0

الرحيمبسم هللا الرحمن

The National Ribat University

Faculty of Graduate Studies

And Scientific Research

Seroprevelance of Torch among pregnant women

attending maternity hospitals in Khartoum state.

A thesis submitted in fulfillment of the requirement of M.Sc. Degree in

Medical Laboratory Sciences

(Microbiology)

By:

Ebtihal Abd Alhaleem Mohamed Ahmed

B.Sc (2010) Medical Laboratory Sciences (National Ribat

University)

Supervisor

Prof. Abubakr Ibrahim Ahmed

2017

I

II

اآليت

.) لبل حعبن: )وفي أفضكى أفل حبصزو

صذق هللا انعظيى

{12االيت انذاريبث } –صىرة

III

Dedication

This thesis is dedicated to: The sake of Allah, my Creator and my Master, My great

teacher and messenger, Mohammed (May Allah blesses and grants him), who taught us

the purpose of life

I also dedicate my dissertation study to my family. A special feeling of gratitude to my

loving parents, my husband Sulaiman, Prof: Abubakr, Prof: Shamsoon, My sisters:

Shatha and my brother Mohamed Who have never left my side and are very special

To friends who encouraged and supported me, Amal, Zainab, Arwa, Heba, Amna, and

all the people in my life who touched my heart, I dedicate this research.

IV

Ebtihal

Acknowledgements

In the name of Allah, the Most Gracious and the Most Merciful ( Alhamdulillah), all

praises to Allah for the strengths and His blessing in completing this thesis.

At the end of this thesis I would like to thank all those people who made this thesis

possible and an unforgettable experience for me. At the end of this thesis, it is a pleasant

task to express my thanks to all those who contributed in many ways to the success of

this study and made it an unforgettable experience for me.

At this moment of accomplishment, first of all I pay homage to my guide,

Prof: Abubakr Ibrahim Ahmed. This work would not have been possible without his

guidance, support and encouragement. Under his guidance I successfully overcame many

difficulties and learned a lot, he used to review my thesis progress, give his valuable

suggestions and made corrections. His unflinching courage and conviction, will always

inspire me. I can only say a proper thanks to him through my future work.

I am also extremely indebted to Dr: Fathia, for providing necessary infrastructure,

samples and resources to accomplish my research work. Very much thankful to her for

picking me up as a student at the critical stage of the master. I gratefully acknowledge

her encouragement and personal attention which have provided good and smooth basis

for the master degree.

My thanks are due to Dr. Arwa for valuable suggestions and untried help during blood

collection.

V

My heartfelt thanks and appreciation are due to all the staff of Medical Laboratory

Sciences, National Ribat University, who offered me the chance to do this research.

Most of the results described in this thesis would not have been obtained without a close

collaboration with few friendships. I owe a great deal of appreciation and gratitude to

Dr. Zainab, and Dr. Jafar.

VI

Abstract

Infections caused by TORCH complex (Toxoplasma gondii, Rubella virus,

Cytomegalovirus (CMV), and Herpes simplex virus (HSV) in pregnant women.

Generally the infection of TORCH is mild for mothers but can be more disastrous to the

fetus. The degree of severity depends on the gestational age when infected, the virulence

of the organisms can damage the fetus in the developmental stages and also increase the

severity of maternal disease. The aim of this study was to evaluate the seroprevelance of

TORCH infections in pregnanant women. This study reports the prevalence of

Toxoplasma gondii, Rubella virus, Cytomegalovirus, and Herpes simplex virus I-II

infections in randomly selected 200 pregnant women by demonstrating the presence of

immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies using western blot

technique. Immunoglobulin M antibodies for Toxoplasma gondii were positive in 41

(20.5%), Nil for Rubella virus, for Cytomegalovirus were positive in 16 (8%), for Herpes

simplex virus-I were positive in 2 (1%), and Herpes simplex virus -II were positive in

1(0.5%). Immunoglobulin G antibodies for Toxoplasma gondii were positive in 50

(25%), for Rubella virus were positive in 70 (33%), for Cytomegalovirus were positive in

59 (30%), and for Herpes simplex virus-I were positive in37 (19%), and for Herpes

simplex virus-II were positive in 15(8%).

VII

مستخلص البحث :

، فيزوس انحصبت األنبيت، انفيزوس انضخى مىصت انمذيت ان هي ي انفيزوصبث ىعتانخي حضببهب يج يزاضاال

( عذ انضبء انحىايم. عىيب حكى اإلصببت خفيفت في األو HSV)( ٢و١)انبضيظ حل(، وفيزوس انCMVنهخليب )

يك أ نجي. عذ انصببيا. درجت انخطىرة حعخذ عه عز حم ثيت عه انجيركب حكىونك يك أ

حذيذ ب انهذف ي هذ انذراصت هى حيزض األيهبث. وك، وكذنك حزيذ ي شذة االون يضز انجي في يزاحم انى

نضخى نهخليبانفيزوس اوانحصبت األنبيتانمىصت انمذيتويم في واليت انخزطىو.ىافي انضبء انح اإلصببت يضخىي

ايزأة حبيم ع طزيك إثببث وجىداألجضبو انضبدة 122بشكم عشىائي ( ونمذ اخخزب٢و١)انبضيظ حلوفيزوس ان

Mانغهىبىني انبعيضبت . كبجعيبث دو عشىائيت ببصخخذاو Gوانغهىبىني انبعي Mانغهىبىني انبعيي فئت

انفيزوس انضخى نهخليب و( ٪8) 16انحصبت األنبيت وهمىصت انمذيت ن٪( 12.2) 12انضبدة إيجببيت في األجضبو

ايزأة 22إيجببيت في G٪(. كبج األجضبو انضبدة انغهىبىني انبعي 2.2)2(١)انبضيظ حلوفيزوس ان٪( 2( )1)

ههزبش ٪( ن25) 30و نفيزوس يضخى انخليب٪( 32) 25و هحصبت األنبيتن ٪(33) 02 نهمىصت انمذيت و ٪(12)

. ( ٢)انبضيظ حلهن٪( 8) 22و (١انبضيظ )

VIII

List of Content:

No Subject pages

I اآلية

Dedication II

Acknowledgments III

Abstract V

Abstract (Arabic) VII

Chapter one

1.1 Introduction 1

1.1.1 Toxoplasma gondii 1

1.1.2 Rubella virus 4

1.1.3 Cytomegalovirus 8

1.1.4 Herpes simplex virus 10

1.2 Literature review 12

1.3 Rationale 15

1.4 objectives 16

1.4.1 General objective 16

1.4.2 Specific objectives 16

Chapter two

2.1 Materials and Methods 17

2.1.1 Study design 17

IX

2.1.2 Study area 17

2.1.3 Study duration 17

2.1.4 Study population 17

2.1.4.1 Inclusion criteria 17

2.1.4.2 Exclusion criteria 17

2.1.5 Sample size 18

2.1.6 Tools of data collection 18

2.1.7 Ethical clearance 18

2.1.8 Specimen collection 18

2.1.9 Sample processing 18

2.1.9.1 Preparation and stability of the reagents of western

blot technique for IgM and IgG

19

2.1.9.2 Preparation and stability of patient sample 20

2.1.9.3 Procedure of western blot technique 20

2.1.9.4 Evaluation and interpretation of the results obtained by

the TORCH profile EUROLINE

21

2.1.9.5 Preparation and stability of the reagents for ELISA

technique for IgM

2.1.9.6 Procedure of ELISA technique

2.1.9.7 Data analysis 21

Chapter three

3. Results 22

X

Chapter four

4.1 Discussion 29

4.2 Conclusion 31

4.3 Recommendation 32

List of Tables:

Table 3.1 Distribution of IgM and IgG antibodies against the TORCH

complex among the studied group of pregnant women

24

Table 3.2 Distribution of IgM antibodies against the TORCH

complex among the studied group of pregnant women

25

Table 3.3 Age distribution of patient positive for IgG antibodies

against the pathogen that cause the TORCH

26

Table 3.4 Distribution of the studied population positive for IgM antibodies

against TORCH pathogen according toTrimesters of pregnancy

27

Table 3.5 Distribution of the studied population positive for IgG antibodies

against TORCH pathogen according toTrimesters of pregnancy

28

XI

Chapter one

Introduction

1

1.1 INTRODUCTION AND LITRATURE REVIEW

TORCH T is a group of viral and protozoan infections that gain access to the fetal

bloodstream transplacentally via the chorionic villi. Hematogenous transmission may

occur at any time during gestation or occasionally at the time of delivery via maternal-to-

fetal transfusion [1].

TORCH screening is a group of blood tests check for several different infections.

TORCH stands for toxoplasmosis, rubella infection, cytomegalovirus infection, and

herpes simplex virus infection, TORCH infections can lead to severe fetal loss. When

pregnant women became infected this may cause abortions, history of intrauterine fetal

death, intrauterine growth retardation, stillbirth, early neonatal death, and/or congenital

diseases [2].

TORCH infections in the mother are transmissible to fetus during the birth process and

cause a group of symptomatic birth defects. Many sensitive and specific tests are

available for serological diagnosis of TORCH complex [3].

1.1.1Toxoplasma gondii:

Toxoplasma gondii is an obligate intracellular parasite, Domestic cats is definitive host,

humans and animals are intermediate hosts, Toxoplasmosis is caused by infection with

the protozoan Toxoplasma gondii, and the infection produces a wide range of clinical

syndromes in humans, land and sea mammals, and various bird species. Toxoplasma

gondii has been recovered from locations throughout the world. There are 3 major

genotypes (type I, type II, and type III) of Toxoplasma gondii. Type II genotype is

responsible for most cases of congenital toxoplasmosis. Individuals at risk for

toxoplasmosis include fetuses, newborns, and immunologically impaired patients.

Toxoplasma gondii was first described in 1908 by Nicolle and Manceaux in Tunisia, and

independently by Splendor in Brazil.[4]

Splendor reported the protozoan in a rabbit, while

2

Nicolle and Manceaux identified it in a North African rodent, the gundi (Ctenodactylus

gundi).[5]

In 1909 Nicolle and Manceaux differentiated the protozoan from Leishmania.[4]

And named it Toxoplasma gondii after the curved shape of its infectious stage (Greek

root „toxon‟= bow).

Toxoplasma gondii was first identified as a human pathogen in 1939.[4] Wolf, Cowen

and Paige identified Toxoplasma gondii infection in an infant girl delivered full-term by

Caesarean section.[5]The infant developed seizures and had chorioretinitis in both eyes at

three days. The infant then developed encephalomyelitis and died at one month of age.

Wolf, Cowen, and Paige isolated Toxoplasma gondii from brain tissue lesions.

Intracranial injection of brain and spinal cord samples into mice, rabbits and rats

produced encephalitis in the animals.[4] Wolf, Cowen and Page reviewed additional cases

and concluded that Toxoplasma gondii produced recognizable symptoms and could be

transmitted from mother to child [5].

In 1948, a serological dye test was created by Sabin and Feldman based on the ability of

the patient‟s antibodies to alter staining of Toxoplasma gondii.[4] The Sabin Feldman

Dye Test was the gold standard for identifying Toxoplasma infection. It is transmitted to

humans mainly by ingestion food or water that are contaminated with oocyst shed by cats

or eating undercooked meat or raw meat containing tissue cyst. Primary infection

acquired during pregnancy may result in severe damage to the fetus. Manifestation of

congenital toxoplasmosis include mental retardation ,seizures, blindness, and death

.Congenital disease may become apparent at birth or not until the second or third decade

of life .Acute and latent Toxoplasma gondii infection during pregnancy are mostly

diagnosed by serological testes including detection of antiToxoplasma gondii specific

IgM and IgG antibodies ,and avidity of Toxoplasma gondii specific IgG antibodies

,amplification of specific nucleic acid sequences (polymerase chain

reaction),histological demonstration of parasite or its antigen ,or by isolation the parasite

3

in culture media. Report epidemiological studies indicate that prevelance if Toxoplasma

gondii infection in pregnant women varies substantially among countries [6].

Pregnant women, especially, should avoid contact with cats, soil and raw meat. Pet cats

should be fed only dry, canned, or cooked food. The cat litter box should be emptied

every day, a task to be avoided by pregnant women. Gloves should be worn while

gardening. Vegetables should be washed thoroughly before eating because they may

have been contaminated with cat feces. Expectant mothers should be aware of the

dangers of toxoplasmosis [6].

At present there is no vaccine to prevent toxoplasmosis in humans. Toxoplasmosis in

human was identified in Sudan in 1966 used the Dye test, and reported the prevalence of

61% in four different states in the country [7]. Another study done in Khartoum State

using ELISA IgG in Sudanese pregnant women and recorded 34.1% [8]. In 1991 in Al

Geizera area in the central of Sudan about 200 km distance from capital reported the

prevalence of 41.7% using the Latex agglutination test [9]. Also, there was study done in

North Geizera in childbearing age women and the prevalence was 73.1% [10].

1.1.2 Rubella virus:

Rubella virus is the pathogenic agent of the disease rubella, and is the cause of congenital

rubella syndrome when infection occurs during the first weeks of pregnancy. Rubella

virus is single stranded RNA (+). That belongs to the genus Rubivirus, The name rubella

is derived from a Latin term meaning "little red"[11]. In 1814, George Maton, first

recognized that a mild illness characterized by rash, adenopathy, and little or no fever

was a discrete entity. Henry Veale, in 1866, named the disease rubella. The illness

attracted little attention until 1942, when Norman Gregg noticed that first-trimester

maternal rubella caused serious birth defects. The full spectrum and impact of rubella

4

embryopathy remained unclarified until rubella virus was isolated in tissue culture in

1962 by two independent groups: Parkman, Buescher, and Artenstein; and Neva and

Weller. Using the new tools of the virus Isolation[12].

Rubella is generally a benign communicable exanthematous disease. Nearly one half of

individuals infected with this virus are asymptomatic. Clinical children are characterized

by mild constitutional symptoms, rash, and suboccipital adenopathy; conversely, in older

children, adolescents, and adults, Rubella may be complicated by arthralgia, arthritis, and

thrombocytopenic purpura. Rare cases of rubella encephalitis have also been described in

children [11].

The major complication of rubella is its teratogenic effects when pregnant women

contract the disease, especially in the early weeks of gestation. The virus can be

transmitted to the fetus through the placenta and is capable of causing serious congenital

defects, abortions, and stillbirths. Fortunately, because of the successful immunization

program initiated in the United States in 1969, Rubella infection and congenital rubella

syndrome are rarely seen today [11].

The molecular basis for the causation of congenital rubella syndrome are not yet

completely clear, but in vitro studies with cell lines showed that rubella virus has an

apoptotic effect on certain cell types[11]

. Rubella (also known as German measles and

three-day measles) is a contagious illness spread by coughing and sneezing. A person

with rubella can transmit the virus anytime from about seven days prior to the onset of

the rash to seven days after the onset. Even if rubella symptoms never develop, a person

can spread rubella if he or she becomes infected with rubella virus [12]. The route of

Rubella virus entry into the host cell is not well understood. There is some evidences to

suggest that Rubella virus enters cells via the endocytic pathway.

5

The risk to a fetus is highest in the first few weeks of pregnancy and then declines in

terms of both frequency and severity, although there is still some risk in second trimester.

The virus infects the placenta and then spreads to the fetus [12].Virus from congenital

infections persists after birth. Those with congenital infections can infect others after

birth for a year or more. Virus occurs in nasopharyngeal secretions, urine and feces.

Later on, patients with congenital rubella syndrome may develop additional

complications including diabetes mellitus (up to 20%), thyroid dysfunction, growth

hormone deficiency, ocular complications [12]. The sequelae of congenital rubella

syndrome are hearing loss ,ophthalmic problems ,congenital heart ,neurologic problems

,intrauterine growth retardation ,thrombocytopenic purpura , hepatomegaly,

splenomegaly )[12].

The diagnosis of Rubella virus based on clinical signs and symptoms is unreliable

because there are many other causes of rash that may mimic rubella infection and up to

50% of rubella infections may be subclinical [12]. A laboratory may use one or more of

the following methods to provide evidence of infection. Detection of rubella IgM

antibodies in an approved or certified laboratory except if the case has received a rubella-

containing vaccine eight days to Eight weeks before sample collection and there has been

no evidence of rubella transmission in the community and no history of travel, or IgG

seroconversion or a fourfold or greater rise in titer to rubella virus (Where the second

serum sample is collected at least 10 days after the first, Acute sample) except if the case

has received a rubellacontaining vaccine Eight days to eight weeks before sample

collection and there has been no evidence of rubella transmission in the community and

no history of travel. Paired sera must be tested in parallel or detection of rubella virus

genome in an appropriate specimen [12, 13].

Rubella virus can be isolated from nasal, blood, throat, urine and cerebrospinal fluid

specimens from rubella and tissues from biopsy or autopsy for confirmation of

6

congenital rubella syndrome (CRS) cases. Virus may be isolated from the pharynx in one

week before and up to two weeks after rash onset. Although isolation of the virus is

diagnostic of rubella infection, viral culture is demanding and labor-intensive. Serology

tests include the demonstration of rubella IgM antibodies or the persistence of antibodies

beyond the predicted decay of passively transmitted maternal rubella IgG antibodies. In

congenital rubella syndrome cases IgM antibodies are sometimes found for up to one

year after birth, and persistence of IgG antibodies beyond six months of age has been

detected in 95% of cases [12, 13].False-positive serum rubella IgM tests have occurred in

persons with parvovirus B19 infections, with a positive heterophile test for infectious

mononucleosis, or with a positive rheumatoid factor [12, 13, 14].

The rubella vaccine is usually given as a combined measles-mumps-rubella inoculation,

which contains the safest and most effective form of each vaccine. Doctors recommend

that children receive the MMR or MMRV vaccine between 12 and 15 months of age, and

again between four and six years of age before entering school. It's particularly important

that girls receive the vaccine to prevent rubella during future pregnancies. Usually babies

are protected from rubella for six to eight months after birth because of the immunity

passed on from their mothers. If a child requires protection from rubella before 12

months of age for example, for certain foreign travel the vaccine can be given as early as

6 months of age. But children who are vaccinated early still need to be vaccinated at the

recommended ages later [12].

Moreover, growing body of study conducted in Sudan in 2011 reported that, the

prevalence of Rubella was 65.3%, accordingly, 34.7% of the pregnant women are at risk

of Rubella infection besides their unborn babies which are vulnerable to congenital

Rubella syndrome [15], in 2013 also reported that seroprevelance of rubella IgM 5.4% and

for IgG was 51.6% [16].

7

1.1.3 Cytomegalovirus:

Human Cytomegalovirus (CMV) is one of the major causes of congenital infections its

clinical manifestation range from asymptomatic forms (90% of cases) to severe fatal

damage and in rare cases death due to abortion. Furthermore 10%_15% of the children

who are asymptomatic at birth may develop late sequel, a member of the herpesviridae

family, it is a double-stranded DNA virus with a protein coat and lipoprotein envelope.

Similar to other herpes viruses, Cytomegalovirus is icosahedral and replicates in the

host's nucleus. Replication in the host cell typically manifests pathologically with large

intranuclear inclusion bodies and smaller cytoplasmic inclusions, and is accompanied by

presence of Cytomegalovirus viral particles in the plasma [17].

Intranuclear inclusions typical of cytomegalovirus infections were first noticed in 1881

by German scientists who thought they represented protozoa. After viruses were grown

in cell cultures, Weller, Smith and Rowe independently isolated and grew

Cytomegalovirus from man and mice in 1956–1957. Antibodies in 30–100% of normal

adults indicate not only a past infection, but the presence of a present latent infection.

The presence of Cytomegalovirus DNA in tissues and most organs surveyed indicates the

ubiquity of latent infection [17]. Cytomegalovirus infection is commonly acquired from

breastfeeding or transplacentally [18].

The most common clinical findings of congenital Cytomegalovirus infection include of

hearing loss (sensor neural hearing), cognitive, motor impairments in newborns,

petechiae, jaundice, microcephaly, and small size for gestational age. Common

laboratory abnormalities include hyperbilirubinemia, increased levels of hepatocellular

enzymes, thrombocytopenia, and increased CSF protein levels. Studies have shown that

asymptomatic children with neurological findings are more likely to have

cytomegalovirus IgM antibody. Many cases of hearing loss in children may be caused by

8

cytomegalovirus infection. Cytomegalovirus excretion is common in children with

congenital infection and may represent a reservoir for infection in other children and day

care workers [19]. Can be diagnosed by Viral isolation from urine or saliva in first three

weeks of life in cell culture(cytopathic effect(CPE), Viral load and DNA copies can

assessed by PCR, Serologically by detection of antibody IgM and IgG by and antigen.

Previous study conducted at Omdurman Maternity Hospital revealed that the

seroprevelance of Cytomegalovirus IgG antibodies among pregnant women was 95[20]. A

recent study conducted at El-Rahad hospital in Western Sudan reported that the

seroprevelance of Cytomegalovirus among pregnant women was 72.2 and 2.5% for

Cytomegalovirus IgG and Cytomegalovirus (IgM) respectively [15].

1.1.4 Herpes simplex virus 1&2:

Infection with Herpes simplex virus is one of the most common sexually transmitted

infections. Because the infection is common, Women in reproductive age it can be

contracted and transmitted to the fetus during pregnancy. Herpes simplex virus is

important cause of neonatal infection, it occurs frequently during the delivery. The

greatest risk of transmission to the fetus and newborn occurs in second half of pregnancy

and it can be fatal, Herpes simplex virus1and 2 belong to the family of Herpesviridae.

Two types exist: herpes simplex virus type 1 (Herpes simplex virus-1) and type 2

(Herpes simplex virus-2). Both are closely related but differ in epidemiology.

Herpes simplex virus-1 is traditionally associated with orofacial disease while Herpes

simplex virus-2 is traditionally associated with genital disease; however, lesion location

is not necessarily indicative of viral type [21]. Herpes simplex virus has been around for a

very long time, in recent years, genital herpes has become an increasing common

sexually transmitted infection. From the late 1970s, HSV-2 seroprevelance has

increased by 30%, resulting that one out of five adults is infected, although it has

9

only been fully understood in the last hundred years. It is one of the oldest sexually

transmitted infections known to human, Herpes simplex virus is transmitted via close

personal contact. Herpes simplex virus infection occurs via inoculation of virus into

susceptible mucosal surfaces (oropharynx, cervix, and conjunctiva) or through small

cracks in the skin[22, 23]. Herpes simplex virus type1. Primary herpes can affect the lips,

and the ruptured vesicles may appear as bleeding of the lips. Up to 80% of herpes

simplex infections are asymptomatic. Herpes simplex virus-1 is transmitted chiefly by

contact with infected saliva, whereas Herpes simplex virus-2 is transmitted sexually or

from a mother's genital tract infection to her newborn. However, lesion location does not

always indicate viral type.

Herpes simplex virus (HSV) infection is best confirmed by isolation of the virus in cell

culture is the preferred virology test for patients who seek medical treatment for

genital ulcers or other mucocutaneous lesions and allows differentiation of the type

of virus (HSV-1 versus HSV-2) [24].Characteristic cytopathic effect with rounding of

cells and cell death are observed, and death of the entire monolayer of cells may be rapid.

Immunofluorescent staining of the tissue culture cells can be used to quickly identify

HSV and can distinguish between types 1 and 2. The characteristic cytologic changes

induced by Herpes simplex virus can be demonstrated in Tzank smears. Multinucleated

giant cells and epithelial cells containing eosinophilic intranuclear inclusion bodies

distinguish the lesions of herpes viruses. Detection of Herpes simplex virus DNA in

clinical specimens is possible with polymerase chain reaction (PCR) techniques provides

increased sensitivity over culture and may ultimately replace culture as the

standard of care for diagnosis [25]. Serological test can be demonstrated for IgM and

IgG antibody. There study conducted in Sudan in 2013 reported that the prevelance oh

Herpes simplex virus was 45positive IgG [26].

11

1.2 Literature review:

In these, there was study in Timisoara (India) conducted in 2008 to assess the TORCH

complex infection among 660 gravidas, only308 was positive and 352 were negative [27].

In another study in dehradun (India) in 2010 was conducted to screen the TORCH in

women with bad obstetric history, it included 70 pregnant women with bad obstetric

history and 35clinically normal women with previous term deliveries. The

seropositivity of cytomegalovirus was 21(42%), Toxoplasmagondii 15(71%), Rubella

virus and Herpes simplex virus Were 8(57 %), while in control group, seropositivity for

Toxoplasma gondii and Cytomegalovirus was2(85% ), and for Herpes simplex virus

5(71%). The highest seropositivity in cases of repeated abortions was seen With

Cytomegalovirus (23.33%), followed by Toxoplasma gondii (20%). [28].

In Bangalore (India), there was study in seroprevelance of TORCH infection and

adverse reproductive outcome in current pregnancy with bad obstetric history in 2013,

included 87 pregnant women with bad obstetric history, The IgM/IgG sero positivity to

Toxoplasma gondii was 5.8(8.0%), Rubella virus was 4.6(90.8%), Cytomegalovirus

was9.2(95.4%) and Herpes simplex virus-2 was2.3(5.8%) respectively. Adverse

outcome occurred in, 66.8% and 43.1% cases of IgM and IgG seropositives. Maximum

number of IgM seropositives cases of abortion (33.4%) were associated with Toxoplasma

gondii, Rubella virus and Cytomegalovirus infection , congenital malformations (25%)

were associated with rubella virus and Cytomegalovirus infection, intrauterine fetal death

(8.4%) was associated with Cytomegalovirus infection and the IgG sero positives

showed abortion(31.3%),intra uterine death(7%), congenital malformations (3.5%) and

still birth(1.2%) [29].

Another study was conducted in kocaely (turkey) 2008, in seroprevelance of Toxoplasma

gondii, Rubella virus, and Cytomegalovirus infection among pregnant women, included

1972 pregnant women, seropositivity for IgG Toxoplasma gondii was 952 (48.3%),

11

Rubella virus was 1896 (96%),and Cytomegalovirus was1900(96.4%),The seropositivity

for IgM for Toxoplasma gondii was 8 (0.4%),Rubella virus was

4(0.2%),Cytomegalovirus was 13(0.7), The seropositivity for IgG and IgM for

Toxoplasma gondii was 31(1.6%), Rubella virus was 35(1.8%) ,and Cytomegalovirus

was 37(1.9%) [30].

This study in Doha (Qatar) 2010,to Toxoplasma gondii seropositivity and Co-Infection

with TORCH pathogen in high risk patient in Qatar among 823 women of childbearing

age, seropositivity for IgM Toxoplasma gondii 5.2%,and for IgG 35.1%, Three infants ≤

6 months of age (0.8% of 353) were congenitally infected. Factors associated with

Toxoplasma gondii IgG seropositivity included older age, East Mediterranean or African

nationality, positive cytomegalovirus (CMV) and herpes simplex virus (HSV)-1

serostatus, and negative rubella IgG results. The decreasing prevalence of IgM antibodies

between 2005 and 2008 suggested that exposure to T. gondii from food or environmental

sources declined over this period in Qatar [31].

In the city of Kirkuk in Iraq in 2014,there was study to assess the TORCH infection in

women with bad obstetric history, it include 252 with bad obstetric history, the

seropositivity of Toxoplasma gondii was 26.68%, Rubella virus was

21.03%,Cytomegalovirus was 34.92% Herpes simplex virus-II 16.6% respectively, with

high frequency of stillbirths 94.11%, incompletemiscarriages74.07% specially (preterm

labour 88.88%) P< 0.0. Relationship between women age and TORCH distribution was

significant specially women aging from 15 to25 years with high occurrences of Herpes

simplex virus-2 13.13% and Toxoplasma gondii 7.14% respectively. Frequency of

TORCH positive according to gestational periods of pregnancy was significant especially

high rate of Toxoplasma gondii IgM 27.77% in first trimester and 66.66% of herpes

simplex virus-2 IgM in third trimester of pregnancy [32].

12

Finally in alrahad city in Sudan, there study was conducted in 2011 to see the

seropositivity of 231 pregnant women, The seropositivity for Cytomegalovirus IgM 6

(2.5%) and Rubella virus IgM 8 (3.4%), Cytomegalovirus IgG 167 (72.2%) [15].

1.3Rationale:

In the last few years, some major health problems were raised by pregnant women with

toxoplasmosis ,rubella infection ,cytomegalovirus infection ,and herpes simplex (1&2)

infection which cause abortion, history of intrauterine, fetal death, intrauterine growth,

retardation, stillbirth, early neonatal death, and/or congenital disease, thus the

investigation need to be performed to assess the seroprevelance of TORCH to determine

exposure of mother and infants and necessary precaution be taken.

13

1.4 Objectives:

1.4.1 General objective:

To determine the seroprevelance of Toxoplasma gondii, Rubella virus, Cytomegalovirus,

Herpes simplex virus 1 and 2 (TORCH) among pregnant women attending selected

hospitals in Khartoum.

1.4.2 Specific objectives:

To detect IgM and IgG antibody against Toxoplasma gondii, Rubella virus,

Cytomegalovirus and Herpes simplex virus 1 and 2 in the blood of the study

population of pregnant women.

To determine the risk factors for TORCH complex microorganisms transmission.

14

Chapter two

Materials & Methods

14

2.1Materials and methods:

2.1.1 Study design:

This is a cross sectional study.

2.1.2 Study area:

The Study was conducted at 3 selected hospitals at Khartoum state including Ribat

University Hospital located in Khartoum city, it provide medical facilities for police men

and families and used to train the students in national ribat university, Military Hospital

located in Omdurman city specialized for military and their families, and Dream

specialized Hospital in Khartoum city, it specialized in obstetrics and gynecology.

2.1.3 Study duration:

The study was conducted during the period from March 2015 to 2017.

2.1.4 Study population:

The pregnant women attending in the study areas during 2015 to 2017 in Khartoum

state whose ranged between 18-44 years.

2.1.4.1 Inclusion Criteria:

All pregnant women came to hospital as out patients at the time of study.

2.1.4.2 Exclusion Criteria:

All pregnant women under age of 18 and more than 45 years old were excluded from this

study.

15

2.1.5 Sample size:

In the estimation of the sample size, statistical formula for sample size calculation was

considered as a basis. The sample size was calculated with a prevalence of 50% and

A total of 200 pregnant women were included in the study.

n = Z2P (1 − P)/d2

Where n = sample size

Z = critical value at 5% level (1.96)

P = prevalence (50%)

d = margin of error (5%)

2.1.6 Tools of Data collection:

Questionnaire: data were collected using well structured questionnaire makes with

patients face to face (age, stage of pregnancy, residence, gynecological and medical

history, and abnormal babies).

Laboratory investigation: Result of the laboratory examination of the blood samples.

2.1.7 Ethical clearance:

All participants in the study had to sign consent for participation and the results of

investigations were to be conveyed to their treating doctors who will discuss results with

them and provide the necessary treatment.

Permission of the ethical committee of the National Ribat University was obtained.

2.1.8 Specimen collection:

Approximately 10 ml of venous blood was collected from each participant.

16

2.1.9 Sample Processing:

The blood samples were drawn to plain containers. Then after 3to 4 hours centrifuged at

3600 rpm for 5 minutes and the serum was separated, and stored at -20°C until

analyzed, serum samples were tested by western blot technique (euro immune )

Germany.

2.1.9.1Preparation and stability of the reagents for western bolt technique for IgM

and IgG:

All reagents were brought to meet the room temperature (+18°C to+25°C)

approximately 30 minutes before use.

Coated test strips: Ready for use once the package was opened after reaching the

room temperature to prevent condensation on the strips. After removal of the strips, the

package was sealed tightly and stored at +2°C to +8°C.

Positive control: the control was a 50X concentrate of IgM (human) for preparation of

control, the amount needed was taken by using clean pipette and diluted 1:50with IgM

sample buffer containing IgG /RF absorbent.

Universal buffer: the universal buffer was 10X concentrate of citrate phosphate buffer.

Before using, the universal buffer was shaken in the bottle. The amount needed was

taken using clean pipette and diluted 1:10 with deionised or distilled water.

Enzyme conjugate: the enzyme conjugate was a 10X concentrate of alkaline

phosphatase labelled anti-human IgM (goat), For preparation of enzyme conjugate the

amount needed was taken by using clean pipette and diluted 1:10 with the ready

universal buffer.

17

Substrate solution: nitro blue tetrazolium chloride / 5-bromo-4-chloro-3-indolyl

phosphate (NPT/BCIP) was ready for use, the bottle closed immediately after using it

because it‟s sensitive to light.

IgM sample buffer: is sample buffer containing IgG/RF absorbent was ready for use

and used only for the dilution of the sample and the positive control.

2.1.9.2 Preparation and stability of patient sample:

Sample material: pregnant women sera were used.

Stability: pregnant women samples were collected and stored at -20°C until required

the number of samples were completed within three months.

Sample dilution: The pregnant women samples for analysis were diluted 1:50 with the

IgG /RF absorbent.

2.1.9.3 Procedure of western blot technique:

The required amount of test strips were taken from the package and placed in each empty

channel, Then the channels filled according to the number of serum samples that have

been tested with 1.5ml of ready universal buffer and incubated for 15minutes at room

temperature on a rocking shaker then all of the liquid aspirated.

30µI of serum sample were added to 1.5IgM sample buffer, then the channel was filled

with 1.5 ml of the diluted serum sample, incubated at room temperature for 30 minutes

on a rocking shaker, all of the liquid from each channel aspirated, then washed them 3

times for 5 minutes by 1.5mlof working strength universal buffer on a rocking shaker.

1.5ml diluted of enzyme conjugate (alkaline phosphatase conjugated anti human IgM)

were added in each channel, incubated for 30 minutes at room temperature on a rocking

shaker, all off the liquid from each channel aspirated ,then washed for 1 time.

18

Finally,1.5ml substrate solution were added into the channels of the incubation tray,

Incubated for 10 minutes at room temperature on a rocking shaker, the fluid was

aspirated from each channel then washed them 3 times for 1minute with deionised or

distilled water.

Then the test strips were placed on the evaluation protocol.

2.1.9.4 Evaluation and interpretation of the results obtained by the TORCH

profile EUROLINE:

Handling: for the evaluation of incubated test strips, generally recommended the

EUROLINESCAN software. After stopping the reaction using deionised or distilled

water, the incubated test sealed into the adhesive foil of the green work protocol using a

pair of tweezer.The position of the test strips can be corrected while they are wet. As

soon as all test strips have been placed on to the protocol, they should be pressed hard

using filter paper and left to air dry. After they have dried, the test stripe will be stuck to

the adhesive foil. The dry test strips are then scanned using a flatbed scanner

(EUROIMMUN AG) and evaluate with EUROLinescan, we have used the

EUROLinescan in our analysis by following all the steps mentions above.

19

2.1.9.5 Preparation and stability of the reagents for ELISA technique for IgM:

Toxoplasma gondii IgM Micro well Plate : Micro well plate coated with anti-human

IgM antibodies

Toxoplasma IgM Conjugate: Recombinant Toxoplasma gondii antigens bound to

peroxidase, Preservative: 0.1%.

Concentrated Wash Buffer (25x): Tris-HCl buffer containing 0.1% Tween 20;

Preservative 0.1%.

Specimen Diluents: Tris buffer; Preservative: 0.1%.

Substrate A: Citrate-phosphate buffer containing hydrogen peroxide; Preservative:

0.1%.

Substrate B: Buffer containing tetramethylbenzidine (TMB).

Stop Solution: 2M Sulfuric acid.

Toxoplasma gondii IgM Cut-Off Calibrator : Diluted human serum weakly reactive

for Toxoplasma IgM antibodies.

Toxoplasma gondii IgM Positive Control: Diluted human serum highly reactive for

Preservative: 0.1%.

21

2.1.9.6 Procedure of ELISA technique:

Prepare Working Wash Buffer by diluting the Concentrated Wash Buffer 1:25.

Pour the contents of the bottle containing the concentrated wash buffer in a

graduated cylinder and fill it with freshly distilled or deionized water to 1250 mL

for 96 wells/plate testing, or 625 mL for 48 wells/plate testing.

Concentrated Wash Buffer was diluted 1:25, added 100 μL of Negative Control in

well F1. (Blue Reagent) , 100 μL of Cut-Off Calibrator in wells C1, D1and E1.

(Blue Reagent), 100 μL of Positive Control in wells G1.

From H1, 100 μL Specimen Diluent were added, 5μL specimen were added

unused stripswas removed and stored at 2-8°C 3. The microwell plate on a flat

bench was mixed for 30 seconds.

The micro well plate covered by Plate Sealer and incubated in a water bath or an

incubator at 37°C ± 2°C for 30 minutes ± 2 minutes,each well was washed 5 times

with 350 μL of Working Wash Buffer per well. The liquid was removed, Turned

the microwell plate upside down on absorbent tissue for a few seconds. Ensure that

all wells have been completely washed and dried. (Improper washing may cause

false positive results) 5

100 μL of Conjugate were added to each well except for the Blank well. (Red

Reagent) • Covered the microplate plate with the Plate Sealer and incubated in a

water bath or an incubator at 37°C ± 2°C for 30 minutes ± 2 minutes, washed 5

times and dried ,

50 μL of Substrate A to each well were added. (Clear Reagent), and 50 μL of

Substrate B were added to each well. (Clear Reagent) Then a blue color developed

21

in wells containing Positive specimens, 50 μL of Substrate A to each well • Add 50

μL of Substrate B to each well 9

Mixed then covered microwell plate with Plate Sealer and incubated at 37°C for 10

min 1 0 • Remove the Plate Sealer.

50 μL of Stop Solution were added to each well. (Clear Reagent) Then a yellow

color developed in wells containing Positive specimens.

Micro well plate readied at 450 nm, but it is strongly recommended to read it at

450/630- 700 nm for better results. • Read at 450/630-700 nm within 30 min

AUTOMATED PROCESSING Automatic EIA microplate processors may be used

2.1.9.7 Data analysis:

Data were analyzed using computerized program SPSS.

22

Chapter three

Results

22

3. Results:

The result obtained showed that out of 200 pregnant women examined

Anti Toxoplasma gondii IgM was detect in 41(21%) and IgG in 50(25%)in western blot,

but it was 2.5 in Toxoplasma gondii IgM ELISA.

Anti Rubella virus IgM was not detect and IgG was detected in 70(33%)

anti Cytomegalovirus IgM was detected in 16(8%) and IgG in 59(30%).

anti Herpes simplex-1IgM was detected in 2(1%)and IgG in 37(19%) IgG positive.

Also anti Herpes simplex-2 was detect in 1(0.5) and IgG in 15(8%).

These result showed age distribution of pregnant women for IgM and IgG antibodies

against pathogen that cause TORCH

In this study the seroprevelance of Toxoplasma gondii, Rubella virus, Cytomegalovirus,

and Herpes simplex virus (1and 2) infection with respect to age. In the age of 18 to 26, it

was detect in Anti Toxoplasma gondii IgM was 15(71%) and in IgG was 12 (20%), Anti

Rubella virus IgM was zero and IgG was17(29%), Anti Cytomegalovirus IgM was

5(23%) and IgG was 13(22%), Anti Herpes simplex virus-1 IgM was 1(4.7%) and IgG

was 12 (20%), Anti Herpes simplex virus-2 IgM was zero and IgG was 4 (7%).

from 27 to 34 it was detect in Anti Toxoplasma gondii IgM was 17(65%) and in IgG was

24(23%), Anti Rubella virus IgM was zero and IgG was 35(22%)

, Anti Cytomegalovirus IgM was 9(3.6%) and IgG was 27(13%), Anti Herpes simplex

virus-1 IgM was zero and IgG was 12 (11%), Anti Herpes simplex virus-2 IgM was zero

and IgG was 6 (6%)

23

Finally, from 35 to 44 it was detect in Anti Toxoplasma gondii IgM was 4(44%) and in

IgG was 14(20%), Anti Rubella virus IgM was zero and IgG was 18(26%)

, Anti Cytomegalovirus IgM was 2(22%) and IgG was 19(27%), Anti Herpes simplex

virus-1 IgM was 1(11%) and IgG was 13(19%), Anti Herpes simplex virus-2 IgM was

2(1%) and IgG was 5 (7%) (Table 3.3, 3.4)

In the current study the seroprevelance of Toxoplasma gondii, Rubella virus,

Cytomegalovirus, and Herpes simplex virus 1&2 infection with respect to stages of

pregnancy.

At first trimester Anti Toxoplasma gondii IgM was 12(66%) and in IgG was 13(20%),

Anti Rubella virus IgM was zero and IgG was 21(33%)

, Anti Cytomegalovirus IgM was 5(27%) and IgG was 16(25%), Anti Herpes simplex

virus-1 IgM was 1(5%) and IgG was 10(15.6%), Anti Herpes simplex virus-2 IgM was

zero and IgG was 4(6%)

In seconed trimester Anti Toxoplasma gondii IgM was 11(68.7%) and in IgG was

17(20%), Anti Rubella virus IgM was zero and IgG was 26(31%)

, Anti Cytomegalovirus IgM was 5(31%) and IgG was 22(26%), Anti Herpes simplex

virus-1 IgM was zero and IgG was 10(83%), Anti Herpes simplex virus-2 IgM was zero

and IgG was 8(9%)

Finally, In Third trimester Anti Toxoplasma gondii IgM was 18(66%) and in IgG was

20(24%), Anti Rubella virus IgM was zero and IgG was 23(28%)

, Anti Cytomegalovirus IgM was 6(22%) and IgG was 21(25%), Anti Herpes simplex

virus-1 IgM was 1(4%) and IgG was 15(18%), Anti Herpes simplex virus-2 IgM was 2

(1%) and IgG was 3(3.6%)(Table 3.5, 3.6).

24

Table 3.1: distribution of IgG and IgM antibody against the TORCH complex among pregnant women .

Type of

organisms

IgM antibody IgG antibody

Frequency percentage frequency percentage

Toxoplasma

gondii

41

21% 50 25%

Rubella virus 0 0% 70 33%

cytomegalovirus 16 8% 59 30%

herpes simplex

virus 1

2

1% 37 19%

herpes simplex

virus 11

1 0.5% 15 8%

25

Table 3.2: age Distribution of patients for IgM antibodies against the pathogen that cause TORCH infection.

Age

group

(years)

total Toxoplasma

gondii

Rubella virus cytomegalovirus Herpes simplex

virus- 1

Herpes simplex virus-

2

Age in

year

frequen

cy

percen

tage

freque

ncy

percent

age

frequenc

y

percent

age

frequ

ency

percenta

ge

frequenc

y

percentage

18-26 21 15 71% 0 0% 5 23% 1 4.7% 0 0%

27-34 26 17 65% 0 0% 9 34.6% 0 0% 0 0%

35-44 9 4 44% 0 0% 2 22% 1 11% 2 22%

Total 36 18% 0 0% 16 8% 2 198 2 1%

P.value 1.9 0.0 2.19 1.55 3.9

26

Table 3.3: Age Distribution of patients for IgM antibodies against the pathogen that cause TORCH

Age

group (years)

total Toxoplasma gondii Rubella virus cytomegalovirus Herpes simplex

virus- 1

Herpes simplex virus-

2 frequency percentage frequency percent

age

frequency percentage frequency percentage frequency percentage

18-26 58 12 20% 17 29% 13 22% 12 20% 4 7%

27-34 104 24 23% 35 22% 27 26% 12 11% 6 6%

35-44 69 14 20% 18 26% 19 27% 13 19% 5 7%

Total 36 18% 0 0% 16 8% 2 198 2 1%

p. value 0.782 0.521 0.2 0.769 2.046

27

Table3.4: Distribution of the studied population positive for IgM antibodies against TORCH pathogen according to trimesters of

pregnancy.

Trimester

s

total Toxoplasma gondii Rubella virus cytomegalovirus Herpes simplex

virus- 1

Herpes simplex

virus-2 frequency percenta

ge frequenc

y percent

age frequency percentag

e frequenc

y percenta

ge frequenc

y percentag

e

First

trimester

18 12 66% 0 0% 5 27% 1 5% 0 0%

Second

trimester

16 11 68.7% 0 0% 5 31% 0 0% 0 0%

Third

trimester

27 18 66% 0 0% 6 22% 1 4% 2 1%

Total 159 18% 0 0% 16 8% 2 198 2 1%

p.value 2.96 0.0 0.359 10.293 3.8

28

Table 3.5: Distribution of the studied population positive for IgG antibodies against TORCH pathogen according to trimesters of

pregnancy .

Trimester

s

total Toxoplasma gondii Rubella virus cytomegalovirus Herpes simplex

virus- 1

Herpes simplex

virus-2 frequency percenta

ge frequenc

y percent

age frequency percentag

e frequenc

y percenta

ge frequenc

y percentag

e

First

trimester

64 13 20% 21 33% 16 25% 10 15.6% 4 6%

Second

trimester

83 17 20% 26 31% 22 26% 10 83% 8 9%

Third

trimester

82 20 24% 23 28% 21 25% 15 18% 3 3.6%

Total 50 18% 70 0% 59 8% 37 198 15 1%

p.value 0.782 0.521 0.2 0.769 2.046

29

Chapter four

Discussion

31

4.1 Discussion

Maternal infections play a critical role in pregnancy. Their occurrance in pregnant

women constitute significant risk factor as these infectious cause fetal and neonatal

mortality they are also important contributors for early and later childhood morbidity.

Viral pathogens usually cause a primary maternal vireamia which may infect the

placenta and emerge to the fetus with the exception of Herpes simplex virus type s1and

2 cause an ascending infection via genital tract to fetal membranes and then to the fetus .

In this study, the seroprevalence of Toxoplasmagondii IgM and IgG antibodies in

pregnant women were 20.5% western blot and 2.5 (ELISA)) and 25% respectively. A

similar study in Iraq showed higher rate [33] but it was similar to the result obtained in

women in Nagpur (India) [34]. A study done in Bangalore (India) sowed lower IgM and

IgG level [29].The variation in the result of Toxoplasma gondii IgM between the two

methods is possibly attributed to the following factors: different antigenic labeled,

whole parasite crud antigens in western blot, white it was only single recombinant

antigen, the specimen was tested later after long time storage under unstable electric

current, its well known that multiple thawing and freezing affect the titer of IgM.

The seroprevalence of the Rubella virus IgM was 35% and IgG was 99%. Studies

conducted in India, Iraq, and sudan (Al rahad and Omdurman) were lower than the

current study in IgM. This were in agreement with that reported in India and AL rahad

city in Sudan in IgG, but Rehab is lower than our study [29, 33, 15, 35 ].

In the current study the seroprevelance of Cytomegalovirus IgM and IgG antibodies

were found to be 8% and 29.5% respectively, there were found to be in agreement with

that reported in Bangalore (India), and in Omdurman(Sudan) in IgM antibody, in

Alrahad (Sudan) a lower seroprevelance in IgM antibody were reported.[ 29, 35, 15]

31

In Cytomegalovirus IgG antibody were reported in Bangalore (India) and Alrahad

(Sudan) showed a higher seroprevelance than in this study, but ther reported by Sadik

were lower than in this study.[,36]

The seroprevelance of Herpes simplex virus type 1 for IgM and IgG antibodies were

found to be 1% and 18.5%, respectively. The sero prevelance of Herpes simplex virus

type 2 for IgM and IgG antibodies were 0.5%and 7.5%, the seroprevelance of IgM is in

agreement with that reported from Bangalore and Hyderabad (India), and in Iraq IgM

antibody was reported lower in IgG antibody [ 29, 36, 33].

32

4.2 Conclusion:

Serodiagnosis showed high titter for toxoplasmosis and cytomegalovirus, with

low rate were found in Rubella virus and Herpes simplex virus

33

4.3 Recommendations:

Women can be advised to screen TORCH before and during pregnancy.

As most of those infections are through food intake ,the standard of hygiene

need to be adequate.

34

Reference

34

5. Reference:

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38

Material required:

Tourniquet

Disposables plastic syringe.

Epindorf tube.

Plain container.

Cotton.

Doctor aid.

Blue &yellow tips

Automatic pipette (10Ml -100ML) ,(100ML-1000ML).

Electric centrifuge.

Kits of IgM & IgG Western Blot (TORCH)

39

Principle of TORCH IgM:

The EUROLINE test kit provides a qualitative assay for human antibodies of the IgM

class to different TORCH antigens Toxoplasma gondii ,rubella ,Cytomegalovirus

,Herpes simplex virus (1&2) . in the first reaction step , diluted patient samples are

incubated with the immunoblot strips. In the case of positive samples ,the specific IgG

&IgM antibodies (also IgA and IgG ) will bind to the corresponding antigenic site. To

detect the bound antibodies,a second incubation is carried out using an enzyme labeled

antihuman IgM (enzyme conjugate) catalyzing a colorreaction.

Principle of TORCH IgG in western blot technique:

The EUROLINE test kit provides a qualitative assay for human antibodies of the IgM

class to different TORCH antigens Toxoplasma gondii ,rubella ,Cytomegalovirus

,Herpes simplex virus (1&2) . In the first reaction step , diluted patient samples are

incubated with the immunoblot strips. In the case of positive samples, the specific IgG

&IgM antibodies (also IgA) will bind to the corresponding antigenic site. To detect the

bound antibodies, a second incubation is carried out using an enzyme labeled

antihuman IgG (enzyme conjugate) catalyzing a color reaction.

Principle of Toxoplasma IgM in ELISA technique:

The Toxoplasma IgM EIA Test Kit is a solid phase enzyme immunoassay based on

immunocapture principle for the qualitative detection of IgM antibodies to Toxoplasma

in human serum or plasma. The microwell plate was coated with anti-human IgM

antibodies. During testing, the specimen diluent and the specimens are added to the

antibody coated microwell plate and then incubated. If the specimens contain IgM

antibodies to Toxoplasma, it will bind to the antibodies coated on the microwell plate to

41

form immobilized anti-human IgM antibody-Toxoplasma IgM antibody complexes. If

the specimens do not contain IgM antibodies to Toxoplasma, the complexes will not be

formed. After initial incubation, the microwell plate is washed to remove unbound

materials. The enzyme-conjugated recombinant Toxoplasma antigens are added to the

microwell plate and then incubated. The enzyme-conjugated recombinant Toxoplasma

antigens will bind to the immobilized anti-human IgM antibody-Toxoplasma IgM

antibody complexes present. After the second incubation, the microwell plate is washed

to remove unbound materials. Substrate A and substrate B are added and then incubated

to produce a blue color indicating the amount of Toxoplasma IgM antibodies present in

the specimens. Sulfuric acid solution is added to the microwell plate to stop the reaction

producing a color change from blue to yellow. The color intensity, which corresponds to

the amount of Toxoplasma IgM antibodies present in the specimens, is measured with a

microplate reader at 450/630-700 nm or 450nm.

41

Appendixes

42

Questionnaire

Patient name…………………………..Age……………..

Residence ……………………………………….. State……………………………………………………………..

Para……………………… Gravida ……………………………Abortion……………

(Miscarriage Stillbirth )

GYNECOLOGICAL HISTORYLMP………………………………..EDD………………………

Type of delivery :(Normal delivery , C.s ).

Normal baby

Abnormal baby : (Hydrocephalus , Microcephalus , Convulsion Mental

retardation ) .

Medical history : DM , Asthma , Fits/convulsion

Comment

……………………………………………………………………………………………………………………………………

………………………………………………………………………………

43

Informed consent

The study has been explained to me and I understand the objective and condition

of the study.

I had the possibility to ask question.

All my questions have been answered to my satisfaction.

My participation in the study is voluntary.

Obtained information on my person stays strictly confidential and will not .appear

in publication.

I agree that my blood is taken to diagnose TORCH and to determine the sero

prevelance of TORCH among pregnant women.

I agree that my blood will be used for further research purposes.

I agree the anonymous data will be stored in an electronic research data base.

I agree the publication of the results of this project in anonymous form.

I declare that I agree to participate to this study.

Name and signature (or fingerprint) of the patient:

Name (or fingerprint) : signature:

Name and signature (or fingerprint) of the responsible accompanying person if the

above person is not capable to sign:

Name (or fingerprint) : signature:

Name and signature of the medical personnel who explained the study:

Name: signature:

44

Table 3.7: Pregnancy outcome:

stages Frequency Percentage

Still Birth YES 10 5

NO 190 95

Total 200 100

Normal Pregnancy YES 199 99.5

NO 1 0.5

Total 200 100

NON 2 1

Once 51 25.5

Twice 48 24

Third 49 24.5

Fourth 28 14

Fifth 10 5

Sixth 5 2.5

Seventh 3 1.5

Eighth 3 1.5

Ninth 1 0.5

Total 200 100

Miss Carriage NON 112 56

Once 54 27

Twice 21 10.5

Third 8 4

Fourth 4 2

Fifth 1 0.5

Total 200 100

45

Toxoplasmagondii Rubella virus Cytomegalovirus Herpes simplex virus(1)

Herpes simplex virus(2)

Normal pregnancy

positive negative positive negative positive negative positive

negative positive negative

non 0 2 0 2 1 1 0 2 0 2

once 14 37 14 37 14 37 9 44 1 52

twice 8 41 18 30 13 35 11 37 3 45

third 10 39 20 29 21 28 10 39 6 43

fourth 13 15 11 17 7 21 5 23 2 26

fifth 2 8 1 9 2 8 1 9 1 9

sixth 2 3 4 1 1 3 0 5 1 4

seventh 1 2 1 2 1 2 1 2 1 2

eighth 0 3 1 2 2 1 0 3 0 3

ninth 0 1 0 1 0 1 0 1 0 1

total 50 150 70 130 59 141 37 163 15 185

miscarriage

non 22 90 39 73 35 77 22 90 10 102

once 17 37 1935 14 40 10 44 12 52

twice 8 13 8 13 4 17 3 18 2 19

third 1 7 2 6 3 5 2 6 2 19

fourth 2 2 1 3 2 2 0 4 0 4

fifth 0 1 1 0 1 0 0 1 0 1

total 50 150 70 130 59 141 37 163 15 185

stillbirth

Non 50 140 15 75 54 136 35 155 15 175

yes 0 10 0 10 5 5 2 8 0 10

total 50 150 70 130 59 141 37 163

15 185