Detection of Fungal Infection in the Product of Conception in Khartoum Bahri Teaching Hospital Athesis Submitted in Partial Fulfillment of the Requirements for M .Sc

ﺑﺴﻢ اﷲ اﻟﺮﲪﻦ اﻟﺮﺣﻴﻢ ALNEELIN University College of Postgraduate& Scientific Research Medical Laboratory Science Department of Histopathology and Cytology

Detection of Fungal Infection in the Product of Conception in Khartoum Bahri Teaching Hospital Athesis Submitted in Partial Fulfillment of the Requirements for M .Sc. Degree in Histopathology Presented By Sara Salih Musa Bakhiet B.Sc of medical laboratory sciences

Supervisor: Ageeb Mohammed Hassan Abdellah M D Clinical pathology Assistant Professor Alribat National University

May 2018

ﺑﺴﻢ ﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﯿﻢ

اﻵﯾــــﺔ

ﻗﺎل ﺗﻌﺎﻟﻲ :

َ . ﱠﷲُ ُﻧورُ ﱠاﻟﺳ َﻣ َﺎو ِات َو ْاﻷ ْر ِض َﻣ َﺛلُ ُﻧ ِور ِه َﻛ ِﻣ ْﺷ َﻛ ٍﺎة ِﻓ َﯾﮭﺎ ِﻣ ْﺻ َﺑ ٌﺎح ْاﻟ ِﻣ ْﺻ َﺑﺎحُ ِﻓﻲ ُز َﺟ َﺎﺟ ٍﺔ ﱡاﻟز َﺟ َﺎﺟ ُﺔ َﻛﺄَ ﱠﻧ َﮭﺎ َﻛ ْو َﻛ ٌب ُد ﱢر ﱞي َﯾُوﻗ ُد ِﻣ ْن َﺷ َﺟ َر ٍة َﻣُﺑ َﺎر َﻛ ٍﺔ َز ْﯾ ُﺗ َوﻧ ٍﺔ َﻻ َﺷ ْر ِﻗ ﱠﯾ ٍﺔ َو َﻻ َﻏ ْر ِﺑ ﱠﯾ ٍﺔ َﯾ َﻛ ُﺎد َز ْﯾ ُﺗ َﮭﺎ ِﯾُﺿﻲءُ َو َﻟ ْو َﻟ ْم َﺗ ْﻣ َﺳ ْﺳﮫُ َﻧ ٌﺎر ُﻧ ٌور َﻋ َﻠﻰ ُﻧ ٍور َﯾ ْﮭ ِدي ﱠﷲُ ﻟِ ُﻧ ِور ِه َﻣ ْن َﯾ َﺷﺎءُ َو َﯾ ْﺿ ِ ربُ ﱠﷲ ُ َ ْاﻷ ْﻣ َﺛ َﺎل ﻟِ ﱠﻠﻧ ِﺎس َو ﱠﷲُ ِﺑ ُﻛ ﱢل َﺷ ْﻲ ٍء َﻋﻠِ ٌﯾم (35)

ﺻﺪق ﷲ اﻟﻌﻈﯿﻢ ﺳﻮرة اﻟﻨﻮر

Dedication

Those who do not thank people do not thank God My mother, spring of tenderness, source of happiness My brethren the eye of the sun My friends and companions People like me and as much as I can My great family Supported me Every Body Support me For the patient and encouragements Our Lord appreciates me and I will make you happy I give you my research

Acknowledgements

Thanks to all members of University of Alnelain and deans of medical laboratory science and Khartoum North Technical Hospital

Abbreviations

- H&E; Haematoxylin and Eosin

- PAS; Periodic Acid Schiff's reagent

- GS; Giemsa stain

- GHS; Grocott Hexamine Silver.

- Spp; Species.

Abstract

Background:

Candida guilliermondii is saprophytic opportunistic pathogenic yeast considered to have low virulence. It is related to local or systemic host immune system, as consequence, disease develops from massive contamination of uterus during gynecological procedures, leading to ascending of infection up through the cervix to placenta then abortion induced.

Aim of the study:

This study aimed to detect the presence of fungi in the product of conception and abortion.

Materials and Methods:

Hundred formalin fixed paraffin blocks (FFPB) of concepts were enrolled from samples of married women from the age between 19 and 45 years, the samples were cut into 4 µm thickness by rotary microtome then stained by two types of special stains PAS& Grocott hexamine silver.

Results:

The results no fungi found.

Conclusion:

This study concludes that there is no relation between Candida infection and abortion but not may be found.

اﻟﻤﺴﺘﺨﻠﺺ

ﺧﻠﻔﯿﺔ:

اﻟﻔﻄﺮﯾﺎت ﻏﯿﻠﯿﺮﻣﻮﻧﺪي ھﻮ اﻟﺨﻤﯿﺮة اﻻﻧﺘﮭﺎزﯾﺔ اﻟﻌﺪوى اﻻﻧﺘﮭﺎزﯾﺔ ﺗﻌﺘﺒﺮ ﻣﻨﺨﻔﻀﺔ اﻟﻀﺮاوة . وﺗﺮﺗﺒﻂ ب اﻟﺠﮭﺎز اﻟﻤﻨﺎﻋﻲ اﻟﻤﻀﯿﻒ اﻟﻤﺤﻠﻲ أو اﻟﻨﻈﺎﻣﯿﺔ، وﻧﺘﯿﺠﺔ ﻟﻠﻌﻼﻗﺔ اﻟﺠﻨﺴﯿﺔ ، ﯾﺘﻄﻮر اﻟﻤﺮض ﻣﻦ اﻟﺘﻠﻮث اﻟﻀﺨﻢ ﻟﻠﺮﺣﻢ أﺛﻨﺎء اﻹﺟﺮاءات اﻟﻨﺴﺎﺋﯿﺔ، ﻣﻤﺎ ﯾﺆدي إﻟﻰ ﺗﺼﺎﻋﺪي اﻟﻌﺪوى ﺣﺘﻰ ﻋﻨﻖ اﻟﺮﺣﻢ إﻟﻰ اﻟﻤﺸﯿﻤﺔ ﺛﻢ اﻹﺟﮭﺎض اﻟﺘﻲ ﯾﺴﺒﺒﮭﺎ.

اﻟﮭﺪف ﻣﻦ اﻟﺪراﺳﺔ:

ﺗﮭﺪف ھﺬه اﻟﺪراﺳﺔ إﻟﻰ اﻟﻜﺸﻒ ﻋﻦ وﺟﻮد ﻋﻼﻗﺔ ﺑﯿﻦ اﻟﻔﻄﺮﯾﺎت واﻹﺟﮭﺎض.

اﻟﻤﻮاد واﻷﺳﺎﻟﯿﺐ:

ﺗﻢ اﺳﺘﺨﺪام ﻣﺎﺋﺔ ﻛﺘﻠﺔ ﻣﻦ اﻟﺒﺎراﻓﯿﻦ اﻟﺜﺎﺑﺘﺔ ﻓﻲ اﻟﻔﻮرﻣﺎﻟﯿﻦ ﻣﻦ ﻋﯿﻨﺎت ﻣﻦ اﻟﻨﺴﺎء اﻟﻤﺘﺰوﺟﺎت اﻟﻼﺗﻲ ﺗﺘﺮاوح اﻋﻤﺎرھﻦ ﺗﺘﺮاوح ﺑﯿﻦ 19 و 45 ﻋﺎﻣﺎ، ﺛﻢ ﺗﻢ ﻗﻄﻊ اﻟﻌﯿﻨﺎت إﻟﻰ 4 ﻣﯿﻜﺮون ﺳﻤﻚ ﺑﻮاﺳﻄﺔ ﺷﺮاﺋﺢ زﺟﺎﺟﯿﺔ ﺛﻢ ﺻﺒﻐﮭﺎ ﻣﻦ ﻗﺒﻞ ﻧﻮﻋﯿﻦ ﻣﻦ اﻟﺼﺒﻎ اﻟﺨﺎﺻﺔ وھﻲ اﻟﻔﻀﺔ واﻟﺒﯿﺮودك اﺳﺪ ﺷﻒ

اﻟﻨﺘﺎﺋﺞ:

وﻛﺎﻧﺖ اﻟﻨﺘﺎﺋﺞ ﺳﺎﻟﺒﺔ.

اﻟﺨﻼﺻﺔ:

ﺗﺨﻠﺺ ھﺬه اﻟﺪراﺳﺔ إﻟﻰ ﻋﺪم وﺟﻮد ﻋﻼﻗﺔ ﺑﯿﻦ اﻻﺟﮭﺎض و اﻟﻔﻄﺮﯾﺎت وﻟﻜﻦ ھﺬا ﻻ ﯾﻨﻔﻲ اﻧﮫ ﻗﺪ ﺗﻮﺟﺪ ﻋﻼﻗﺔ.

Contents I.……………………………………………….………………………………………………………………………اﻵﯾﮫ

Dedication ...... II Acknowledgements ...... III Abbreviations ...... IV

Abstract ...... V VI.…………………………………………………………………………..اﻟﻤﺴﺘﺨﻠﺺ

Chapter One

1-Introduction and literature review……………………………………………….2

1-1. Female Genital System……………………………...…………………..……2

1-1-1. Vagina……………………………………………………………………....2

1-1-2. Cervix…………………………………………………………………….…3 1-1-3. Uterus…………………………………………………………………….…3

1-1-4. Fallopian tube………...……………………………………………….….....3

1-1-5. Ovaries……..…………………………………………………………….…4 1-2.External organ …………………….…………………………………….…….4

1-2-1.Vulva………………………………………………………………….……..4

1-3.Clinical significance…………...……………………………………………....5

1-3-1. Vaginitis……………...…………………………………………..…….……5

1-3-2. Bacterial vaginosis……...………………………………………………...…5

1-3-3. Yeast infection...…………………...…………………….…….…5 1-4.Female genital mutiations………...………………………………………...….6

1-5.Genital surgery….………...………………………………...…………7

1-6.Placental specimens and products of conception………….………..9

1-7. Classification………………………………….………………………...…...10

1-8. Rationale…..…………..,………………………………………………….…10

1-9. Hypothesis……………..…………………………...……………...………....10

1-9-1.Null hypothesis……..………………………………...…………………….10

1-9-2.Alternative hypothesis……..……………………...………………...……...10

1-10. Objectives…..…….…………………………………………………………10

1-10-1. General objective…..………………………………………………...... …10

1-10-2.Specific objectives……………..…………………………………..….…..11

Chapter Two

2- Material and methods………………………………………………………….13

2-1. Materials…………….……………...……………………………………….13

2-2.Methodology…………………………………………………………………13

2-2-1.Study design……………………………………………………………….13

2-2-2.Study area………………………………………………………………….13

2-2-3.Study sample, sample size…………………………………………………13

2-2-4. Sample processing…………………………………………………………13

2-2-5. staining…………………………………………………………………….13

2-2-5-1. Haematoxlyin and Eosin………………….……………………………..14

2-2-5-2.PAS……..…………………………………..………….…………….…..14

2-2-5-3. GHS ………………………………………………….…………….……14

2-6.Result Interpretation…………………………………………………..……...15

2-7.Data analysis……………………………………………………………….....15

Chapter Three

3-1. Results..………..……………………..……………………………………...17

3-2. Tables..……………………………………….………………………....18__20

Chapter four

4-1. Discussion……………………………………………………………………22

4-2. Recommendations…………………………………………………….…...... 23

References..…………………………………………………………………....24-27

CHAPTER ONE

1. Introduction and Literature Review

1.1. Female Genital System:

The female reproductive system (or female genital system) is made up of the internal and external sex organs that function in human reproduction. The female reproductive system is immature at birth and develops to maturity at puberty to be able to produce gametes, and to carry a fetus to full term. The internal sex organs are the uterus and Fallopian tubes, and the ovaries. The uterus or womb accommodates the embryo which develops into the fetus. The uterus also produces vaginal and uterine secretions which help the transit of sperm to the Fallopian tubes. The ovaries produce the ova (egg cells). The external sex organs are also known as the genitals and these are the organs of the vulva including the labia, clitoris and vaginal opening. The vagina is connected to the uterus at the cervix (Juniordoctors13thed).

The female internal reproductive organs are the vagina, uterus, Fallopian tubes, and ovaries.

1.1.1. Vagina: the vagina is the canal between the hymen (or remnants of the hymen) and the cervix, while, legally, it begins at the vulva (between the labia) It may be that the incorrect use of the term vagina is due to not as much thought going into the anatomy of the female genitals as has gone into the study of male genitals, and that this has contributed to an absence of correct vocabulary for the external female genitalia among both the general public and health professionals. Because of this

12 and because a better understanding of female genitalia can help combat sexual and psychological harm with regard to female development, researchers endorse correct terminology for the vulva (Kirkpatrick M (2012).

1.1.2. Cervix: The cervix is part of the female reproductive system. Around 2–3 centmeters' (0.8– 1.2 in) in length, it is the lower narrower part of the uterus continuous above with the broader upper part—or body—of the uterus. The lower end of the cervix bulges through the anterior wall of the vagina, and is referred to as the vaginal portion of cervix or ectocervix while the rest of the cervix above the vagina is called the supravaginal portion of cervix.[3] A central canal, known as the cervical canal, runs along its length and connects the cavity of the body of the uterus with the lumen of the vagina (Sáenz2014).

1.1.3. Uterus: The uterus or womb is the major female reproductive organ, the cervix is part of the female reproductive system. Around 2–3 centimetres (0.8–1.2 in) in length, it is the lower narrower part of the uterus continuous above with the broader upper part—or body—of the uterus.[3] The lower end of the cervix bulges through the anterior wall of the vagina, and is referred to as the vaginal portion of cervix (or ectocervix) while the rest of the cervix above the vagina is called the supravaginal portion of cervix. A central canal, known as the cervical canal, runs along its length and connects the cavity of the body of the uterus with the lumen of the vagina (Grayet al., 1995).

1.1.4. Fallopian tube: The Fallopian tubes are two tubes leading from the ovaries into the uterus. The tube allows passage of the egg from the ovary to the uterus. When an oocyte is

13 developing in an ovary, it is encapsulated in a spherical collection of cells known as an ovarian follicle. Just prior to ovulation the primary oocyte completes meiosis I to form the first polar body and a secondary oocyte which is arrested in metaphase of meiosis II. This secondary oocyte is then ovulated. The follicle and the ovary's wall rupture, allowing the secondary oocyte to escape. The secondary oocyte is caught by the fimbriated end and travels to the ampulla of the uterine tube where typically the sperm are met and fertilization occurs; meiosis II is promptly completed. The fertilized ovum, now a zygote, travels towards the uterus aided by activity of tubal cilia and activity of the tubal muscle. The early embryo requires critical development in the Fallopian tube (Li et al., 2017).

1.1.5. Ovaries: The ovary is an organ found in the female reproductive system that produces an ovum. When released, this travels down the fallopian tube into the uterus, where it may become fertilized by a sperm. There is an ovary (from Latin ovarium, meaning egg/nut) found on the left and right sides of the body. The ovaries also secrete hormones that play a role in the menstrual cycle and fertility. The ovary progresses through many stages beginning in the prenatal period through menopause. It is also an endocrine gland because of the various hormones that it secretes (Colvin et al., 2013).

1.2. External organs: 1.2.1. Vulva:

The main structures of the vulva are: the mons pubis, the labia majora and labia minora, the external parts of the clitoris – the clitoral hood and the glans, the urinary meatus, the vaginal opening and hymen, and Bartholin's and Skene's

14 vestibular glands. Other features include the pudendal cleft, pubic hair, sebaceous glands, the vulval vestibule, and the urogenital triangle (Tortora et al., 1987)

1.3. Clinical significance:

1.3.1. Vaginitis:

Vaginitis is inflammation of the vagina and largely caused by an infection. It is the most common gynecological condition presented. It is difficult to determine any one organism most responsible for virginities because it varies from range of age, sexual activity, and method of microbial identification. Vaginitis is not necessarily caused by a sexually transmitted infection as there are many infectious agents that make use of the close proximity to mucous membranes and secretions. Vaginitis is usually diagnosed based on the presence of vaginal discharge, which can have a certain color, odor, or quality (Zaino"Diseas of the Vagina").

1.3.2. Bacterial vaginosis:

This is a vaginal infection in women. It is differs from vaginitis in that there is no inflammation. Bacterial vaginosis is polymicrobial, consisting many bacteria species. The diagnosis for bacterial vaginosis is made if three of the following four criteria are present: (1) Homogenous, thin discharge, (2) a pH of 4.5 in the vagina, (3) epithelial cells in the vagina with bacteria attached to them, or (4) a fishy odor. It has been associated with an increased risk of other genital tract infections such as endometritis (Zaino"Diseas of the Vagina").

1.3.3. Yeast infection: Vaginal yeast infection:

This is a common cause of vaginal irritation and according to the Centers for Disease Control and Prevention at least 75% of adult women have experienced one at least once in their lifetime. Yeast infections are caused by an overgrowth of fungus in the vagina known as Candida. Yeast infections are usually caused by an imbalance of the pH in the vagina, which is usually acidic. Other factors such as pregnancy, diabetes, weakened immune systems, tight fitting clothing, or douching can also be a cause. Symptoms of yeast infections include itching, burning, irritation, and a white cottage-cheese-like discharge from the vagina. Women have also reported that they experience painful intercourse and urination as well. Taking a sample of the vaginal secretions and placing them under a microscope for evidence of yeast can diagnose a yeast infection. Treatment varies from creams that can be applied in or around the vaginal area to oral tablets that stop the growth of fungus (Zaino"Diseas of the Vagina")

1.4. Female genital mutations:

Also known as female genital cutting and female circumcision, is the ritual cutting or removal of some or all of the external female genitalia. The practice is found in Africa, Asia and the Middle East, and within communities from countries in which FGM is common. UNICEF estimated in 2016 that 200 million women living today in 30 countries—27 African countries, Indonesia, Iraqi Kurdistan and Yemen—have undergone the procedure (UNICEF 2016).

Typically carried out by a traditional circumciser using a blade, FGM is conducted from days after birth to puberty and beyond. In half the countries for which national figures are available, most girls are cut before the age of five. Procedures differ according to the country or ethnic group. They include removal of the clitoral hood and clitoral glans; removal of the inner labia; and removal of the

16 inner and outer labia and closure of the vulva. In this last procedure, known as infibulation, a small hole is left for the passage of urine and menstrual fluid; the vagina is opened for intercourse and opened further for childbirth (Abdulcadir et al., 2011)

1.5. Genital surgery:

Genitoplasty:

Genitoplasty refers to surgery that is carried out to repair damaged sex organs particularly following cancer and its treatment. There are also elective surgical procedures which change the appearance of the external genitals (Mahadevanet al., 2013)

Abortion defines an "abortus" as a fetus or embryo removed or expelled from the uterus during the first half of gestation-20 weeks or in the absence of accurate dating criteria, fetuses weighing less than 500g are usually not considered as births (Cunningham et al., 2010)

Fungi have been isolated from the penis and from seminal fluid in human males, suggesting that infection of urethra or seminal vesicles may occur. Males thus infected main asymptomatic carriers (Horowitz et al., 1987).

Limited observations(Arendrup et al.,2010).Widely distributed in nature on human skin or other sides of the body Mostly recovered in cancer, hematology and dermatology services Pseudo-outbreak in pediatric patients in Brasil2 (Servolo et al., 2007).

The fungi in Sudan cause many diseases, but in the analysis the incidence in frozen equine semen show Candida in (57.1%) of cases, (42.8%) of Candida incospicua,

(14.3%) Candida guilliermondii that due to transmission by artificial semen from males to females (CoronaAet al., 2009) Males are asymptomatic carrier (Horowitzet ai.,1987).

(77%) of fetal candidiasis associated with ICUD were systemic (heart, brain, liver, gastrointestinal gland and lung) compared to (33%) of cases not associated with IUCD (J Perinat1999).

Causes may be due to genetic, immumocompromise disease, rhesus incompatibility and Infectious disease like bacteria, fungi, and parasite.

There is different type of fungi but some is very important. It is widely distributed in nature isolated form soil, insect, plant, seawater, saprophyte human, skin and mucosal surface (un common causative agent of disease). Candida albicans species is non Candida guilliermondii virulence (Savini et al., 2010).

Candida guilliermondii is saprophytic opportunistic pathogenic yeast considered to have low virulence. It is related to local or systemic host immune system, that disease from massive contamination of uterus during gynecological procedures, leading to ascending of infection up through the cervix to placenta then abortion induced (Blanchard et al.,1992).

The fungi from focal origin or skin are source of causing disease of reproductive system. Those are some types of fungi that can identify from urine in culture with Candida species and aspergillums species. Candida albicans is the first pathogen isolated from the reproductive tract (Blanchard et al., 1992).

In different study, some types of Candida spp cause endometritis or abortion like: Cryptococcus and Aspergillus was reported (Blanchard et al., 1992-2001).

In the Arab mare, Candida guilliermondii is the most fungi spp that can cause abortion reported, more than other type of fungi.

Recently, the ratio of abortion increase due to many factors. Many studies reported the direct relationship between different types of fungi and some parasitic agents like toxoplasma. Undoubtedly, there are several risk factors such as poor uterine contractility, anatomical malformations and inefficient uterine defense mechanism. This may explain why fungal endometritis is commonly associated with history of uncorrected anatomical defects that lead to pneumovagina, recurrent persistent post- breeding endometritis and frequent intrauterine antibiotic therapy (Coutinhodaet a., 2011).

1.6. Placental specimens and products of conception:

Products of conception and placental specimens are submitted for the investigation of septic abortion and listeriosis. Listeria monocytogenes may cause serious infection in pregnant women, neonatal infants and patients who are immunocompromised (Schuchat et al., 1991)(Mylonakis et al., 2002). In pregnant women septicaemia caused by L. monocytogenes presents as an acute febrile illness that may affect the fetus(Mylonakis et al., 2002) This may lead to systemic infection (granulomatosis infantisepticum), stillbirth and neonatal meningitis. Products of conception, placenta and neonatal screening swabs should be examined for this organism. Routine culture of vaginal swabs for L. monocytogenes is not usually performed although it may be useful in suspected cases. Blood cultures are indicated. Serological investigations have no place in the diagnosis of listeriosis

(Schuchat et al., 1991).

Septic abortion may result in serious maternal morbidity and may be fatal. Uterine perforation, presence of necrotic debris, and retained placental products can lead to

19 infection. Most infections are polymicrobial and involve anaerobes. Clostridial sepsis complicating abortion is potentially lethal. Clostridium species are part of the normal vaginal flora in some women.(Sedgh et al.,2012).

1.7. Classification:

Candida, which belongs to the Kingdom: Fungi, phylum: Ascomycota, subphylum: Saccharomycotina, class: Saccharomycetes, order: Saccharomycetales, family: Saccharomycetacae, genus: Candida (Hajjeh et al., 2004). About 20 species are known to cause infections in humans (Moris et al., 2008). Includes the species C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. guilliermondii, C. lusitaniae, and C. krusei, C. dubliniensis, C. pelliculosa, C. kefyr, C. norvegensis, C. haemulonii, and Saccharomyces cerevisiae (Aittakorpi et al., 2012).

1.8. Rationale:

The incidence of antenatal complications and abortion in increasing worldwide due to infectious diseases (fugal). Nevertheless further thorough studies should be done in this era to reflect the magnificence of this problem nationally.

1.9. Hypothesis:

1.9.1. Null hypothesis:

There is no relation between abortion occurrence and fungal infections.

1.9.2. Alternative hypothesis:

There is relation between presence of fungal infections and abortion occurrence.

1.10. Objectives:

1.10.1. General objective:

- To detect the presence of fungi infections in the product of conception.

1.10.2. Specific objectives:

- To detect the presence of fungal infection in the product of conception by using PAS and Grocott hexamine silver stains.

- To correlate fungal infection with associated disease and number of abortions.

-To compare between PAS and silver in detection of fungal infection.

CHAPTER TWO

Material and Methods

2.1. Material:

Tissue paraffin blocks obtained from the product of conception were be used in this study.

2.2. Methodology:

2.2.1. Study design:

Analytical retrospectivedescriptivecase study.

2.2.2. Study area:

This study is conducted in Khartoum Bahri Teaching Hospital, during January 2016 to January 2017.

2.2.3. Study sample and sample size:

One hundred tissue paraffin blocks from concept of abortion previously collected from Bahri hospital was selected for this study randomly.

2.2.4. Sample processing:

All samples proceeded via Lieca tissue processing and then make three section from each block with thickness 3-4u were cut by rotary microtome.

2.2.5. Staining:

I used three types of stains routine stain (H&E) and special stain

PAS periodic acidSchiff's reagent the main use of it to demonstration of glycogen and other use help in demonstration of micro organism.

Grocott hexamine silver use to identification of fungi.

I used H&E before that special stain.

2.2.5.1. Haematoxlyin and Eosin:

Sections were dewaxed in xylene, hydrated through graded alcohols (100%, 90%, 70%) to tap water,stained the sections by Mayer’s haematoxlyin for 5 minute, washed by tap water for 5 minute ,stained by eosin for 2 minute, dehydratedthrough alcohols (70%, 90%, 100%),cleared the slides, and mounted in D.P.X mounting media(John et al.,1996)

2.2.5.2. PAS (periodic acid Schiff's reagent): Sections were dewaxed in xylene, hydrated through graded alcohols (100%, 90%, 70%) to tap water, stained the sections with periodic acid for 5 minute, washed by tap water, covered by Schiff's reagent for 20 -30 minute, put the sections in jar under tap water for 5-8 minute, after that stained by Mayer's for 5 minute, then washed, dehydrated through alcohols (70%, 90%, 100%),then blotted by filter paper , cleared the slides, and mounted in D.P.X mounting media(John et al.,1996) 2.2.5.3. Grocott hexamine silver: Sections were dewaxed in xylene , hydrated through graded alcohols (100% , 90% ,70%) to water then oxidized in 5 % aqueous chromium trioxide ( chromic acid ) for 1 hour, washed in tap water , rinsed in 1% sodium metabisuphide, then washed in tap water for 5 minute then rinsed in distilled water , placed in preheated (56ºc) incubating solution in dark , up to 1 hour , rinsed well in distill water then fixed in 3% sodium thiosulphate for 5 minute , covered with counter stain 15-30 seconds

24 then blotted , dehydrated , cleared and mounted in D .P.X mounting media (John et al.,1996).

2.6. Result Interpretations:

Results obtained were detected by researcher and confirmed by tow histopathologists.

2.7. Data analysis:

The collected data were analyzed using Statistical Package of Social Science version 22.0. Frequencies, mean and standard deviation were calculated for comparison and presented in form of tables.P-value is used to assess the significance of the results.

CHAPTER THREE

3.1. Results:

Table-1 : showsThe age of study subjects was between 19 - 45 years, I were divided age into two groups less than 30 and above 30 years old, high percentage in above 30 and low percentage in less 30 years old.

Figure (1) shows number of abortions which represent in the people 1 time (7%) samples, 2 (58%) samples, 3 (33%) samples and 4 (2%) samples.

Table-2: shows PAS stain among samplestudy.

Table-3: Shows the distribution of sliver stain among study samples,

Table-4: Shows the final result of two types of stain with Candidathere is no fungi detected by any type of stain.

Table (1): Percentage of abortion among age group

Age frequency percent

Less than30 35 35 More than30 65 65

Total 100 100.0

Figure (1): Number of abortions

Missing 4

Table-2: Distribution of PAS stain among study samples

PAS Frequency Percent

negative 100 100 positive - -

Total 100 100.0

Table -3: Distribution of sliver stain among study samples

GHS Frequency percent

negative 100 100 positive - -

Total 100 100.0

Table-4 : The result of 3 types of stain with Candida

Result Frequency Percent

negative 100 100 positive - -

Total 100 100.0

CHAPTER FOUR

4.1. Discussion:

In Sudan, there is little data about vaginal infections in pregnant women; a cross- sectional study of pregnant women was conducted at Omdurman Maternity Hospital, Khartoum, Sudan, found Candida albicans was detected in (16.6%). Higher infection rates were recorded among subjects in the third trimester (71.6%) than in the second trimester of gestation (28.4%) (Abdelazizet al., 2014).

Our study results showed , there is marked increase in abortion cases among females of age group more than 30, the majority of cases diagnosed in age over30, that may be due to different genetic and environmental factors especially pathogens and genetic factors and our behavior.

Candida- associated abortion occurs rarely but is often associated with the presence of an intar-uterine conception device (IUCD) that may ascend infection from contaminated external genitalia (Maralli et al.,1996)

I used two types of stain Periodic acid Schiff's reagent PAS and Grocott's hexamine silver stain. They highlight the wall of the fungus and thus are useful for screening the tissue sample, can be combined with H&E in such a way that the fungus and the host reaction can be clearly observed.

PAS demonstrated only living fungi and GHS for both living and dead fungi (Carson et al., 2009).

Periodic Acid Schiff's (PAS) Detects glycogens in tissues, fungal walls contain large amounts of glycogen and thus PAS can be used for screening for fungal organisms (Sangoi A. R., et al. 2009).

4.2. Conclusionand recommendation:

- This study concludes that there is no relation between Candida infection and abortion but not may be found.

- All sample of the product of conception must be use one of special stain that help in diagnosis of causes of abortion especially women aborted more than two times, and age above 30 years

- Increase the sample size to detect any more finding that may help in the diagnosis.

- Further investigations:

• Immunohistochemistry. • PCR study.

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