffering from malignancies, diabetes mellitus and those on immunosuppressive drugs (cytotoxic and steroids) had lower levels of immunity to diphtheria compared to other persons. A study of the placentae of sickle cell disorder patients showed areas of infarction and fibrosis.92 It was noted in this study that these changes may suppress placental transfer of antibodies. Another study reported that maternally acquired immunity may

be reduced in newborns from HIV-infected women.93 However, only specific IgG antibodies to polio and measles viruses were assayed for in this study.93

In newborns, immunity to diphtheria is dependent on the immune status of their mothers and the amount of anti-diphtheria antibodies that was successively transferred to them via the placental route.83, 94 Consequently, factors that affect utero-placental functions would affect the status of immunity to diphtheria in newborns.94 Maternal conditions that may reduce utero-placental functions include; anaemia, hypertensive and cardio-pulmonary diseases.95 Fetal and newborn conditions that may be associated with reduced utero-placental functions include; prematurity, post-maturity, immune and non-immune hydrop fetalis, multiple gestation, perinatal asphyxia and intra-uterine growth restriction.94, 95

Immunity to Diphtheria in Females of Reproductive Ages, Mothers and their Newborns

Studies on immunity to diphtheria indicate that adolescents and adults (including females of child-bearing ages) have waned immunity to diphtheria.84, 91 In a review of European studies on population immunity to diphtheria, Galazka and Robertson84 found that the overall proportion of adults with antibody titres below the level considered to be protective varied from 23% in Finland to 26% in Denmark, 38% in the UK, 49% in France and to 53% in Poland. In Germany, Moldova and Russia, those aged 20-40 years had the lowest levels of anti- diphtheria antibodies.84

A 1980 German hospital based study assessed the immunity to diphtheria in 185 mothers and their newborns.96 Using a serum concentration cut off of 0.1IU/ml, adequately protective anti-diphtheria antibodies were found in only 29.2% of mothers and 27% of their babies. However in this study, the passive

33 haemagglutination technique was used for antibody assay. The results from this technique have been shown to be unreliable and have poor correlation with the reference methods for testing immunity to diphtheria.29, 97, 98

A subsequent German study using ELISA technique in 1993,35 however had similar findings. This age-stratified study on immunity to diphtheria was carried out in 400 healthy individuals including newborns, children and adults. The study subjects were divided into 8 subgroups of 50 persons each. Exclusion criteria for newborn included premature birth and major organ system defects. Newborns and persons over 50 years of age constituted the least protected groups, with significantly lower median antitoxin titre than the other age groups. Only 30% of newborns had adequate protection to diphtheria while 14% had no protection at all.35 It was concluded in this study that the lack of adequate immunity to diphtheria in a significant proportion of newborns reflected inadequate protection in women of reproductive ages. While this inference may be valid, as 20% of women in the reproductive ages in the study had inadequate protection, it would have been better proven if the mothers of the babies had been assayed.

There are also studies on immunity to diphtheria in Africa.38, 82, 91 In a population based age-stratified study by Kriz et al38 between 1967 and 1973, 6740 sera were collected from Nigeria, Niger, Kenya, Togo, Burma, Algeria, Tunisia, Afghanistan, Mongolia and Yugoslavia. In each of these countries, 674 persons between the ages of 1 year and 40 years were consecutively recruited. Newborns were not included in this study. The in-vitro neutralization in microcell culture technique was used. In Nigeria and Kenya, it was shown that 85-90% of individuals aged 15-40 years had protective titres of anti-diphtheria antibody. There was no gender related difference in antitoxin titres in this age group. However the cut-off for determining protective antibody titre in this study was

0.01IU/ml. This obviously would have caused overestimation of those with adequate immunity as full immunity to diphtheria is currently determined to be derived from titres ≥ 0.1IU/ml.80 Secondly, the study was conducted before formal vaccination with diphtheria toxoid was commenced in these countries. In Kenya vaccination with diphtheria toxoid under EPI was introduced in 1980, and the coverage of DPT3 increased to as high as 74% in the 1990s.82, 99 In Nigeria, vaccination with diphtheria toxoid was commenced in 1979.40 DPT3 coverage has progressively increased in Nigeria, from 5% in 1984 to 72% in the year 2006.41 Odusanya et al100 reported that the DPT 3 coverage in a rural Nigerian community (Sabongidda-Ora, Edo State) in 2006, was 80.8%. The DPT3 coverage in Edo State in 2010 was 72%.101 Consequently, the findings of Kriz et al38 may no longer be representative of these countries. In a subsequent study in Kenya in 1990, 70 of 84 individuals (83%) had anti- diphtheria antibody greater than the protective level.82 Some 84 individuals of ages greater than 7 years were randomly selected over a period of one month. The antibody was assayed using in-vitro neutralization technique. Levels greater than 0.01IU/ml were considered protective. The study reported that, 100% of females aged 18-45 years had protective titres (> 0.01IU/ml) of anti-diphtheria antibody. If the more appropriate cut-off for protective titre of 0.1IU/ml were to be used, based on the results provided in the study report, the proportion of females aged 18-45 years with protective titres would have been 53%. Also noteworthy is the non- inclusion of newborns in this study and the small sample size in this population based study.

A 2005 report from Egypt showed that only 22% of adults including females of reproductive ages had full immunity (antibody ≥ 0.1IU/ml) to diphtheria.91 As many as 30% of females of reproductive ages had no protection (antibody <

0.01IU/ml) to diphtheria.91 This was an age-stratified population based study that recruited 709 individuals aged between 2 months – 105 years from six regions of Egypt. Newborns were not included in this study. The ELISA technique was used in this study. DPT3 vaccine coverage has remained high in Egypt over the last 2 decades; 84% in 1980, 87% in 1990, 98% in 2000 and 97% in 2010.102 This study91 thus recommended booster diphtheria toxoid vaccines for the adult population. However the effect of the low anti-diphtheria antibody titres in women within the reproductive ages on the passive immunity acquired by newborns was not assessed.

In 1980 in Mali, immunity to diphtheria was assessed in 85 mothers and their newborns using passive haemagglutination technique.103 Cut-off for protective titre was 0.1IU/ml. 100% of mothers and 81% newborns had protective levels of antibodies. None of the mothers had been vaccinated with diphtheria toxoid. Hence it was concluded that recurrent subclinical cutaneous diphtheria may be responsible for maintaining the high levels of protection in these mothers. However, a significant proportion (19%) of newborn did not demonstrate protective titres. This re-enforces the need for mothers to have very high antibody titres (possible by giving diphtheria toxoid booster doses during the reproductive ages or during pregnancy) to compensate for any significant attrition in the amount of antitoxin passively transferred to their newborn. However, the sample size of this study was small. Secondly, the method of assay (passive haemagglutination) is fraught with disadvantages that render its results unreliable as this technique has a low sensitivity and may have been unable to detect lower antibody titres in the newborns. 29, 97, 98 Furthermore, the findings of this study may no longer be applicable as there has been a progressive increase in the coverage of diphtheria toxoid (DPT3) vaccination in Mali (as in many other African countries including

Nigeria). The DPT3 coverage in Mali has increased from 42% in 1990, to 92% in 2010.102

Relationship between Maternal and Newborn Anti-diphtheria Antibody Titres

It has been documented that antibodies to diphtheria toxin pass freely from mother to the foetus.29, 37 The placental transfer of anti-diphtheria antibodies is very efficient.37 As high as 95% of newborns will acquire levels of passive immunity to diphtheria that are similar to their mothers’ levels of immunity to diphtheria.37 In a Romanian study by Durbaca,37 protective immunity to diphtheria was reported in 79.6% and 77.1% of mothers and their babies respectively. This good correlation between maternal and newborn titres of anti-diphtheria antibody was supported by findings from another study carried out by Ehrengut and Tegeler in Germany.96 In this study, 29.2% of mothers and a corresponding 27% of their newborns had protective titres of antibody.96 In both studies, the percentage of immune women that failed to transfer corresponding levels of immunity to their infants ranged between 3 and 7.5%.37, 96 Allerdist et al103 however reported differently in Mali, as up to 19% of immune women failed to transfer corresponding levels of immunity to their infants. While 100% of mothers in this study103 had protective titres of anti- diphtheria, only 81% of their newborns had corresponding levels of protective antibody titres.

In an Iranian study,83 88 parturient women who had diphtheria toxoid (dT) vaccination during pregnancy, were followed up till delivery. Using the ELISA technique, analysis of blood samples collected from these mothers at delivery showed that 100% of them were fully immune to diphtheria. However, only 89% of their babies were fully immune to diphtheria. Also in this study, a control group

37 of parturient women who were not vaccinated were assessed. Of the 22 women who constituted this group, 73% of them were fully immune to diphtheria while only 50% of their babies had full immunity to diphtheria. Twenty three percent of immune mothers failed to transfer corresponding levels of immunity to their infants.82 As in the European studies, 37, 96 the Iranian83 and Malian103 studies controlled for factors that may affect placental transfer of antibodies. However, making comparisons and deductions from these studies 37, 96, 83, 103 is difficult because of the small sample sizes of the Iranian83 and Malian103 studies compared to that of the European studies.37, 96

Generally, the relationship between maternal and newborn anti-diphtheria antibody titres is such that not all mothers with adequate immunity to diphtheria will transfer the same amount of immunity to their infants.37, 96, 103 This reinforces the need for assessment of immunity to diphtheria concurrently in both babies and their mothers as inadequate immunity to diphtheria in a newborn may not always indicate a non-immune mother.

Measurement of Immunity to Diphtheria

The various methods described for the measurement of immunity to diphtheria are reviewed below:

The Schick test29 The standard procedure to detect immunity to diphtheria toxin in early studies was the Schick test, first described by Béla Schick, a Hungarian-born American paediatrician.104 A small amount of toxin was injected intradermally. A positive reaction was characterized by inflammation appearing after 24 to 36 hours, and this signified lack of antitoxin, while a negative result (lack of

38 inflammation) indicated presence of antitoxin. A control test with inactivated toxin was performed to exclude allergic reactions to toxin. Although Schick test results usually correlated well with serum antitoxin levels, this technique is no longer used due to technical difficulties in performing intradermal injections, the requirement for two visits, ethical problems in causing painful inflammatory lesions or sometimes ulcers in children when the result is positive, and unreliability in cases of skin anergy (often found in newborns and young infants) where negative results can be erroneously interpreted as evidence for immunity.105

Neutralization test on animals29 Neutralization test, like the Schick test, is mainly of historical interest. It was usually performed on the depilated skin of rabbits or guinea-pigs. 106 Different dilutions of serum mixed with fixed amounts of diphtheria toxin were injected into the depilated skin of the animal, and the antitoxin concentration was estimated based on the presence or absence of an inflammatory reaction. If an inflammatory reaction is present it signifies lack of immunity while the absence of an inflammatory reaction signifies the presence of immunity. Results of the neutralization test may differ depending on the avidity of the antibody tested, the concentration of toxin used in titration, and the specie of the laboratory animal used. The test is laborious, time-consuming and expensive, and requires suitable animals.

Neutralization test on microcell culture29 The neutralization test on microcell culture is based on the observation that the survival of mammalian cells in culture is inhibited by diphtheria toxin.107 This effect is neutralized when diphtheria antitoxin is present in serum samples.107 The titration of the antitoxin in the serum samples is done in plastic micro-tissue culture

39 plates, in which dilutions of test sera are mixed with challenge toxin. After a short incubation, Vero (green monkey renal epithelium) or HeLa (cell suspension in a special culture medium) is added. After incubation for four-five days at 37°C, results are read as a change in the colour of the reagents in micro-titre plate wells. The colour change is due to the normal metabolic formation of acid in surviving cells, resulting in changes in the pH of the medium and a visible colour change from red to yellow. The absence of an acidic colour change signifies massive cell deaths and thus lack of antitoxin in the patient’s serum. Vero cells are more sensitive to diphtheria toxin since they have large numbers of binding sites (receptors) and take up the toxin in a highly specific, time- and temperature- dependent manner.108 When a serum dilution contains antitoxin, the cells continue to grow, and the medium undergoes colour changes ranging from red to yellow (depending on the amount of antitoxin in the serum).108 This was the method used by Kriz et al38 in determining the level of diphtheria immunity in Nigeria. This technique is the designated gold standard for determining antitoxin levels and it is very sensitive (minimum detectable level 0.005IU/mL).29 However, it is expensive, complex and not easily reproducible. It takes up to five days to carry out each test.82 Furthermore, it requires staff with special skills in tissue culture techniques and specialized laboratory equipments that may not be routinely accessible.

Passive haemagglutination29 The passive haemagglutination test uses an in vitro technique. This test makes use of reagent kit that contains sheep, turkey, horse, or human red cells that are pre-coated with diphtheria toxoid. These cells are then incubated with the patient’s serum. If a patient’s serum contains diphtheria antibodies, the red cells

40 are agglutinated by the antitoxin. The degree of agglutination is dependent on the amount of antitoxin present in the serum. The test is inexpensive and can be performed in a modestly equipped laboratory. The passive haemagglutination test is rapid (results available in one hour), and reproducible. However, the test tends to underestimate low concentrations of diphtheria antibody.97 Furthermore, the results of the test for anti-diphtheria antibodies can be distorted by non-specific agglutinins in the sera of patients. Consequently, results from this test have been found to be often discrepant rendering interpretation of the test very risky for the individual patient.97, 98 Overall there is relatively poor correlation of the passive haemagglutination test with contemporary toxin neutralization tests considered as standard reference methods. ELISA29 The enzyme-linked immunosorbent assay (ELISA) also uses an in-vitro technique that involves the binding of antigen to polystyrene tubes. Exotoxins, such as diphtheria toxin, have a highly lipophilic moiety in their molecule, and thus, coat the tubes efficiently.109 The test is carried out using commercially available test kits. The specific components of each test kit may differ depending on the manufacturer. However, each kit is usually validated against reference standards and supplied with an instruction manual. Commonly, the test kits include test wells that are coated with diphtheria toxin. This constitutes the solid phase. The subject’s serum is added to a well and any antibodies specific for the antigen present will bind to the solid phase (forming an immune complex). After removal (by washing with water) of unbound material, anti-human IgG conjugated to an enzyme - alkaline phospatase (provided with the test kit), is then added. This reacts with the immune complex. After removal of excess conjugate by washing, a substrate - para-nitrophenylphosphate (also

41 provided with the kit) is added. This reacts with the conjugated enzyme to produce a coloured derivative of the substrate. The colour intensity is proportional to the amount of specific antibody bound and can be quantified by photometry. For detecting protective levels of antibodies, the results of the ELISA test correlate well with results of the in vivo neutralization test in guinea-pigs110 and the results of the neutralization test in tissue culture.111 It was shown that, results from ELISA technique coincided in 85.7% (correlation coefficient: 0.964) with the reference method.111 Svenson et al109 noted that ELISA technique was at least ten times as sensitive as the in vivo rabbit skin test. ELISA technique has several advantages. It is simple, economical and rapid.29 Results from ELISA are highly reproducible and precise (as it can measure class-specific diphtheria antibodies such as IgG).109, 111 The ELISA technique is a very practical method for sero-epidemiological studies.29 Thus this method was used for determining diphtheria antibodies (IgG) levels in this study. Laboratory Diagnosis of Diphtheria

The first step in the laboratory diagnosis of diphtheria is to obtain appropriate clinical specimens from the patient.113 WHO guidelines for the collection of specimens on swabs from suspected diphtheria cases and for the transportation of the swab have been published.113

A clinical specimen should be obtained as soon as possible when any form of diphtheria is suspected, even if treatment with antibiotics has already begun.114 Multiple site sampling should always be considered in a suspected case as this may increase the organism recovery rate and hasten laboratory results.114 If possible, a

42 specimen should be taken from under the membrane, where bacteria are concentrated.114

The diagnosis of diphtheria based on direct microscopy of a smear is not advisable as false positives and false negatives may occur.113, 114 The use of selective media, such as Hoyle’s Tellurite, is advised.113 Furthermore, swabs with specimens from asymptomatic carriers or contacts may contain only small numbers of organisms, which may be obscured by the overgrowth of normal throat flora. 114 Tellurite-containing media inhibit the growth of normal oral flora and C. diphtheriae reduces the tellurite salts, producing characteristic black colonies.114

The test for toxigenicity, which detects the potent exotoxin, a phage-encoded protein, is the most important test and should be done without delay on any suspect isolate that is found by routine screening or while investigating a possible case of diphtheria. 114 The methods described for detection of toxigenicity are reviewed below:

The in-vivo virulence test:114 It involves inoculation of guinea pigs with suspected infected tissue and then looking out for the characteristic skin reactions (sometimes death) in these animals. Although, it was widely used in several countries, the expense, slowness, risk of accidental self-inoculation, and the increasing unacceptability of in-vivo tests in many countries led to a decline in its use.

The Elek immunoprecipitation test:70 This test is based on the double diffusion of diphtheria toxin and antitoxin in an agar medium. A sterile, antitoxin-saturated filter paper strip is embedded in the culture medium, and C. diphtheriae isolates are streak-inoculated at a 90° angle to the filter paper. The production of diphtheria

43 toxin can be detected within 18 to 48 hours by the formation of a visible toxin- antitoxin precipitin band in the agar.

Polymerase chain reaction (PCR):115 In recent years, the use of PCR for the detection of the diphtheria toxin structural gene (tox) has been described. Most assays that use pure bacterial cultures have focused on the detection of sequences that code for the biologically active (fragment A) subunit of the toxin.

In order to enhance the identification of diphtheria cases and ensure early administration of treatment, the current recommended case definition for diphtheria is as shown below;116

Probable case: In the absence of a more likely diagnosis, an upper respiratory tract illness with

 an adherent membrane of the nose, pharynx, tonsils, or larynx; and  absence of laboratory confirmation; and  lack of epidemiologic linkage to a laboratory-confirmed case of diphtheria.

Confirmed case: An upper respiratory tract illness with an adherent membrane of the nose, pharynx, tonsils, or larynx; and any of the following:

 isolation of Corynebacterium diphtheriae from the nose or throat; or  histopathologic diagnosis of diphtheria; or  epidemiologic linkage to a laboratory-confirmed case of diphtheria.

Treatment should be administered to probable cases while awaiting laboratory confirmation.116, 117

Treatment of diphtheria

WHO117 recommends immediate administration of diphtheria antitoxin and antibiotics following clinical diagnosis. Worsening outcomes have been associated

44 with delays in administration of the antitoxin.117 The antitoxin has been recommended to be given at an empirical dose based on the degree of toxicity, site and size of the membrane, and duration of illness.80 Similar doses are given to adults and children.3 The parenteral route is preferred.80 The role of antimicrobial therapy is to halt toxin production, treat localized infection, and prevent transmission of the organism to contacts.80 C. diphtheriae is usually susceptible to various agents in vitro, including penicillins, erythromycin, clindamycin, rifampin, and tetracycline.4 Resistance to erythromycin is common in populations if the drug has been used broadly.4 Only erythromycin or penicillin is recommended;80 erythromycin is marginally superior to penicillin for eradication of nasopharyngeal carriage.4 The WHO noted that untreated patients remain infectious for 2-3 weeks but become non-infectious 24 hours following commencement of antibiotic treatment.117 Erythromycin has been recommended to be given orally at a dose of 40–50 mg/kg/day divided every 6 hr for 14 days.4 Elimination of the organism should be documented by at least 2 successive negative cultures from the nose and throat (or skin) obtained 24 hours apart after completion of therapy.4 Re-treatment with erythromycin is recommended if either culture yields C. diphtheriae.3

In a Nigerian report by Sadoh et al,17 none of the diphtheria cases that were managed received diphtheria antitoxin. This was attributed to the unavailability of the antitoxin in Nigeria.17 Each of the patients however received intravenous crystalline penicillin. The fatality rate in this report was however high – 40%.17

Recovery from the myocarditis and neuritis is often slow but usually complete.4 Corticosteroids do not diminish these complications and are not recommended.4 Recommended supportive care includes respiratory isolation.3 This should be continued until the cultures taken after cessation of therapy are

45 negative.4 Cutaneous wounds are cleaned thoroughly with soap and water.4 Nasogastric tube feeding may be required for patients with palatal/pharyngeal paralysis.3 All suspected diphtheria cases should be reported to local and state health departments.17

Prevention of Diphtheria

According to the WHO,117 the most effective method for the prevention of diphtheria epidemic is mass immunization of the entire population. Immunization to diphtheria diminishes transmission of the organism and provides herd immunity when at least 70-80% of a population is immunized.4 The National Programme on Immunization for Nigeria recommends three doses of diphtheria, pertussis, tetanus (DPT) vaccine to be administered to infants at age of six weeks and then given at four weekly intervals.118 It is recommended that developing countries like Nigeria should aim at coverage of 90%.29 Current statistics indicate that the coverage for three doses of DPT in Nigeria is 74% and 72% in Edo State, which is clearly short of the 90% target.41, 101

The methods described for the prevention of diphtheria in specific groups of individuals are reviewed below:

Convalescent Patients The WHO117 reported that unless immunized, children and adults may repeatedly succumb to diphtheria. Vaccination of patients with an age-appropriate diphtheria toxoid vaccine following recovery from diphtheria was thus recommended.117

Asymptomatic Case Contacts

It has been recommended 3, 4 that all household contacts and those who have had intimate respiratory or habitual physical contact with a patient are closely monitored for illness through the 7-day incubation period. Cultures of the nose, throat, and any cutaneous lesions are performed. Antimicrobial prophylaxis is presumed effective and is administered regardless of immunization status using erythromycin for 7 days or a single injection of benzathine penicillin G.4 Diphtheria toxoid vaccine, in age-appropriate form, is given to immunized individuals who have not received a booster dose within 5 yr.4 Those who have received fewer than 3 doses of diphtheria toxoid or who have uncertain immunization status are immunized with an age-appropriate preparation on a primary schedule.4

Asymptomatic Carrier When an asymptomatic carrier is identified, the following steps are recommended:3  Antimicrobial prophylaxis should be administered for 7-10 days  An age-appropriate preparation of diphtheria toxoid should be immediately administered if the patient has not received a booster injection within 1 year.  Individuals should be placed on strict isolation (respiratory tract colonization) or contact isolation (cutaneous colonization only) until at least 2 subsequent cultures taken 24 hours apart after cessation of therapy demonstrate negative results. Repeat cultures should be performed at a minimum of 2 weeks after completion of therapy in patients and carriers; if results are positive, an additional 10-day course of oral erythromycin should be administered and follow-up cultures performed. 3, 4

Prevention of Maternal and Neonatal Diphtheria It has been recommended in some countries like the USA and Iran, that dT or Tdap vaccines should be administered to women of reproductive ages and pregnant women.75, 83 This is meant to boost their immunity to diphtheria (as well as tetanus and pertussis) and consequently prevent these diseases not only in these women but also in their infants.75 An Iranian study83 assessed the effectiveness of diphtheria toxoid vaccination in pregnancy and recommended as part of routine antenatal care, the administration of diphtheria-tetanus (dT) vaccine intramuscularly at the 4th and 6-7th month of gestation . Additionally, it has been noted that in the absence of high/adequate passively acquired diphtheria antibodies (< 0.1IU/ml), most infants would develop significant immune response to an additional diphtheria toxoid (DPT) dose given in the newborn period.29 There is however paucity of data on the immunogenicity of diphtheria toxoid vaccine administered to newborns particularly African (including Nigerian) neonates.

Justification for the Study

Diphtheria, a well known killer of children, is re-emerging.9, 12 This resurgence of diphtheria in several countries worldwide has drawn attention to the need for routine evaluation of the status of immunity against the disease in the different age groups in these countries.

Respiratory diphtheria during pregnancy has been reported.19-21 The mortality rate of the disease occurring during pregnancy can be as high as 50%.75 Diphtheria in pregnancy can become complicated by fetal loss or premature birth in up to a third of mothers who survive the disease.75 Parturient women with respiratory diphtheria can transmit C .diphtheriae through the airborne route to their newborns.19 Neonatal diphtheria, though an uncommon condition, has also been reported.23-26

Studies on immunity to diphtheria indicate that adolescents and adults (including females of child-bearing ages) have waned immunity to diphtheria.82, 84, 91, 119 In a Kenyan study82 only 53% of women of reproductive ages demonstrated adequate immunity to diphtheria. In Egypt as much as 30% of females within the reproductive ages had no protection (antibody < 0.01IU/ml) to diphtheria.91

Klouche et al35 reported low and inadequate level of anti-diphtheria antibodies in neonates, suggesting waning immunity in their mothers. A similar finding was reported by Nathenson and Zakzewski in the Bronx, New York.36 Ehrengut et al96 studied 185 mothers and their newborns and noted that only 29.2% of the mothers and 27% of their newborns had protective levels of anti-diphtheria antibodies in their sera.

In Nigeria there are no recent serological studies on immunity to diphtheria and no report has so far assessed the amount of anti-diphtheria antibody being passively transferred from Nigerian mothers to their newborns (to the best knowledge of this researcher). It is not known if, as in other parts of the world, adolescents and adults (including women of reproductive ages) have waned anti- diphtheria immunity. This is possible given the improvement in the immunization coverage and improvement in hygiene and social status resulting in fewer opportunities for the boosting of immunity through contact with cutaneous diphtheria.39, 41 Amongst the child population, neonates are particularly vulnerable since their immunity depends solely on what immunity they obtain from their mothers.29 Nigerian mothers are part of an older/adult population that may be similar to those that have been widely reported to have waning anti-diphtheria immunity. The thrust of this research therefore was to determine the level of antibodies against diphtheria in mothers and their newborns delivered in UBTH, Benin City, Nigeria. It is envisaged that, findings from this study will help to fill the identified knowledge gap.

AIMS AND OBJECTIVES

GENERAL OBJECTIVE:

To evaluate the status of immunity to diphtheria in mothers and their newborns delivered in the University of Benin Teaching Hospital (UBTH), Benin City, Nigeria

SPECIFIC OBJECTIVES:

1) To assay the IgG antibodies to diphtheria in mother-baby pairs seen in UBTH Benin City. 2) To determine the relationship between the levels of IgG anti-diphtheria antibodies in mothers and their babies in UBTH Benin City. 3) To identify any associations between the level of anti-diphtheria IgG antibodies in mothers and their babies and selected socio-demographic factors such as mothers’ age, immunization status, ethnicity and place of abode (urban versus rural) as well as family size and the family socio- economic class.

SUBJECTS AND METHODS

Study Design

This was a cross-sectional analytical study.

Ethical Considerations

Ethical approval for the study was obtained from the Ethics and Research Committee of UBTH, Benin City (appendix III). Informed written consent was obtained from subjects (appendix I).

Study Site

The study was carried out at the Antenatal Care unit and Labour Ward of the University of Benin Teaching Hospital (UBTH), Benin City, Edo State.

Edo State has a population of about 4 million and has a land mass of 19,794km square.120 The State lies approximately within 05 44N and 07 34N latitudes, 05 4E and 06 45E longitudes.120 Benin City, the capital of Edo State is situated in a moist tropical rainforest zone and lies about 80 metres above sea level.121 UBTH, founded in 1973 is a 700 bedded tertiary centre that provides primary, secondary and tertiary health care services to the people living in the immediate five Local Government Areas (Egor, Ikpoba-Okha, Oredo, Ovia North East and Ovia South West) of the state. It also serves as referral centre to the whole of Edo state and its neighbours; Delta, Ondo, Kogi and Anambra States. These states cut across South-South, South-West, North-Central and South-Eastern geo- political zones of Nigeria.

The Antenatal Care unit of UBTH records an average of 60 newly registered pregnant women weekly. The ANC holds on Mondays, Tuesdays, Thursdays and Fridays. About 42 babies are delivered weekly in the Labour Ward.

Sample Size

Sample size was calculated using the formula for determining minimum sample size described by Araoye122 using 95 percent confidence interval;

n = Z2pq

d2 where n= the desired sample size (for population size that is greater than 10,000)

Z = the standard deviation set at 1.96 which corresponds with 95% confidence interval p = the proportion in the target population (newborns) estimated to have protective levels of anti-diphtheria antibodies (≥ 0.1IU/ml). The prevalence of protective anti- diphtheria immunity in newborns in Mali was 81%.103

Therefore, P = 0.81

q = 1- p = 1 – 0.81 = 0.19

d = degree of accuracy was taken to be 5.0% or 0.05

Therefore n = (1.96)2 x 0.81 x 0.19 = 236

(0.05)2

The calculated sample size of 236 was for a population size greater than 10,000.

53 nf = the desired sample size for the study if the population was less than 10,000.122 nf = n 1+ (n/N)

N= the estimate of population size of babies delivered annually in the Labour ward of UBTH from the year 2000- 2008 (available from the delivery records in the Labour ward) = 2196

Therefore, nf= 236 ÷ 1+ (236/2196) = 213

A minimum sample size of 213 was calculated from the above formula.

However, to accommodate for serum samples that may be spilled and hence lost during assay, an attrition rate of 10% (that is, a response rate of 90%) was allowed for.122 Thus, the enlarged minimum sample size Ns was 237 using:

Ns = nf (90/100)

Where nf = the minimum calculated sample size – 213

Therefore, Ns = 213 0.9 = 237

Study Subjects

Pregnant women in their third trimester of pregnancy were enrolled during their ANC visits and informed consent obtained. Both mothers and their babies who met the inclusion criteria were then recruited into the study immediately after delivery in the Labour Ward of UBTH.

Inclusion Criteria

Mothers

1. Parturient women who registered in UBTH for antenatal care (ANC) and who received adequate ANC follow-up (a minimum of 4 visits and at least 1 in each of the 3 trimesters of pregnancy was deemed adequate123). This was necessary because information on mothers’ state of health and medication was sourced from their ANC records. 2. Mothers who delivered in UBTH 3. Mothers who gave informed consent Newborns 1. Babies born in UBTH labour ward 2. Babies whose mothers received regular ANC in UBTH 3. Healthy term newborns (that is, babies whose gestational age at delivery was within 37 to 41 completed weeks). 4. Babies with birthweights within 2500gm – 3999gm.

Exclusion Criteria

1. Mothers with the following characteristics: (a) Mothers whose pregnancies were not registered at nor received antenatal care at UBTH (since information on mothers’ state of health and medications was sourced from their ANC records). (b) Mothers booked in UBTH but with inadequate ANC follow-up (for the same reason as stated above). (c) Mothers with history or clinical/laboratory information suggestive of medical conditions that may have affected utero-placental

functions and/or maternal immunity, for example, maternal anaemia, cardio-pulmonary diseases, hypertensive diseases, HIV/AIDS infection, malignancies, diabetes mellitus, Sickle cell disorder. Clinical findings such as maternal antenatal blood pressure records and results from antenatal laboratory work-up like packed cell volume, genotype and retroviral screening were used to identify the presence of exclusion criteria. (d) Mothers on medications that may have affected utero-placental functions and/or maternal immunity such as, cytotoxic drugs and steroids, and those who received blood or blood products during the index pregnancy.

2. Babies with features suggestive of illnesses like perinatal asphyxia, neonatal sepsis, immune or non-immune hydrops fetalis, twin-twin transfusion or disseminated intravascular coagulopathy. 3. Babies of multiple gestation and those delivered via Caesarean section.

Informed Consent

The purpose of the research was explained to prospective mothers in their third trimester of pregnancy. A written consent was obtained prior to delivery (Appendix I).

Recruitment of Patients

Subjects who met the inclusion criteria were recruited consecutively until the sample size was met.

Data collection

The study proforma (Appendix II) was used to obtain information from mothers recruited for the study. It sought information on the mother’s bio-data, immunization history and socio-demographic data. The socio-economic status of each child’s family was determined using the Olusanya et al124 classification.

Socio-Economic Classification

Using the scoring system described by Olusanya et al124 socio-economic classification was determined as follows:

A. Father’s occupation was assigned score ranging from 1 – 3 as follows;

1) Professionals, top civil servants, politicians and businessmen 2) Middle level bureaucrats, technicians, skilled artisans and well-to-do traders 3) Unskilled workers and those in general whose incomes were at or below the minimum wage

B. Mother’s highest level of educational attainment was assigned a score of 0 – 2 as follows;

0 Education up to the University level 1 Secondary or tertiary level below the University (for example, College of Education, School of Nursing) 2 No schooling or up to primary level only.

Each baby’s family social class was obtained by adding the respective scores in A and B above. A score of 1 represented social class I. A score of 2 represented social class II. A score of 3 represented social class III. A score of 4 represented social class IV, while a score of 5 represented social class V. Social classes I and II

57 represented upper class, class III the middle class, while IV and V belonged to the lower class.

For example, where the father of a baby was a Secondary school teacher (score = 2) and the educational attainment of the mother was primary six (score = 2), the combined score for this family was 4. This family was of social class IV and was classified as belonging to the lower class.

Household size Classification

Household sizes were classified into 2 categories; small/average and large sized households based on a national average household size of 5.0persons/household.125 A household with less than 6 persons was classified as a small/average household, while one with 6 or more persons was classified as a large household.

Classification of Place of Abode

The subjects’ places of abode were classified into urban and rural settings in line with the Ministry of Lands, Housing and Survey, Benin City, Edo State which classified communities in the state into urban and rural using the following criteria;126, 127

 Population density  Predominance of agricultural related livelihood  Availability of infrastructural services.

A rural community was identified by the presence of low population density, predominance of agricultural-related livelihood and poor infrastructural services while urban communities were identified following exclusion of the rural areas.126, 127

Clinical Evaluation

Each baby recruited for the study was examined. For each baby, the gestational age at birth was determined from both the date of the mothers’ Last Menstrual Period (LMP) and the Dubowitz and Dubowitz (D&D)128 gestational age estimation chart. Where the estimated gestational ages derived from the LMP and D&D differed by more than a week that derived from D&D was used.128 The sex was recorded while the weight was measured using Waymaster® baby weighing scale which had a degree of accuracy of 50g. The scale was calibrated prior to use. Babies were weighed naked. The occipito-frontal circumference (OFC) was measured using a measuring tape that was evenly calibrated, inelastic and made of shrink-resistant material (degree of precision – 0.1cm). The head circumference is the longest measurement around the head usually in the occipito- frontal plane.129 The tape was applied firmly over the glabella and supraorbital ridges anteriorly and that part of the occiput posteriorly that gave the maximum circumference.129 As a precaution, the measuring tape used for measurement was checked daily against a standard ruler.129

The length was measured, using an infantometer. The infantometer used was Seca Infantometer® which has a measuring range of 33cm to 100cm and a limit of accuracy of 0.1cm. It consists of a fixed head piece, a horizontal backboard, an adjustable foot piece and a measurement indicator attached to the adjustable foot piece. The midwife attending to the baby assisted during measurement of the length. The baby with all clothing removed was placed lying prone on the backboard of the infantometer. The baby was positioned such that the feet were toward the foot piece and the head was towards the fixed head piece. With the help of the assistant, the baby’s head was supported at right angle to the head piece, so

59 that the inner and outer canthi of the eyes were in the vertical plane. 129 Gentle traction was applied to the baby’s neck to bring the top of the head in contact with the fixed head piece and to straighten out excessive lumbar lordosis. The assistant secured the head of the baby in this position by lightly cupping her hands over the ears of the baby. The examiner then aligned the child’s legs by placing his hand gently but with mild pressure over the knees and with the other hand slide the foot piece to rest firmly at the sole of the feet. Care was taken to ensure that the toes point directly upward with both soles of the feet flexed perpendicular against the foot piece. The examiner then noted and documented the reading of the indicator as the length of the baby. The values of the babies weight in kilogramme as well as the OFC and lengths in centimeters were recorded (Appendix II).

Specimen Collection:

About 3ml of blood was collected from all enrolled mothers using aseptic procedure, into a plain universal bottle. Samples were collected from mothers immediately after delivery. About 3ml of cord blood was collected from babies immediately following birth.

Procedure for Specimen Collection130

A. Venepuncture (mothers): 1. The sampling procedure was explained to the mother 2. Plain universal bottle was appropriately labeled with her name and date of sampling and tagged with a unique serial number and the alphabet “A” 3. Her arm (that of her upper limb that is free of IV line for fluid and/or drugs administration) was held in a fully extended position with the palmar surface upward (for antecubital access).

4. Using a 21-G needle attached to a 5ml syringe, 3ml of blood was withdrawn. 5. With the needle detached from the syringe, the blood sample was emptied into the pre-labeled universal bottle. 6. Sharps were discarded appropriately B. Cord blood (babies): 1. A Universal bottle was pre-labeled with a serial number similar to that of the mother’s but with the alphabet “B” included. 2. Using a 5ml syringe and 21-G needle, 3ml of blood was withdrawn from large veins on the foetal side of the placenta immediately after delivery.94 3. With the needle detached from the syringe, the blood was emptied into the pre-labeled universal bottle 4. Sharps were discarded appropriately.

The sera of both the mother’s and baby’s blood were separated by centrifugation. The sera were stored in a refrigerator at 4°C and analyzed within 72 hours of collection.112

Laboratory Methods

Laboratory testing of specimens was carried out at the University of Benin Teaching Hospital Chemical Pathology laboratory. The ELISA technique was used for the study. Serion ELISA Classic Diphtheria IgG®, a commercially available kit, was used to assay anti-diphtheria antibody titres. It is based on a quantitative immunoassay technique that is specific for diphtheria IgG antibody.112

Materials

The materials used for the laboratory tests included;

1. Six Serion ELISA Classic Diphtheria IgG® Kits

Components of each kit;

 12 Break-apart microtiter test strips each with 8 antigen coated single wells (altogether 96 test wells).  Standard serum (ready-to-use) – for positive control  Negative control serum (ready-to-use)  Anti-human-IgG – conjugate (ready-to-use)  Washing solution concentrate (Sodium chloride with Tween 20, 30 mM Tris), sufficient for 1000ml after dilution.  Dilution buffer (Phosphate buffer with protein and Tween 20)  Stopping solution (1.2 N sodium hydroxide)  Substrate (Para-nitrophenylphosphate) - ready-to-use  Standard curve and Evaluation table

2. Photometer for microtiter plates reading (at 405nm wavelength). 3. Incubator (37°C) 4. Moist chamber 5. Distilled water

Assay Method112

 The subject’s serum was diluted using the dilution buffer solution in a 1:100 dilution ratio. To achieve this, 10µl of each subject’s serum was added to 1000µl of dilution buffer. This mixture is gently shaken for 2 minutes to ensure adequate mixing and a homogenous solution.

 Using a pipette, 100µl of the diluted serum was added into the micro-test wells pre-coated with diphtheria toxin (supplied with the kit).  The test well with the sample was incubated for 60mins at 37°C in a moist chamber. Any antibodies in the serum that are specific for the antigen (diphtheria toxin), formed an immune complex with the antigen.  After incubation, the micro-test well were washed with the washing solution  Then using a pipette, 100 µl of anti-human-IgG conjugate was added to the micro-test well  The test well was again incubated for 30mins at 37°C in a moist chamber  After incubation, the micro-test well was washed with the washing solution  Then using a pipette, 100 µl of substrate (Para-nitrophenylphosphate) solution was added to the micro-test well  The micro-test well was then incubated for 30mins at 37°C in a moist chamber. There was a resultant colour change of the contained solution. The intensity of the colour is dependent on the amount of bound anti-diphtheria antibodies.  After incubation, 100 µl of stopping solution was added to the micro-test well  Within 5mins of adding the stopping solution, the micro-test well was inserted into a corresponding micro socket of Stat Fax 303/Plus photometer® (a micro-titre ELISA reader). The optic reading range of the photometer was 405-630nm wavelength with a photometric accuracy of +/- 1%.  The photometer wavelength was set at 405nm and the optic density of the content of the micro-test well was read and recorded.

A batch of a maximum of 8 samples were worked on and read by the photometer at a time. It took an average of 3 hours to run a batch of samples.

Interpretation of results

The interpretation of the optic density readings derived from the above laboratory procedure is based on the principle behind the test. On addition of the substrate solution, the test solution either retained its straw colour if there were no IgG anti-diphtheria antibodies in the patient’s serum or developed a blue colour change if such antibodies were present. The intensity of the blue colour is proportional to the amount of antibodies that had been bound to the microtest well. Thus, the optic density is proportional to the amount of anti-diphtheria antibodies in the patient’s serum. For the test evaluation a standard curve is included in the test kit (using the positive and negative control sera provided with the kit). On the vertical or y-axis are optic density (OD) values while on the horizontal or x-axis are the corresponding antibody concentration in IU/ml. After completion of the test procedure, the OD of each test sample was derived using the photometer. Then these OD-values (photometer readings) of the patients’ samples were then assigned to their corresponding antibody activity in IU/ml using the standard curve. The range of reliability of the test was 0.005-2.0IU/ml.

The test results for each mother and baby was recorded into the appropriate proforma (Appendix II).

Quality Control

The laboratory procedures were carried out by the researcher with assistance from two laboratory scientists in the Chemical Pathology laboratory of UBTH. Prior to the commencement of the study, the researcher underwent a 2 months

64 training (June 1st –July 30th, 2010) in the relevant laboratory methods under a senior laboratory scientist at the Chemical Pathology department of UBTH.

In order to maintain reliable test results, every 10th assay of mother-baby paired sera carried out by the researcher was repeated by the senior laboratory scientist in the chemical pathology unit and the results compared.

DATA ANALYSIS

Data was entered into an SPSS spreadsheet (version 18.0). The mean antibody titres for mothers and babies were determined. Subjects’ immunity to diphtheria as determined from the laboratory tests was classified using WHO guidelines80 as follows;

Serum anti-diphtheria antibodies (IU/ml) Evaluation  < 0.01 No protection  0.01 - < 0.1 Minimal protection  0.1 - < 1.0 Safe protection  > 1.0 Long term protection A serum antibody titre of at least 0.1IU/ml was the cut off for protective anti- diphtheria immunity in the study. Thus babies and mothers were also classified as follows;

Serum anti-diphtheria antibodies (IU/ml) Evaluation  < 0.1 Inadequate protection  ≥ 0.1 Adequate protection The proportion of mothers and babies with different levels of immunity was recorded as percentages.

Association of socio-demographic factors with the levels of protection against diphtheria were assessed using Chi square (with Yate’s correction and Fisher’s exact test where appropriate). Differences between means were assessed using the Student’s t test (and Analysis of Variance, ANOVA where appropriate). The paired t test was used to compare the antibody titres of mother-baby pairs.

Correlation between maternal and newborn antibody titres was determined using Pearson’s correlation test. In all statistical tests, p-value <0.05 was considered statistically significant.

RESULTS

The study was carried out over a period of 8 months (September 2010 – April 2011). One thousand two hundred and eighty four women received antenatal care during the study period. Of these, 684 eligible women in their third trimester were followed up till delivery. Only 572 of those followed up gave birth to their babies in the Labour ward during the study period. Of those who delivered in the Labour ward, 240 mother-baby pairs met the inclusion criteria and were recruited. However, 9(3.8%) mother-baby pairs were excluded from analysis on account of spillage during laboratory analysis. Thus, a total of 231 mother-baby pairs were analyzed in the study. Socio-demographic characteristics of the study population

The mean age of mothers was 29.6 ± 4.54 years with a range of 16 – 46 years. The highest proportion of mothers, 110/231 (47.6%) were in the age range 26 – 30 years. The age distribution of the mothers studied is as shown in Figure 1.

Figure1: Age distribution of mothers

The other socio-demographic characteristics of the subjects are shown in Table I. The subjects places of residence were in the following Local Government Areas; Egor, Oredo, Ovia North East and Ovia South West (all in Edo State). Places of abode were urban in 208(90.0%) cases while 23(10.0%) were from rural areas. Majority 95(41.1%) were Bini while none was of Hausa ethnicity. The mean number of persons per household was 4.2 ±1.39 with a range of 2 – 8 persons.

Table I: Socio-demographic characteristics of the mothers.

Characteristics Mothers n %

Place of abode Rural 23 10.0 Urban 208 90.0 Total 231 100.0

Ethnicity Bini 95 41.1 Ibo 48 20.8 Esan 33 14.3 Yoruba 16 6.9 Etsako 15 6.5 Urhobo 10 4.3 Others 14 6.1 Total 231 100.0

Family size (persons/household) Small/Average (≤ 5) 215 93.1 Large (≥ 6) 16 6.9 Total 231 100.0

Socio-economic status of the family Socio-economic class I & II (Upper class) 103 44.6 Socio-economic class III (Middle class) 79 34.2 Socio-economic class IV & V (Lower class) 49 21.2 Total 231 100.0

Immunization history Card showed fully immunized 4 1.7 Card showed incomplete DPT immunization 1 0.4 Fully immunized, by history 93 40.3 Incompletely immunized, by history 29 12.6 Immunization status unknown 103 44.6 Told not immunized 1 0.4 Total 231 100.0

Of the 231 mother-baby pairs 103 (44.6%) belonged to the upper social class, 79(34.2%) to the middle social class and 40(21.2%) to the lower social class. Majority of the mothers, 103(44.6%) did not know their vaccination status. Of the 231 babies, 97(42.0%) were male and 134(58.0%) were female, giving a male: female ratio of 1:1.4. The mean birth weight was 3.2±0.4kg with a range of 2.5 – 3.9kg. Table II shows the characteristics of the babies. Most of the babies 101(43.7%) weighed between 3.0 and 3.4kg while the majority of the babies were born at gestational ages of 38 weeks 69(29.9%) and 39 weeks 61(26.4%).

Table II: Characteristics of babies.

Characteristics Babies n %

Sex Male 97 42.0 Female 134 58.0 Total: 231 100.0

Birth weight (kg) 2.5 – 2.9 53 23.0 3.0 – 3.4 101 43.7 3.5 – 3.9 77 33.3 Total: 231 100.0

GestationalAge (weeks) 37 40 17.3 38 69 29.9 39 61 26.4 40 46 19.9 41 15 6.5 Total: 231 100.0

Diphtheria immunity levels in mother-baby pairs The mean anti-diphtheria antibodies titre in the mothers was 0.23±0.29 IU/ml with a range of 0 – 1.00IU/ml while that of the babies was 0.19 ± 0.24 IU/ml with a range of 0 – 1.00IU/ml. Majority of the mothers 133(57.6%) and babies 123(53.2%) had safe protection. However, as much as 69 (29.9%) mother-baby pairs had no protection from diphtheria. Prevalence of the different diphtheria immunity levels in mother-baby pairs is shown in Table III.

Table III: Immunity status for diphtheria in mother-baby pairs.

Immunity status (antibody titre) Mothers Babies n(%) n(%)

No protection (<0.01IU/ml) 69(29.9) 69(29.9)

Minimal protection (0.01 - <0.1IU/ml) 21(9.1) 38(16.5)

Safe protection (0.1 - <1.0IU/ml) 133(57.6) 123(53.2)

Long term protection (>1.0IU/ml) 8(3.4) 1(0.4)

Total 231(100.0) 231(100.0)

Figure 2 shows the adequacy of protection from diphtheria in mothers and their babies (inadequate immunity = antitoxin titre < 0.1IU/ml and adequate immunity = titre ≥ 0.1IU/ml). One hundred and forty one (61.0%) mothers and 124(53.7%) babies had adequate immunity to diphtheria.

70.0% 61.0% 60.0% 53.7%

50.0% 46.3%

39.0% 40.0% Mothers 30.0% Babies

20.0%

10.0%

0.0% Adequately protected Inadequately protected

Figure 2: Prevalence of protective immunity in mother-baby pairs.

The mean antibody titre of male babies was 0.20±0.25IU/ml while that of females was 0.19±0.24IU/ml. The difference in the mean titres of male and female babies was not statistically significant (t = 0.31, p = 0.76). Using antibody titre of 0.1IU/ml as cut-off level for adequate protection, a higher proportion 79/134 (59.0%) of female babies were adequately protected compared to that of males 45/97 (46.4%). This difference was also not significant (p = 0.06 Fischer’s exact, 95% CI = 0.55 to 1.01). Table IV shows the relationship between babies’ characteristics and their anti-diphtheria immunity. Table IV: Association between Babies’ Characteristics and their anti- diphtheria immunity.

Characteristics Mean antibody titres(IU/ml) Immunity status Total

Adequate Inadequate n(%) n (%) n (%)

Sex (n)

Male (97) 0.20±0.25 45(46.4) 52(53.6) 97(100.0) Female (134) 0.19±0.24 79(59.0) 55(41.0) 134(100.0) Total (231) 231 p-value 0.76 0.06

Birth weight in kg(n)

2.5-2.9 (53) 0.19±0.24 28 (52.8) 25 (47.2) 53(100.0) 3.0-3.4 (101) 0.15±0.22 47 (46.5) 54 (53.5) 101(100.0) 3.5-3.9 (77) 0.25±0.27 49 (63.6) 28 (36.4) 77(100.0) Total (231) 231 p-value 0.03 0.08

Gestational age in weeks(n)

37 (40) 0.16±0.20 20 (50.0) 20 (50.0) 40(100.0) 38 (69) 0.17±0.25 37 (53.6) 32 (46.4) 69(100.0) 39 (61) 0.20±0.26 32 (52.5) 29 (47.5) 61(100.0) 40 (46) 0.24±0.25 29 (63.0) 17 (37.0) 46(100.0) 41 (15) 0.17±0.26 6 (40.0) 9 (60.0) 15(100.0) Total (231) 231 p-value 0.53 0.56

The mean antibody titres of babies with different ranges of birthweight were significantly different (ANOVA: F = 3.98, p = 0.02). The highest mean antibody titre 0.25±0.27IU/ml was observed in babies with birthweights within the range 3.5 – 3.9kg while the lowest 0.15±0.22IU/ml was observed in those with birthweights within 3.0 – 3.4kg. However, no significant relationship was observed in the mean titre values with increasing or reducing ranges of birth weight. (Regression, r2 = 0.07, p = 0.22). Relationship between maternal and baby anti-diphtheria immunity The mean antibody titre 0.23±0.29 IU/ml of mothers was not significantly different from the mean titre 0.19±0.25 IU/ml of their babies (t=1.59, P = 0.11, 95% CI = −0.09 - 0.01). However, there was a significant positive correlation between the levels of immunity to diphtheria in mothers and their babies (“r” = 0.982, p = < 0.0001). As much as 124 (87.9%) babies of 141 mothers with adequate immunity were equally adequately protected while all the 90 (100%) babies of mothers with inadequate immunity were themselves inadequately protected. Figure 3 shows the positive correlation between maternal antibody titres and those of babies

1.400

1.200 y = 1.155x + 0.0122 R² = 0.9644

1.000

0.800

0.600

0.400 Babies' Antibody Babies' (IU/ml) titres

0.200

0.000 0.000 0.200 0.400 0.600 0.800 1.000 1.200 Mothers' Antibody titres (IU/ml)

Figure 3: Relationship between antibody titres in mother-baby pairs.

However, 17/141 (12.1%) mothers who had adequate immunity gave birth to babies with inadequate protection. The mean antibody titre of mothers who successfully transferred adequate immunity to their babies, 0.40±0.30IU/ml was significantly higher than the mean, 0.14±0.13IU/ml of those mothers who failed to transfer adequate immunity to their babies (t = 6.27, df =44, p < 0.0001). The minimum maternal antibody titre at which adequate immunity was transferred to babies was 0.10IU/ml. However, 14 of the 17 mothers who failed to transfer their adequate immunity to their babies, had antibody titre of 0.1IU/ml. The remaining 3 mothers had titres up to 0.65IU/ml. All mothers who had titres > 0.65IU/ml successfully transferred adequate immunity to their babies.

Association between the level of anti-diphtheria IgG antibodies and some socio-demographic factors

Maternal Age: No significant association was observed between maternal age and maternal antibody titres: r2 = 0.005. Further analysis however showed that mothers in the age range of 41 years and above had the lowest mean anti- diphtheria antibody titre of 0.08±0.00IU/ml while mothers in age range of 26-30 years had the highest mean value of 0.26±0.33IU/ml. The difference between the means of the different age range were not statistically significant (ANOVA: F=1.40, p = 0.23). Table V shows the association between maternal age groups and the level of anti-diphtheria immunity in mothers and their babies. Babies of mothers within the age range of 41years and above had the lowest mean antibody titre of 0.08 ±0.00IU/ml while babies of mothers in the age range of 26-30years had the highest mean of 0.22 ±0.28IU/ml. The difference in mean antibody titres of babies of mothers from the different age range were also not statistically significant (ANOVA: F = 1.37, p = 0.24).

Table V: Association between maternal age groups and anti-diphtheria immunity in mother-baby pairs.

Mothers Babies

Age in years(n) Mean titres(IU/ml) Immunity status Mean titres(IU/ml) Immunity status

Adequate Inadequate Adequate Inadequate

n (%) n (%) n (%) n (%)

16-20(7) 0.21±0.31 3(42.9) 4(57.1) 0.19±0.29 2(28.6) 5(71.4) 21-25(28) 0.11±0.13 18(64.3) 10(35.7) 0.09±0.10 14(50.0) 14(50.0) 26-30(110) 0.26±0.33 69(62.7) 41(37.3) 0.22±0.28 61(55.5) 49 (44.5) 31-35(64) 0.25±0.27 39(60.9) 25(39.1) 0.20±0.23 37(57.8) 27(42.2) 36-40(20) 0.21±0.26 12(60.0) 8(40.0) 0.18±0.23 10(50.0) 10(50.0) ≥ 41 (2) 0.08±0.00 0(0.0) 2(100.0) 0.08±0.00 0(0.0) 2(100.0) Total (231) 141 90 124 107 p-value 0.23 0.50 0.24 0.42

Maternal childhood vaccination status: Table VI shows the association between maternal childhood vaccination status and the level of anti-diphtheria immunity in mothers and their babies. Mothers with evidence of complete primary series of diphtheria toxoid vaccination had a lower mean anti-diphtheria antibody titre of 0.08±0.06IU/ml compared to the mean value 0.24±0.27IU/ml of those who were not aware of their vaccination status. However, the difference between the mean titres of mothers from the different categories of vaccination status was not statistically significant (ANOVA: F = 0.42, p = 0.74).

Table VI: Association between maternal vaccination status and anti- diphtheria immunity in mother-baby pairs.

Mothers Babies

Immuni- Mean titres(IU/ml) Immunity status Mean titres(IU/ml) Immunity status zation Adequate Inadequate Adequate Inadequate status(n) n (%) n (%) n (%) n (%)

A (4) 0.08±0.06 2(50.0) 2(50.0) 0.06±0.04 2(50.0) 2(50.0) B (1) 0.50 1(100.0) 0(0.0) 0.40 1(100.0) 0(0.0) C (93) 0.24±0.30 53(57.0) 40(43.0) 0.20±0.26 43(46.2) 50(53.8) D (29) 0.22±0.36 14(48.3) 15(51.7) 0.18±0.29 12(41.4) 17(58.6) E (103) 0.24±0.27 70(68.0) 33(32.0) 0.19±0.23 65(63.1) 38(36.9) F (1) 0.10 1(100.0) 0 (0.0) 0.10 1(100.0) 0(0.0) Total (231) 141 90 124 107 p-value 0.74 0.29 0.73 0.10

A = card evidence of full vaccination, B = card evidence of incomplete DPT vaccination, C = fully vaccinated, by history, D = incompletely vaccinated, by history, E = not aware of vaccination status, F = told not vaccinated.

Maternal ethnicity: Mothers of Esan ethnicity had the highest mean anti- diphtheria antibody titre 0.28±0.30IU/ml compared with the urhobos who had the lowest mean titre 0.20±0.35IU/ml. There was however no statistically significant difference between the mean titres of the various ethnic groups (ANOVA: F = 0.26, p = 0.95). Urhobo mothers were more likely (60%) to have babies with inadequate immunity to diphtheria while those mothers of other ethnicity such as the itsekiris, isokos and ijaws were least (14.3%) likely to have babies with inadequate immunity. There was however no significant difference between the prevalence of adequately and inadequately protected babies based on maternal ethnicity (x2 = 10.41, p = 0.11). Table VII shows the association between maternal ethnicity and the level of anti-diphtheria immunity in mothers and their babies.

Table VII: Association between maternal ethnicity and anti-diphtheria immunity in mother-baby pairs.

Mothers Babies

Ethnicity(n) Mean titres(IU/ml) Immunity status Mean titres(IU/ml) Immunity status

Adequate Inadequate Adequate Inadequate n (%) n (%) n (%) n (%)

Bini (95) 0.22±0.29 50 (52.6) 45(47.4) 0.18±0.25 43(45.3) 52(54.7) Esan (33) 0.28±0.30 23(69.7) 10(30.3) 0.23±0.24 18(54.5) 15(45.5) Etsako (15) 0.23±0.33 9(60.0) 6(40.0) 0.19±0.30 9(60.0) 6(40.0) Urhobo (10) 0.19±0.35 4(40.0) 6(60.0) 0.15±0.26 4(40.0) 6(60.0) Ibo (48) 0.22±0.29 31(64.6) 17(35.4) 0.18±0.27 28(58.3) 20(41.7) Yoruba (16) 0.23±0.29 12(75.0) 4(25.0) 0.20±0.25 10(62.5) 6(37.5) Others (14) 0.27±0.23 12(85.7) 2(14.3) 0.21±0.17 12(85.7) 2(14.3) Total (231) 141 90 124 107 p-value 0.95 0.09 0.96 0.11

Maternal place of abode: The association between maternal place of abode and the level of anti-diphtheria immunity in mothers and their babies is shown in Table VIII. Table VIII: Association between maternal place of abode and anti-diphtheria immunity in mother-baby pairs.

Mothers Babies

Place of Mean titres(IU/ml) Immunity status Mean titres(IU/ml) Immunity status abode (n) Adequate Inadequate Adequate Inadequate n (%) n (%) n (%) n (%)

Rural (23) 0.42±0.36 17(73.9) 6(26.1) 0.35±0.30 16(69.6) 7(30.4) Urban (208) 0.21±0.27 124(59.6) 84(40.4) 0.17±0.23 108(51.9) 100(48.1) Total (231) 141 90 124 107 p-value 0.01 0.26 0.01 0.13

Mothers who resided in urban settings had significantly lower mean anti-diphtheria antibody titre 0.21±0.27IU/ml than the mean 0.42±0.36IU/ml of those who resided in rural settings (t = 2.71, df = 24, p = 0.01, 95%CI = -0.37 to -0.04). Similarly, babies of mothers from rural settings had a significantly higher mean titre 0.35±0.30IU/ml than the mean titre 0.17±0.23IU/ml of babies born to mothers from urban settings (t = 2.79, df = 24, p = 0.01, 95% CI = 0.31 to 0.04)

Household size: Table IX shows the association between household size and the level of anti-diphtheria immunity in mothers and their babies. Mothers from families of small/average household sizes had a slightly lower mean antibody titre 0.23±0.28IU/ml compared to the mean titre 0.24±0.32IU/ml of those from large sized households. The difference in these mean titres was however not significant (p = 0.95). There was no statistically significant difference between the mean titres of babies born to mothers from small/average sized households compared to those from large sized households (t = 0.00, p = >0.99, 95%CI = -0.01 to 0.01). Table IX: Association between household size and anti-diphtheria immunity in mother-baby pairs.

Mothers Babies

Household Mean titres(IU/ml) Immunity status Mean titres(IU/ml) Immunity status

size (n) Adequate Inadequate Adequate Inadequate n (%) n (%) n (%) n (%)

≤5 (196) 0.23±0.28 122 (62.2) 74(37.8) 0.19±0.24 106(54.1) 90(45.9) ≥6 (35) 0.24±0.32 19 (54.3) 16 (45.7) 0.19±0.26 18(51.4) 17 (48.6) Total (231) 141 90 124 107 p-value 0.86 0.45 >0.99 0.86

Family socio-economic status: Mothers from families of the upper socio- economic class had mean antibody titre of 0.25±0.30IU/ml while mean titre 0.20±0.24IU/ml was observed in mothers in the lower class. The differences in the mean titres of mothers from the various socio-economic classes were however not statistically significant (ANOVA: F = 0.50, p = 0.61). The levels of immunity

77 transferred to babies from mothers of different socio-economic class were not significantly different (x2 = 1.80, p = 0.41). Table X shows the association between family socio-economic status and the mothers and babies anti-diphtheria immunity. Table X: Association between family socio-economic status and anti- diphtheria immunity in mother-baby pairs.

Mothers Babies

Socio- Mean titres(IU/ml) Immunity status Mean titres(IU/ml) Immunity status economic Adequate Inadequate Adequate Inadequate class (n) n (%) n (%) n (%) n (%)

Upper (103) 0.25±0.30 63(61.2) 40(38.8) 0.20±0.26 51(49.5) 52(50.5) Middle (79) 0.23±0.30 51(64.6) 28(35.4) 0.19±0.25 47(59.5) 32 (40.5) Lower (49) 0.20±0.24 27(55.1) 22(44.9) 0.17±0.21 26(53.1) 23 (46.9) Total (231) 141 90 124 107 p-value 0.61 0.57 0.78 0.41

DISCUSSION

The result of this study has demonstrated that 61% of mothers and 53.7% of their babies are adequately protected against diphtheria while a significantly high percentage of mothers (39%) and babies (46.3%) are inadequately protected against diphtheria. Similar high prevalence of inadequate immunity to diphtheria has been observed in Europe,35, 84, 96 Egypt,91 and Kenya.82 This may be attributed to waning of immunity in mothers in this study as have been reported in women of similar ages in other studies.35, 82, 91 Waning immunity in older persons such as the mothers in this study has been linked to large-scale immunization of children with diphtheria toxoid which results in high levels of immunity in children and considerable decline in diphtheria incidence.4, 29 As the number of clinical cases of diphtheria declines, asymptomatic carriage of C. diphtheriae becomes rare.84 This leads to decreased opportunity for exposure to the organism with resultant decrease of immunity boosting effect associated with such exposure.29

Reduced exposure to recurrent subclinical infections may also be attributed to documented improvement in personal and environmental hygiene in our locale.28, 39 Subclinical diphtheria infections especially cutaneous forms have been linked to settings of poor personal and environmental hygiene.29 Booster doses of diphtheria toxoid or recurrent subclinical diphtheria infections are required to sustain anti- diphtheria immunity acquired following childhood vaccination into adulthood.29 This may explain why a significant proportion of the subjects had inadequate anti- diphtheria immunity.

Since herd immunity is provided when at least 70-80% of a target population is adequately protected,4 the implication of only 61% of mothers and 53.7% of babies having adequate anti-diphtheria immunity is that these sections of the

79 population may be susceptible to diphtheria epidemics as well as complications of the disease.77

There is a paucity of data on the prevalence of immunity to diphtheria in the Nigerian populace, and particularly that of mothers and their newborns. However, the finding of only 61% of mothers with adequate immunity to diphtheria in this study is in contrast to an earlier Nigerian work38 that reported 85% prevalence of adequate immunity in women of similar ages. The difference may be due to the fact that unlike the earlier Nigerian work,38 this study was conducted after the commencement and widespread coverage of childhood vaccination with diphtheria toxoid in this contry.40, 41 Secondly, since this earlier study38 was carried out, there has been documented improvement in the level of personal and environmental hygiene in Nigeria.39 Poor personal and environmental hygiene have been associated with increased frequency of cutaneous diphtheria. This form of diphtheria is commonly subclinical and is known to boost and maintain anti- diphtheria immunity into adulthood.28, 29, 73 This may explain why the women in this study were significantly less protected than their counterparts of similar ages in the earlier Nigerian study.38

The finding of this study was also in contrast to that in Mali where all (100%) the mothers and 81% of their babies studied had adequate immunity to diphtheria.103 This difference may be attributed to the fact that the Malian study was also conducted before widespread coverage of childhood vaccination against diphtheria. 102

The transplacental transfer of anti-diphtheria antibodies was very efficient in this study. This is supported by the strong positive correlation between the maternal and baby’s antibody titres (correlation coefficient = 0.98). This finding is

80 comparable to what has been observed by other workers.37, 96 This strong correlation has been linked to an active transport mechanism involved in the transfer of antibodies from the mother to the fetus via the placenta.29 The significance of this is that newborns and young infants can effectively be protected against diphtheria by ensuring their mothers maintain adequate levels of immunity to diphtheria through the child-bearing ages. This is particularly important as the benefit of active immunity is achieved only after the second dose of DPT (given at the 10th week of life).84

Some other workers have however reported large differences between maternal and infant anti-diphtheria immunity.83, 103 In Iran for example,83 the difference in the prevalence of adequate immunity to diphtheria in mothers and their babies was 17% compared to the 7.3% observed in this study. This difference may be attributed to the difference in the timing of collection of the infants’ blood samples. While in this study blood samples were collected from the cords, in the Iranian study, samples were collected at 2 months of age, just before commencement of DPT vaccination. Studies have indicated a steady loss of diphtheria antibody after birth.29, 30 It has been reported that the average blood level of antitoxin of 10 day old babies may be as low as half that of the cord blood and subsequently babies lose half their antitoxin every four and half weeks.29, 30 This may explain why a lower proportion of babies in the Iranian study, compared to their mothers, had adequate protection in contrast to the findings of this study. In Mali on the other hand,103 the difference between the prevalence of adequately protected mothers and their babies was 19%. This difference may be attributed to the lower sensitivity of the assay method in the Malian study which may have been unable to detect lower newborns antibody titres.29, 97, 98 Thus, the antibody titres of

81 the babies in the Malian study may have been underestimated in comparison to their mothers’.

Increase in anti-diphtheria antibody titres with increasing birth weights was observed. This finding is comparable to that of a Gambian study94 where diphtheria antibody titres in newborns also increased with increasing birth weights. This finding may be explained by the fact that placental transfer of antibodies may be reduced in the presence of such factors as intra-uterine growth restriction that is associated with lower birth weights.94, 95 This study also noted increase in antibody titre with increasing gestational age (up to 40 completed weeks). This finding is similar to that of the Gambian study94 which also noted increase of antibody titres with gestational age at birth. This finding may be linked to increase in birth weight with gestational age as well as increase in the activity of the placental receptors responsible for the transfer of antibodies to the fetus with gestational age.94 However, this is up to a limit as post-maturity is associated with decreased placental functions.95

Waning of immunity to diphtheria with age was observed in this study. Women of older ages groups had lower mean anti-diphtheria antibody titres and were less protected against diphtheria than younger women. This finding is similar to what was noted in other works.29, 35, 91 This has been linked to declining rate of exposure to natural subclinical diphtheria infections with age resulting from increasing coverage of childhood vaccination against diphtheria on one hand and improving personal and environmental hygiene on the other hand. Recurrent subclinical infections or booster doses of diphtheria toxoid vaccines are required to maintain immunity to diphtheria into adulthood.29 The implication of this finding is that babies may be born with inadequate protection from diphtheria as some women may have children at much older age.

History of childhood vaccination history did not significantly influence the level of immunity to diphtheria in the subjects. However, the women studied were born in the period (1969-1994) when documented DPT3 coverage ranged from less than 5% before 1984 to 59% in 1990 and 44% in 1994.41 Low childhood DPT3 coverage has been associated with high anti-diphtheria immunity in the older/adult population due to boosting of immunity from recurrent subclinical infections.29 Furthermore, the large proportion of women who did not know their vaccination status makes drawing deductions from this finding difficult. However, the finding is comparable to that of a Kenyan work, where no significant association was found between previous childhood vaccination and the level of anti-diphtheria immunity in older children and adults.82 This observation may be explained by findings from several works84, 85, 87-89 that have documented limited persistence of diphtheria antibody following the primary series of three DPT vaccinations. In the absence of booster doses of diphtheria toxoid vaccine, the immunity acquired following the childhood vaccination wanes with age. 29, 30, 35, 82 Consequently, a significant proportion of women in the reproductive age group, such as the subjects of this study, residing in a country like Nigeria where booster doses of diphtheria toxoid vaccine are not routinely given could have inadequate immunity to diphtheria.

Further observations from this study suggest that persons who resided in rural settings were more protected against diphtheria than those who resided in urban areas. Women from rural settings suffer from more cutaneous diphtheria infections than their urban counterparts as a result of the prevailing poorer personal and environmental hygiene in these sections of the society as well as the hazards of such activities as farming and fishing that are more commonly practised in the rural areas.127 Frequent cutaneous diphtheria infections have been linked with poor

83 personal and environmental hygiene as well as pre-existing skin lesions.73 Recurrent cutaneous diphtheria infections are known to maintain and boost immunity to diphtheria.28, 29

In this study subjects from larger household sizes tended to have higher anti- diphtheria antibody titres compared to those from smaller household sizes. Overcrowded living conditions has been associated with increased contact rates which was further linked with increased frequency of cutaneous diphtheria infections.62, 63 Recurrent subclinical cutaneous diphtheria is a known means for maintaining and enhancing immunity to diphtheria.3

No significant association between ethnicity and levels of immunity to diphtheria was observed in this study. Documented absence of racial predilection to diphtheria3 may be accounted for by this finding.

In this study, no significant association was observed between socio- economic class and the levels of protection from diphtheria in mothers and their babies. However, some workers have suggested that persons from poorer settings may suffer more episodes of cutaneous diphtheria than others from more affluent settings.62, 63 If persons from the lower socio-economic classes suffer from more episodes of cutaneous diphtheria, it is expected that such individuals should have higher anti-diphtheria antibody titres when compared to the titres of those from higher socio-economic classes. The absence of an association between socio- economic class and anti-diphtheria immunity in the subjects of this study may be attributed to the relatively poor economic situation of most Nigerians regardless of their social class. Having a University degree, no longer guarantees a job. Furthermore, many who are employed receive meager salaries that can hardly pay for living standards that befit their social statuses.

CONCLUSION

Based on the findings of this study, the following conclusions are made;

 A significant proportion of parturient women and their newborns in UBTH

were inadequately protected against diphtheria.

 Among those who had adequate protection, majority 133/141 (94.3%) had

only safe protection meaning that they will require a booster dose of

diphtheria toxoid within 1 year according to WHO recommendation.

 Protection against diphtheria in a newborn was closely dependent on the

adequacy of maternal protection.

 Parturient women and babies from rural areas had significantly higher anti-

diphtheria antibody titres than those from urban areas.

 Anti-diphtheria immunity in parturient women and their newborns was not

significantly affected by maternal age, ethnicity, household size and socio-

economic class.

RECOMMENDATION

From this study, it is hereby recommended that;

 Vaccination of pregnant women, irrespective of their childhood vaccination

history, with booster doses of adult type diphtheria toxoid should be

included in their antenatal care.

LIMITATION OF THE STUDY

 Inability to use the gold standard – neutralization test on microcell culture

technique, for assaying anti-diphtheria antibody titre in this study.

 The large number of mothers who did not know their childhood vaccination

status.

FUTURE LINE OF STUDY

1. Assessment of the rate of loss of passive immunity to diphtheria in Nigerian infants.

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86. Hori H, Afari EA, Akanmori BD, Kamiya Y, Sakatoku H, Nkrumah FK, Kamiya H, Chazono M, Fukai K. A randomized controlled trial of two cellular pertussis-diphtheria-tetanus vaccines in primary immunization in Ghana: antibody response and adverse reactions. Ann Trop Paediatr 1994; 14: 91-6. 87. Dalmatov VV, Gotval’d RN, Tumorina SZ, Klishevich VP, Arzhaeva NG. Serological surveillance of diphtheria infection. J Microbiol Epidermiol Immunol 1986; 122: 43-7. 88. Pichichero ME, Barkin RM, Samuelson JS. Pediatric diphtheria and tetanus toxoids-absorbed vaccine: immune response to the first booster following the diphtheria and tetanus toxoids vaccine primary series. Pediatr Infect. Dis. J. 1986; 5: 428-30. 89. Englund JA, Glozon WP, Barroto L. Controlled study of a new five-component acellular pertussis vaccine in adults and young children. J Infect Dis 1992; 16: 1436-41. 90. Pappenheimer AM. Diphtheria: studies on the biology of an infectious disease. Harvey Lect 1982; 76: 45-73. 91. Redwan EM, El-Awady MK. Status of diphtheria immunity in the Egyptian population. Ann Trop Med Parasitol 2005; 99: 93-9. 92. Rathod KB, Jaiswal KN, Shrivastava AC, Shrikhande AV. Study of placenta in sickle cell disorder. Indian J Pathol Microbiol 2007; 50: 698-701. 93. De Moraes-Pinto MI, Farhat CK, Carbonare SB, Curti SP, Otsubo ME, Lazarotti DS, Campagnoti RC, Carneiro-Sampaio MM. Maternally acquired immunity in newborns from women infected by the human immunodeficiency virus. Acta Paediatr 1999; 12: 1034-8. 94. Okoko JB, Wesumperuma HL, Hart CA. The influence of prematurity and low birthweight on transplacental antibody transfer in a rural West African population. Trop Med Int Health 2001; 6: 529-34.

95. Pursley DM, Cloherty JP. Identifying the high-risk newborn and evaluating gestational age, prematurity, postmaturity, large-for-gestational-age, and small- for-gestational-age infants. In: Cloherty JP, Stark AR. (eds.) Manual of Neonatal Care, 4th ed. Lippincott-Raven 1998:39-40. 96. Ehrengut W, Tegeler J. Immunity against diphtheria in mothers and their newborns in Hamburg. Monatsschr Kindercheilkd 1982; 130: 775-7. 97. Fulthorpe A. Multiple diphtheria antigen-antibody systems by passive haemagglutination techniques. Immunology 1962; 5:30–45. 98. Galazka A, Abgarowicz A. Assays of diphtheria and tetanus antibodies by the passive haemagglutination method. Epidemiol Rev 1967; 21:237–52. 99. Ministry of Health Kenya, Expanded Programme on Immunization. National immunization coverage survey, Nairobi: Government Printers 1992; 80. 100. Odusanya OO, Alufohai EF, Meurice FP, Ahonkai VI. Determinants of vaccination coverage in rural Nigeria. BMC Pub Hlth, 2008; 8:381-7. 101. Ministry of Health, Edo State. DPT3 coverage factsheet, July, 2011. 102. WHO. Vaccine – preventable diseases: monitoring system. 2011 global summary. Immunization, Vaccines and Biologicals. World Health Organization, 2011. 103. Allerdist H, Ehrengut W, Fofana Y. Diphtheria immunity in Mali (mother and their neonates and children under two years of age). Trop Med Parasitol 1981; 32: 274-5. 104. Schick test – Wikipedia, the free encyclopedia. Available from: http://en.wikipedia.org/wik/schick-test. (Accessed 30th May 2010). 105. Papadatos C, Papavangelou G, Koukou D. Diphtheria antitoxin levels and the Schick reaction in mothers and their newborns. Acta Paediatrica Scandinavica 1967; 172: 181–5.

106. Glenny A, Llewellyn-Jones M. The intracutaneous method of testing diphtheria toxin and antitoxin. J Pathol 1931; 34:143–56. 107. Miyamura K, Tajiri E, Ito A, Murata R, Kono R. Micro cell culture method for determination of diphtheria toxin and antitoxin titres using Vero cells II. Comparison with the rabbit skin method and practical application for sero- epidemiological studies. Biologicals 1974 ; 2:203–9. 108. Middlebrook J, Dorland R, Leppla S. Association of diphtheria toxin with Vero cells: demonstration of a receptor. J Biol Chem 1978; 253:7325–30. 109. Svenson S, Larsen K. An enzyme-linked immunosorbent assay for the determination of diphtheria toxin antibodies. J Immunol Methods 1977; 17:249– 56. 110. Knight P, Tilleray J, Queminet J. Studies on the correlation of a range of immunoassays for diphtheria antitoxin with the guinea-pig intradermal test. Dev Biol 1986; 64:25–32. 111. Melville-Smith M, Balfour A. Estimation of Corynebacterium diphtheriae antitoxin in human sera: a comparison of an enzyme-linked immunosorbent assay with the toxin neutralization test. J Med Microbiol 1988; 25:279–83. 112. Serion Diagnostics. Serion ELISA Classic Diphtheria IgG. Available from: http://www.virion-serion.de/uploads/mit_download/product_information_ Diphtheria.pdf (Accessed 20th August 2009). 113. Efstratiou A, Maple PAC. Manual for the laboratory diagnosis of diphtheria. Copenhagen: Expanded Programme on Immunization in the European region of the World Health Organization, 1994; ICP/EPI 038 (C). 114. Efstratiou A, Engler KH, Mazurova IK, Glushkevich T, Vuopio-Varkila J, Popovic T. Current approaches to laboratory diagnosis of diphtheria. J Infect Dis 2000; 181:138-45.

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APPENDICES Appendix I Consent form

S/no:………… Dept of Child Health, U.B.T.H, Benin City. 05/09/2010. Dear parent, I am Dr Cummings Henry, a resident in the department of Child Health, University of Benin Teaching Hospital. I am carrying out a study to determine the status of immunity to diphtheria in mothers and their newborns being delivered in UBTH. Diphtheria is an infectious disease commonly affecting the airway. It may cause cough and difficulty in breathing. In severe cases it may cause death in up to 2 out of 10 affected persons. Newborns and infants in the first few months of life are usually protected by immunity derived from their mothers. Thereafter, they develop their own immunity from vaccines that are given to them from the second month of life. Studies from some centres in Africa and Europe have shown that mothers no longer have adequate amount of immunity to diphtheria, thus their newborns do not receive protective amount of immunity to this deadly disease – diphtheria. This study will be of benefit to you and your baby as you and your baby’s level of immunity will be determined and the results communicated to you. This study would require me asking questions relating to your biodata, previous illnesses and medications, immunization, family and social history. It will also include a general examination of your baby and the collection of a cord blood sample. A small amount (3ml) of blood will be collected from you immediately after the delivery for your own level of immunity to diphtheria.

The result of the tests will be communicated to you at the post-natal clinic and necessary intervention (if any), discussed. Thank you for your anticipated co-operation. Kindly sign below, if you consent to the involvement of yourself and your baby in the study. Name of parent:………………………………………………………….. Signature/Thumbprint of parent: ...... Date:…………. Phone no:……………….. Signature of researcher:………………………………….. Signature/Thumbprint of witness:………………………… Researcher : Dr Cummings Henry (Phone no: 07030318330)

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Appendix II Proforma ASSESSMENT OF IMMUNITY TO DIPHTHERIA IN MOTHERS AND THEIR NEWBORNS AT THE UNIVERSITY OF BENIN TEACHING HOSPITAL BENIN CITY This proforma is to investigate the status of immunity to diphtheria in mothers and their newborns in UBTH, Benin City. The information needed include; biodata, family/Social history, and past immunization history. The information obtained shall be confidential. Date……………………… S/no………………………………………………

A. Family Data 1. Maternal Data: a. Name………………………………………………………………………….. b. Age………………………………………………………………………………. c. Address…………………………………………………………………..……… d. Ethnicity………………………………………………………………………. e. Religion……………………………………………………………………….. f. Occupation……………………………………………………………….. g. Level of Education: None ( ) Primary ( ) Secondary( ) Tertiary ( ) h. Marital status: Single ( ) Married ( ) Co-habiting ( ) Others ( ) i. Vaccination Hx: i. I have vaccination card showing I had full (DPT) vaccination ( ) ii. My vaccination card shows incomplete DPT vaccination ( ) iii. My vaccination card is not available but I was told (by parent/guardian) that I was fully vaccinated ( ) iv. My card is not available and I was told that I was not fully vaccinated ( ) v. My card is not available and I am not aware of my vaccination status( ) vi. I was told that I had no vaccination ( ) 2. Paternal data: a. Occupation………………………………………………… b. Level of Education: None ( ) Primary ( ) Secondary ( ) Tertiary ( ) 3. Others: a. No. of persons in the household……………………………….. b. Housing: Flat ( ) Passage house ( ) Others (specify)………………………… c. Socio-economic status: Upper class ( ) Middle class ( ) Lower class ( ) B. Clinical Evaluation of Baby a. Date of Birth……………………………………….. b. Time of Birth……………………………………….

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c. Sex : Male ( ) Female ( ) d. Gestational Age (weeks) using– Mother’s LMP: - ……………… D&D:- ……………… e. Weight (Kg)……………:- Appropriate for gestational age yes ( ) No ( ) f. Occipito-frontal circumference (cm)……………………………… g. Length (cm)………………………………………………………

C. Diphtheria Antibody Titre

1. Mother a. Date/Time of sample collection…………………………………. b. Date/Time of sample analysis………………………………………. c. Test Result (UI/ml)…………………………………………………….

2. Baby a. Date/Time of sample collection…………………………………. b. Date/Time of sample analysis…………………………………… c. Test Result (UI/ml)………………………………………………

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