Mycological Causes of Diarrhea among Children in

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Republic of Iraq Ministry of Higher Education And Scientific Research University of Diyala College of Medicine

Mycological Causes of Diarrhea among Children in Diyala A Thesis Submitted to the Council of the College of Medicine, University of Diyala in Partial Fulfillment of the Requirements for the Degree of Master of Science in Medical Microbiology

By Ali Riyadh Hameed Majeed B.V.M.S. - College of Veterinary Medicine - University of Diyala

(2014)

Supervised by

2018 A.C.

Assistant professor

Assistant professor

Dr. Luma T. Ahmed

Dr. Sabah M. Ali

College of Medicine University of Diyala

College of Medicine University of Mustansiriyah

1439 A.H.

Supervisor Certification I certify that this dissertation entitled (Mycological Causes of Diarrhea among Children in Diyala ) was prepared by (Ali Riyadh Hameed) under my supervision at the College of Medicine - University of Diyala as a partial fulfillment of the requirements for the Degree of Master of the Science in Medical Microbiology. In view of the available recommendation, I forward this thesis for debate by the examining committee.

Signature Supervisor

Assistant Professor Dr. Luma T. Ahmed College of Medicine Department of Microbiology University of Diyala

Signature Co-Supervisor

Assistant professor Dr. Sabah M. Ali College of Medicine Department of pediatrics University of Mustansiriyah

Signature Assistant Professor Dr. Areej A. Hussein Head of Microbiology Department College of Medicine University of Diyala

Committee Certification We, the examining committee, certify that we have read this thesis entitled (Mycological causes of Diarrhea among children in Diyala) was prepared by “Ali Riyadh Hameed” and as the examination committee examined the student in its content and in our opinion it adequate for award of the Degree of Master of the Science in Medical Microbiology.

Signature: Name: Dr. Jassim M. Karhout Scientific Degree: Professor (Chairman) Date: / /2018

Signature : Name: Wisam A. Mehdi Alsaeed Scientific Degree: Professor (Member) Date: / /2018

Signature : Name: Mehdi SH. Jabr Scientific Degree: Professor (Member) Date: / /2018

Signature : Name: Luma T. Ahmed Scientific Degree: Assistant professor (Member/Supervisor) Date: / /2018

Signature: Name: Sabah M. Ali Scientific Degree: Assistant professor (Member/Co-Supervisor) Date: / /2018

Approved by the Council of the College of Medicine - University of Diyala.

Signature Professor Dr. Talib Jawad Kadhim Dean College of Medicine – University of Diyala Date: / / 2018

Dedication To… My dear mother, who surrounds me with her love and kindness, and never forgets me in her sincere prayer. My father, brother and sister, the source of light in my life. They have shared the moments of laughter and sorrow. All who will benefit from this work even a word. I lovingly dedicate this work

Ali

Acknowledgments First and forever, I would like to thank our Lord Allah the most merciful for his blessings and favors to completing this work at this final shape. My deepest gratitude is to my supervisor, Dr. Luma T. Ahmed and co-supervisor, Dr. Sabah M. Ali for supervision, support,

scientific

guidance

and

encouragement.

Great

appreciations are also due to the College of Medicine and Department of Microbiology, University of Diyala. I also thank the staff of laboratory and specialized doctors at AL-Batool Teaching Hospital. Last not least, I owe true love and gratitude to my big and small family for their kindness, help, encouragement and support. I also would like to thank everyone help me directly or indirectly in performing this research.

Ali

Summary : One hundred stool samples of children less than three years referred to AL-Batool Teaching Hospital, in Diyala province during the period (from 2nd October 2016 to 3rd December 2016). were collected sixty-four samples of them were diagnosed as Candida spp. infections by making a routine and confirmative diagnostic processes by direct microscopic examination of stool , cultured on Sabouraud dextrose agar (SDA) and microscopic examination of colonies. The results revealed that, 24(37.5%) of isolates were Candida albicans;14(21.9%) isolates of Candida globrata; 11(17.2%) isolates of Candida parapsilosis; 8(12.5%) isolates of Candida krusei and 7(10.9%) isolates of Candida tropicalis. The results of PCR study by detect the 25S rDNA showed that 20(83.3%) isolates belonged to the genotype A ; 2(8.3%) isolates belonged for each genotype B and genotype C of the C. albicans. The results showed there are significantly (P0.05),the results showed that Candida infection rate higher in male children compared to female children (66.7% vs. 60.5%).The Candida infection rate in (≥2) months patients high compared with other age groups , actually (77.8%).The rate of the infection was higher among patients who reside in rural areas compared to those residing in urban areas (65.6% vs. 61.1%).The infection rate was higher among patients without previous diarrhea compared to patients with previous diarrhea (64.5% vs. 62.5%). Candida infection rate was higher in children with chronic diarrhea compared to acute diarrhea (80.0% vs. 63.2%).The rate of the infection was higher among those on raw feeding(age more 2 years) (75.0%) compared to those on mixed or bottle or breastfeeding (68.4%, 63.1%, and 50.0%) respectively. Candida infection rate was higher in children who lived in the area open sewage without drainage, actually (67.3%) while children lived in the area open sewage with drainage (52.6%) and close sewage (35.4%). The rate of the infection was among patients whose mothers age is under 20 years (89.4%) compared with other age groups. The rate of the infection among patients whose mothers have primary education level was (76.5%) compared to those with other education level of mothers.

II

List of Contents Item No.

2.1 2.2 2.3 2.4 2.5 2.6 2.6.1 2.6.2 2.6.3 2.6.4 2.6.5 2.6.6 2.6.7 2.7 2.8 2.9 2.1 2.11 2.12 2.12.1 2.12.2

Subjects Summary List of Contents List of Figures List of Tables List of Abbreviations Chapter One: Introduction Chapter Two: Literature Review Gastrointestinal tract infection Etiology of gastrointestinal tract infection History of Candida Taxonomy Genus Candida Candida species Candida albicans Candida glabrata Candida tropicalis Candida Krusei Candida parapsilosis Candida dubliniensis Candida stellatoidea General characteristics of Candida spp. Cell biology and enzymology Candidiasis Candidiasis epidemiology Type of diseases Pathogenesis of Candida infections Superficial infection Mucocotaneous infection III

Page No. I III VII IX XI

4 5 5 6 7 8 8 9 10 10 11 11 11 11 13 15 15 18 19 20 20

2.12.3 2.13 2.14 2.15 2.16 2.17 2.17.1 2.17.2 2.17.3 2.17.4 2.17.5 2.17.6 2.17.7 2.17.8 2.18 2.18.1 2.18.1.1 2.18.1.2 2.18.2 2.18.2.1 2.18.3 2.18.3.1 2.18.4 2.18.5 2.18.6 2.18.7 2.18.7.1 2.19 2.20 2.21 2.21.1 2.21.2 2.21.3

Systemic infection Candida spp. from gut commensal to pathogen Candida-associated with diarrhea Symptoms Risk factors Virulence Factors Polymorphism Adhesins and invasions Biofilm formation Connection sensing and thigmotropism hydrolases secretion Metabolic adaptability Respond to environmental stress Hemolysins Antifungal therapy Azoles Spectrum of activity Mechanism of action Ketoconazole Mechanism of action Clotrimazole Mechanism of action Fluconazole Miconazole Polyenes Nystatin Mechanism of action Mechanisms of resistance to antifungal agents Candida spp. and bacteria interaction Supplements for Candida treatment Probiotics Foods Herbs IV

21 22 25 25 25 26 26 26 27 27 28 28 28 29 29 30 31 31 31 31 32 32 32 32 33 33 33 34 34 36 36 36 37

Chapter Three: Materials and Methods 3.1 Materials 3.1.1 Apparatus and Equipment 3.1.2 Chemicals and Solution 3.1.3 Kits and Markers 3.1.4 Primers and Marker 3.1.5 Culture media 3.2 Methods 3.2.1 Sterilization 3.2.2 Preparation of solutions and stains 3.2.2.1 Solutions I. NaCl solution II. Ethanol III. Lysis buffer 3.2.2.2 Lactophenol Cotton Blue Stain(LPCB) 3.2.3 Preparation of culture media 3.2.3.1 Sabouraud Dextrose Agar (SDA) 3.2.3.2 Corn Meal Agar (CMA) 3.2.3.3 Chromogenic Agar Candida (CAC) 3.2.3.4 Yeast extracts Peptone Dextrose broth (YPD) 3.2.4 Study area and population 3.2.5 Collection of Samples 3.2.6 Direct Microscopical Examination 3.2.7 Culturing of Samples 3.2.8 Isolates Identification 3.2.8.1.1 Morphology 3.2.8.2 Microscopical Examination 3.2.9 Phenotypic Identification 3.2.9.1 Germ Tube Test 3.2.9.2 Chlamydospore Formation Test 3.2.9.3 Chromogenic Agar Candida ( CAC) 3.2.10 Preservation of the Candida isolates 3.2.10.1 Short-term preservation V

38 38 39 39 40 41 41 41 41 41 41 41 41 42 42 42 42 43 43 44 44 44 44 45 45 45 45 45 46 46 46 46

3.2.10.2 3.2.11 3.2.12 3.2.12.1 3.2.12.2 3.2.12.3 1 2 3 4 3.2.13 4.1 4.2 4.3 4.3.1 4.3.2 4.3.3 4.4 4.4.1 4.4.2 4.4.3 4.5 4.5.1 4.5.2 4.5.3 4.5.4 4.5.5 4.5.6 4.5.7 4.5.8 4.5.9

Long-term preservation Questionnaire performance Molecular Methods – based PCR DNA Extraction Concentration and purity of DNA Materials used for thermal cycling Primer selection and preparation PCR working solution Programmable thermal controller Electrophoresis Statistical Analysis Chapter Four: Results Samples collection and Direct microscope examination Sabouraud dextrose agar cultural characteristics Phenotype Identification of Candida spp. Germ tube test Chlamydospore formation test Chromogenic Agar Candida (CAC) Molecular identification depending on Polymerase chain reaction (PCR) Concentration and Purity of DNA extracted from Candida spp. isolates Identification of Candida spp. Identification of Candida albicans genotypes Analysis of questionnaire data Gender Age Resident Previous diarrhea Duration of diarrhea Mode of feeding Sewage Source of water Water sterilization VI

47 47 47 47 49 49 49 49 50 51 53 54 54 55 56 57 58 61 61 61 64 65 65 66 68 69 70 71 72 73 75

4.5.10 4.5.11 4.5.12 4.5.13

6.1 6.2

Bottle sterilization Previous antibiotic Age of mothers Education of mothers Chapter Five : Discussion Discussion Chapter Six: Conclusions and Recommendations Conclusions Recommendations References Appendix

76 77 78 80 82 89 90 91 117

List of Figures Item No.

Subjects

2-1 2-2 2-3 2-4 2-5

Major morphologies of Candida spp. Structure of the C. albicans cell wall Hospital - acquired Candida infections The human mycobiota The steps of C. albicans tissue invasion Virulent factors distribute to C.albicans pathogenicity mechanism The bacteria fungal interaction: the combination of physical associations and molecular interactions Direct microscope stool examination, show Candida budding and candida non-budding (40X) Figure 4-2 : Colonies of Candida spp. (Pure culture) on SDA at 37ºC for 48 hrs. Candida albicans stained with Lactophenol cotton blue (40X) Infections rate Candida spp. among children diarrhea Germ tube formation by C. albicans (40X) Pseudohyphae , Blastospore and Chlamydospores of C. albicans cultured on CMA at 35 ℃ (40X )

2-6 2-7 4-1 4-2 4-3 4-4 4-5 4-6

VII

Page No. 12 15 17 23 24 29 36 54 55 55 56 57 58

4-7

4-8 4-9 A

4-9 B

4-10

4-11 4-12 4-13 4-14 4-15 4-16 4-17 4-18 4-19 4-20 4-21 4-22

Colonies of Candida spp. cultured on chromogenic agar Candida at 37ºC for 48 hrs appeared different colors. Candida spp. isolated from children diarrhea under study Agarose gel electrophoresis(1.5%) for 1.5 hr at 5volt/cm of Candida spp. DNA products generated through universal primer ITS1 and ITS4, stained with Ethidium bromide Agarose gel electrophoresis(1.5%) for 1.5 hr at 5volt/cm of Candida spp. DNA products generated through universal primer ITS1 and ITS4, stained with Ethidium bromide Agarose gel electrophoresis(1.5%) for 1.5 hr at 5volt/cm of Candida genotypes DNA products generated through the primer pairs CA-INT-L and CAINT-R, stained with Diamond nucleic acid The distribution of candidiasis according to gender The distribution of candidiasis according to age The distribution of candidiasis according to residency The distribution of candidiasis according to previous diarrhea. The distribution of candidiasis according to duration of the diarrhea The distribution of candidiasis according to mode of the feeding . The distribution of candidiasis according to sewage state The distribution of candidiasis according to source of the water The distribution of candidiasis according to sterilization of the water The distribution of candidiasis according to bottle sterilization The distribution of candidiasis according to previous antibiotic use The distribution of candidiasis according to age of the mothers VIII

59

60 62

63

64

66 67 68 69 70 71 73 74 75 77 78 79

4-23

The distribution of education of mothers

candidiasis

according to the

81

List of Tables Item No.

Subjects

3-1 A 3-1 B 3-2 3-3 3-4 3-5 3-6

Apparatus utilized in this study Equipment utilized in this study Chemicals and Solution utilized in this study the Kits utilized in this study in this study Primers used in the study Culture media utilized in this study The Mixture of PCR Working Solution. Temperature cycling program for PCR, for identification of Candida spp. through The primer pairs ITS1 and ITS4 Temperature Cycling Program for PCR. identification of C. albicans genotype through the primer pairs CAINT-L and CA-INT-R

3-7

3-8

Page No. 38 38 39 40 40 41 50 51

52

4-1

Candida spp. isolated from children stool

60

4-2

Candida spp. were phenotypic identified depending on the morphological features Candida spp. ,DNA products generated through the primer pairs ITS1 and ITS4 Candida genotype. DNA products generated through The primer pairs CA-INT-L and CA-INT-R Candida spp. infection rate among patients according to gender Candida spp. infection rate among patients according to age Candida spp. infection rate among patients according to residency Candida spp. infection rate among patients according to previous diarrhea Candida spp. infection rate among patients according to duration of the diarrhea

61

4-3 4-4 4-5 4-6 4-7 4-8 4-9

IX

63 65 66 67 68 69 70

4-10 4-11 4-12 4-13 4-14 4-15 4-16 4-17

Candida spp. infection rate among patients according to mode of the feeding Candida spp. infection rate among patients according to sewage state Candida spp. infection rate among patients according to source of the water Candida spp. infection rate among patients according to water sterilization Candida spp. rate among patients according to bottle sterilization Candida spp. rate among patients according to previous antibiotic use Candida spp. infection rate among patients according to age of the mothers Candida spp. rate among patients according to education of the mothers

X

72 73 74 76 77 68 80 81

List of abbreviations Abbreviation AAD AIDS BFIs BSI bp C. albicans C. non albicans CAC Candida spp. CDC CMA Df DM DNA EDTA GIT GT HIV hrs ICU ITS LPCB min M OD PBS PCR pH Rnase rpm rRNA SAP SDA

Meaning Antibiotic associated diarrhea Acquired immune deficiency Bacterial fungal interactions blood stream infections base pair Candida albicans Candida non albicans chromogenic agar Candida Candida species Center for Disease Control Corn Meal Agar degree of freedom demethelase Deoxyribo nucleic acid Ethylenediaminetetraacetic acid) Gastrointestinal Germ tube Human immunodeficiency virus hours Intensive Care Units Internal transcribed spacer Lacto Phenol Cotton Blue minutes Molar (mol/L) Optical Density Phosphate Buffered Saline Polymerase Chain Reaction power of Hydrogen Ribo nuclease rounds per minute Ribosomal RNA Secreted Aspartyl Proteinases Sabouraud Dextrose Agar XI

sec Sig SPSS UV WHO YPD μ

second significant Statistical Package for Social Sciences Ultra violet World Health Organization Yeast extract Peptone Dextrose Micro

XII

Chapter One Introduction

Chapter One

Introduction

1. Introduction Candida species constitute a portion of the natural microbiota of the human mucosal, oral cavity, vagina, and gastrointestinal tract (Moran et al. 2012; Sardi et al.,2013).Several species, including Candida albicans, C. dublinensis, C. glabrata, C. guilliermondii, C. Lusitaniae, C. parapsilosis and C. tropicalis, can be found as part of the normal human commensal flora, especially in all sections of the gastrointestinal tract (Netea et al.,2008; Schulze and Sonnenborn,2009). In normal healthy person, there is a balance between Candida species as a normal flora and the normal defense mechanism of the body (Ferrer, 2000),which will cause opportunistic infection in the presence of any of the predisposing factors like; diabetes mellitus, malnutrition (Conlon and Snydoman,2000), humidity, burn, HIV infection (Roitte et al.,1998), renal failure,

endocrine

disturbance

(Guggenheimer

et

al.,

2000

),cancer,

indiscriminate usage of antibiotics (Daly et al,1981),glucocorticoids and cytotoxic drugs (Roitte et al.,1998). However, in response to improving or disturbance in the sponsor security systems in the gut, like the intestinal microbiota, gut-associated immune system and the mucosal barrier, Candida spp. can convert from safe commensals into pathogens or disturbance in the host defense systems in the gastrointestinal, including the intestinal microbiota, gutrelated immune system, and the mucosal preventive, Candida spp.can convert from harmless commensals into pathogens (Walker et al.,2009, Netea et al.,2008). Candidiasis is primarily caused by C. albicans, while there has been a striking increase in the frequency of non-albicans Candida species in the last few years. The most important species which are considered pathogenic to human are C.albicans, C. tropicalis, C. Kruse, C.glabrata, C.lusitaniae and C.viswanathii (Chander,2002).Candidia Worldwide distributed in nature -1-

Chapter One

Introduction

(Morton and Harris,1975).Colonization of the gastrointestinal genitourinary tract

and

may occur during birth directly from the birth canal

(Winner,1975), at some time during infancy or perhaps later in life, in which the source may be environmental like polluted fresh and marine water (Valdescollazol et al.1987) soil, air (Meyer et al.,1984), plant (Ferrer,2000) , contamination of bedding, air of the hospital, wash basins and could be of human mucous membrane or gastrointestinal tract (Morton and Harris,1975). Candida albicans is the more typical Candida species isolated from human stool. On the other hand, several reports have advised that Candida albicans may cause diarrhea,while other reports

suggested reason that

antibiotic-associated diarrhea (AAD) in young children (Danna et al, 1991). In recent years, the incidence of Candida spp. infections have increased. It has also been shown that C.albicans also causes diarrhea. Candidiasis in neonates does a serious and relatively common cause of late-onset sepsis associated with mortality. The recent study indicates that non-albicans infections are on the rise, which often accounts for more than 50 % of candidiasis found in the infected population.( Saravolatz et al.,2003). In the developing world as a whole, about one-third of infant and child deaths are due to diarrhea. Dehydration causes approximately 70% of diarrheal deaths, the loss of much salts and water from the body, which needs water to maintain blood volume and other fluids to function properly (Gupta and Mahaj,2005). Underlying reasons for the spread of diarrheal are found in poor hygiene and sanitation; limited access to safe drinking water as well as unsuitable education of health care providers and recipients (Thapar and Sanderson,2004; Curtis et al.,2000). Mainly each child will suffer from diarrhea at a certain point, the potency for great dehydration is always concerned with electrolyte abnormalities and hypovolemia in a child with diarrhea. People with diarrhea often have a fever and stomach ache (abdominal cramps).The infectious -2-

Chapter One

Introduction

agents creating diarrhea can be enteric bacterias, parasites, viruses and fungi.Yeast like fungi are usually found in the gastrointestinal system in small numbers since their attachment and habitation to the mucosal surface is prevented by the anaerobic microflora.The prolonged use of antibiotics can cause an imbalance in defensive microbial flora in the gastrointestinal tract, leading to antibiotic-associated diarrhea (Krause et al.,2003). The yeasts have been reported in increased frequency and quantity in the stool of the patients, which can result from antibiotic treatment of diarrhea.(Krause et al., 2001). Although not commonly suspected clinically, such pathogenic yeast-like fungi can raise the severity of diarrhea-causing severe dehydration, malnutrition, and mortality in already debilitated patients especially, immune affected individuals, children, and older patients. Discontinuing the antibiotic use, if required, administration of specific antifungal remedy can lessen morbidity and mortality in such patients (Krause et al., 2003). For the treatment of fungal infection, many antifungal have been used such as compounds of polyenes and azoles. However, the random usage of these antifungal in the last few years helps with the appearance of resistant strains to many antifungal, in addition to side effects (AL-Hadithy, 1998; Cowan, 1999). Aims of the study : 1.To isolation and identification of Candida spp. isolated from children suffering

from

diarrhea

in

Diyala

province

by

routine

laboratory

procedures and molecular techniques based PCR. 2.To identification genotypes distributions of Candida albicans. 3.Study the distribution of the Candida spp. among children and to explore the effect of some relevant factors.

-3-

Chapter Two Literature Review

Chapter Two

Literature Review

2. Literature Review 2.1. Gastrointestinal tract infection Gastrointestinal complications (constipation,impaction,bowel obstruction, radiation enteritis and diarrhea) are common problems for oncology patients (Supportiveand Board,2017). In human, gastrointestinal tract (GIT) is colonized by a wide and diverse community of microbes that are essential to its functions. These microorganisms have evolved in concert with their host to occupy specific regions and niches in the GIT (Simon and Gorbach, 1986). Candida species constitutes a portion of the natural microbiota of the GIT (Sardi et al.,2013, Moran et al.,2012 ).Approximately 85% of the intestinal microflora in a healthy person is beneficial bacteria, and 15% is pathogenic bacteria. Interactions of the microbial contribute to the homeostasis of the gut flora and destabilization of this microorganism which contributes to these ecosystem results in various gastrointestinal disorders (Emily, 2005). .Diarrheal illness, most of it attributable to enteric infection may continue to kill children. About 10 – 15% of cases of childhood diarrhea caused by bacterial pathogens Escherichia coli, Salmonella, Shigella, and others (Rishard, 1987).Candida albicans has been shown to be a cause of diarrhea (Birdsall, 1997).The neglect of the importance of fungi, especially yeast in the digestive system, makes the inability to detect the identification of microorganisms that cause diarrhea of 40% children (Forbes et al.,2001). Candida spp. is

one of the microorganisms found naturally in the

gastrointestinal tract. (Crook, 1984). Infections caused by Candida spp. are commonly referred to as candidiasis (or candidosis or moniliasis) (Bodey et al., 1992). The Candida grows into the rhizome stage that penetrates of the intestines walls, and the endotoxins of the Candida overgrowth begin to the blood stream and invade the rest of the body (Hilton et al, 1992).Candidiasis is the most frequently encountered fungus infection especially in condition with -4-

Chapter Two

Literature Review

depression of immune system. Administration of antibiotics for a long duration and use of immunosuppression agents, and patient with HIV infection leads to high incidence of candidiasis (Heelan et. al., 1998; Herrero et. al., 1999). 2.2. Etiology of gastrointestinal tract infection Candida is a conditionally pathogenic fungus. It is normally found in the skin, mouth, gut, and other mucous membranes. It causes infection when antibiotics or other factors reduce our natural resistance to its overgrowth. generally Candida infections are superficial, limited to mucus membranes. Some of the microorganisms may interact synergistically with Candida albicans to enhance the pathological effect of infection. This phenomenon has been documented for Cytomegalovirus and Candida albicans, Pseudomonas aeruginosin and Candida albicans, Salmonella typhimurium and Candida albicans, and Streptococcus faecalis and Candida albicans(Odds et al, 1988). Other names that have been given to Candida infection include Candidarelated complex, polysystemic candidiasis, chronic candidiasis, candidiasis hypersensitivity syndrome, and moniliasis (Crook, 1997). 2.3. History of Candida The initial name of Candida originates from the Latin term “candidus”, meaning “glowing white”, which relates to the creamy and glistening white special yeasts of colonies on culture media Candida infection, or “candidiasis”, is also known as“moniliasis” or “candidosis” (McCullough et al,1996). In 1665, Pepys Diary reported, “a patient has a fever, a thrush and a hiccup” (Barnett, 2008). Mycologists accepted the idea that oral thrush originates from the host this even late

1900s, Castellani quoted previous accounts of thrush as

pathological secretions of the oral mucosa (Calderone and Clancy, 2002). Yeasts were first identified in the GIT in the nineteenth century (Benham, 1931).Nevertheless, at that time no effort was made to identify and characterize

-5-

Chapter Two

Literature Review

these yeasts. In the early 1900s, the occurrence of yeast in excrement did report in medical research and education tried to link their appearance of GIT diseases, such as dysentery (GIT disease described by malabsorption typically reported in the tropics. Prior the Second World War, isolates specimens of people did name as yeasts of the genus Monilia, which did after that renamed into the genus of the Candida (Barnett, 2008; Langeron and Guerra, 1938). Culturing it from the digestive tract of healthy humans and those with GIT disorder from a zone of examination over the mid-1900s. Candida is more common isolated from stool specimens, although from this importance and appearance in stool , it is also isolated from healthy people (Suhr, 2015). The long known history of yeasts in the gastrointestinal tract and their peculiar presence began to specialize in the post-1980s to suggest theories associating unhealthy life styles to Candida excess in the bowels (Schulze and Sonnenborn, 2009).Yeasts are eukaryotic microorganisms classified in the kingdom Fungi, with the 1,500 species described estimated to be only 1% of all yeast species (Kurtzman and Piškur, 2006(. 2.4. Taxonomy Candida

belongs to the heterogeneous to Kingdom: Fungi, Phylum:

Ascomycota,Subphylum:Ascomycotina,Class:Ascomycetes,Order:Saccharomycet ales,Family:Saccharomycetaceae,Genus:Candida.The genus of the Candida contains approximately 200 species (Gray and Roberts, 1988). The taxonomy of the genus Candida is increases over time to be a reclassification of some Candida spp.

(e.g., Torulopsis glabrata has been

correctly named as C. glabrata), and the discovery of new species such as C. metapsilosis, C. dubliniensis, C. orthopsilosis, C. orthopsilosis, and C. metapsilosis were previously classified as part of the C. parapsilosis complex (Bendel, 2011). Furthermore, some species have been classified under C. albicans, including C. claussenii and C. langeronii. Recently, some new -6-

Chapter Two

Literature Review

Candida species were distinguished from C. albicans, such as C. albicans varafricana, a new sucrose-negative different of C. albicans that are closely related to C. stellatoidea type II (Howell and Hazen, 2011). More than 200 of the Candida spp. have been described, most of which exist as saprophytes organisms. Half of the described species cannot grow at 37°C, making them unsuccessful as human pathogens. Approximately thirty species can infect humans. Candida albicans is the most general species, followed by further pathogenic species which include C. glabrata, C. parapsilosis, C. krusei, C. tropicalis, C. kefyr, , C. guilliermondii, C. lusitaniae, C. inconspicua, C. famata, C., dubliniensis C. rugosa, C. norvegensis, C. lipolytica, C. pelliculosa , C. sake, C. apicola, C. zeylanoides , C. intermedia, C. valida, C. pulcherrima, C. haemulonii, C. stellatoidea, C. humicola, , C. utilis, C. lambica, C. ciferrii, C. colliculosa, C. holmii, C. marina, and C. sphaerica etc. (pfaller et al., 2007). Candida. albicans, and non- albicans spp. Include;C. glabrata, C. tropicalis, C. krusei and C. parapsilosis, and account for 90-92% of all cases of candidiasis (Lockhart,2014; Guinea, 2014). The Candida spp. the pathogenic list is expanding rapidly (pfaller et al., 2007). 2.5. Genus Candida The genus Candida belongs to the kingdom Fungi, division Eumycota (true fungi), which relates to class Deuteromycetes(Fungi imperfection) and the family Saccharomycetaceae (budding yeast) containing different genera of yeast, the genus of Candida which is one of the most common yeasts (Hannula, 2000). Candida is a simple diploid eukaryote organism lacking the sexual cycle. Its cells take different shapes (cocci, ovoid, cylindrical or elongate), which can be stained by Gram's and also by lactophenol blue staining. The ovoid yeast cell is ranging from 3 to 5 μm in size (Lodder, 1974). Candida which is commonly part of the normal flora of mouth, skin, intestinal tract, and vagina, it is a necessary yeast as part of the normal flora for human health (Barefoot and -7-

Chapter Two

Literature Review

Klaenhammer, 1983). Candida is dimorphic yeast exhibits a number of different morphological forms under different environmental conditions; such forms include budding yeast cells (blastoconidia, blastospores), pseudohyphae (elongated

cells

appear

filamentous

cell

chains),

true

hyphae,

and

chlamydospores. Growth requirement of Candida are 20-38°C within the range of pH 2.5 to 7.5 (Odd, 1988). Candida spp. Colonies are cream-colored to yellowish, soft ,glistening , free from moisture and wrinkled based on the species(Larone,1995). Ideal factors enhancing filamentation of Candida (yeast– hypha) are 35°C temperature, pH 7.0, an inoculum of blastospores in addition to the presence of serum, Nacetylglucosamine and proline ,proved that the genus Candida include about154 species. Among them, six species are most frequently isolates from human infections. The most abundant one of these species is Candida albicans. Other species involve Candida tropicalls, Candida glabrata, Candida parapsilosis, Candida krusie, and Candida lusitaniae are also isolated as causative agents of Candida infections(Abi-Said et al., 1997). 2.6. Candida species The genus of the Candida includes about 150 different species; but not whole species of Candida cause infection to humans. In medically significant, C. albicans, that most common species identified (50-60%);Candida glabrata (previously known as Torulopsis glabrata) (15-20%) ; C. parapsilosis (1020%); Candida tropicalis (6-12%) ; Candida krusei (1-3%) ; Candida kefyr (0.05) Candida spp.

Total 24 100.00% 14 100.00% 11 100.00% 8 100.00% 7 100.00% 64 100.00%

Appendix 3: Candida spp. infection among patients according to the age Age(month) ≥2 3-5 6-11 12-23 No. 4 1 12 5 C.albicans % 16.67% 4.17% 50.00% 20.83% No. 0 4 7 2 C. glabrata % 0.00% 28.60% 50.00% 14.30% No. 3 3 2 1 C. parapsilosis % 27.27% 27.27% 18.18% 9.09% No. 0 3 3 1 C. krusei % 0.00% 37.50% 37.50% 25.00% No. 0 3 3 0 C. tropicalis % 0.00% 42.90% 42.90% 0.00% No. 7 14 27 9 Total % 10.94% 21.88% 40.62% 12.50% Df=16;P-value= 0.964 non sig.( P>0.05) Candida spp.

- 118 -

24-36 2 12.50% 1 7.10% 2 18.18% 1 12.50% 1 14.30% 7 10.94%

Total 24 100.00% 14 100.00% 11 100.00% 8 100.00% 7 100.00% 64 100.00%

Appendix Appendix 4: Candida spp. infection among patients according to the residency.

Residency Candida spp.

Urban

Rural

No. 10 % 41.70% No. 5 C. glabrata % 35.70% No. 5 C. parapsilosis % 45.50% No. 1 C. krusei % 12.50% No. 1 C. tropicalis % 14.30% No. 22 Total % 34.40% Df=4;P-value= 0.751 non sig.( P>0.05)

14 58.30% 9 64.30% 6 54.50% 7 87.50% 6 85.70% 42 65.60%

C.albicans

Total 24 100.00% 14 100.00% 11 100.00% 8 100.00% 7 100.00% 64 100.00%

Appendix 5 : Candida spp. infection among patients according to the previous diarrhea. previous diarrhea Yes No No. 7 17 C.albicans % 29.20% 70.80% No. 3 11 C. glabrata % 21.40% 78.60% No. 0 11 C. parapsilosis % 0.00% 100.00% No. 2 6 C. krusei % 25.00% 75.00% No. 3 4 C. tropicalis % 42.90% 57.10% No. 15 49 Total % 23.40% 76.60% Df=4;P-value=0.546 non sig.(P> 0.05) Candida spp.

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Total 24 100.00% 14 100.00% 11 100.00% 8 100.00% 7 100.00% 64 100.00%

Appendix Appendix 6 : Candida spp . infection among patients according to duration of the diarrhea.

Duration of diarrhea acute chronic No. 22 2 C.albicans % 91.70% 8.30% No. 13 1 C. glabrata % 92.90% 7.10% No. 10 1 C. parapsilosis % 90.90% 9.10% No. 8 0 C. krusei % 100.00% 0.00% No. 7 0 C. tropicalis % 100.00% 0.00% No. 60 4 Total % 93.80% 6.30% Df=4;P-value= 0.993 non sig.(P> 0.05) Candida spp.

Total 24 100.00% 14 100.00% 11 100.00% 8 100.00% 7 100.00% 64 100.00%

Appendix 7 : Candida spp. infection among patients according to mode of the feeding. Mode of feeding Brest feeding Bottle feeding Mix feeding Raw food No. 0 18 4 2 C.albicans % 0.00% 75.00% 16.70% 8.30% No. 2 9 2 1 C. glabrata % 14.30% 64.30% 14.30% 7.10% No. 0 8 2 1 C. parapsilosis % 0.00% 72.70% 18.20% 9.10% No. 1 3 3 1 C. krusei % 12.50% 37.50% 37.50% 12.50% No. 1 3 2 1 C. tropicalis % 14.30% 42.90% 28.60% 14.30% No. 4 41 13 6 Total % 6.30% 64.10% 20.30% 9.40% Df=12;P-value= 0.993 non sig.(P> 0.05) Candida spp.

- 120 -

Total 24 100.00% 14 100.00% 11 100.00% 8 100.00% 7 100.00% 64 100.00%

Appendix Appendix 8 : Candida spp. infection among patients according to the sewage state. Sewages State Without drainage With drainage No. 14 2 C.albicans % 58.33% 8.33% No. 7 4 C. glabrata % 50.00% 28.57% No. 5 4 C. parapsilosis % 45.50% 36.40% No. 6 0 C. krusei % 75.00% 0.00% No. 5 0 C. tropicalis % 71.42% 0.00% No. 37 10 Total % 57.81% 15.63% Df=8;P-value= 0.738 non sig.(P> 0.05) Candida spp.

Close 8 33.33% 3 21.42% 2 18.20% 2 25.00% 2 28.87% 17 26.56%

Total 24 100.00% 14 100.00% 11 100.00% 8 100.00% 7 100.00% 64 100.00%

Appendix 9 : Candida spp . infection among patients according to source of the water.

source of the water Tap water Mineral water No. 22 1 C.albicans % 91.70% 4.20% No. 14 0 C. glabrata % 100.00% 0.00% No. 10 1 C. parapsilosis % 90.90% 9.10% No. 7 1 C. krusei % 87.50% 12.50% No. 6 1 C. tropicalis % 85.70% 14.30% No. 59 4 Total % 92.20% 6.30% Df=8;P-value= 0.860 non sig.(P> 0.05) Candida spp.

- 121 -

River 1 4.20% 0 0.00% 0 0.00% 0 0.00% 0 0.00% 1 1.60%

Total 24 100.00% 14 100.00% 11 100.00% 8 100.00% 7 100.00% 64 100.00%

Appendix Appendix 10 : Candida spp. infection patients according to water sterilization.

Sterilization of water no Sterilization sterilization No. 15 9 C.albicans % 62.50% 37.50% No. 10 4 C. glabrata % 71.40% 28.60% No. 5 6 C. parapsilosis % 45.50% 54.50% No. 5 3 C. krusei % 62.50% 37.50% No. 3 4 C. tropicalis % 42.86% 57.14% No. 38 26 Total % 59.38% 40.63% Df=4;P-vaule= 0.895non sig.(P>0.05) Candida spp.

Total 24 100.00% 14 100.00% 11 100.00% 8 100.00% 7 100.00% 64 100.00%

Appendix 11 : Candida spp. infection among patients according to bottle sterilization. Bottle Sterilization sterilization non-sterilization No. 10 14 C.albicans % 41.70% 58.30% No. 3 11 C. glabrata % 21.40% 78.60% No. 2 9 C. parapsilosis % 18.20% 81.80% No. 3 5 C. krusei % 37.50% 62.50% No. 2 5 C. tropicalis % 28.60% 71.40% No. 20 44 Total % 31.30% 68.80% Df=4;P-value=0.832 non sig.(P> 0.05) Candida spp.

- 122 -

Total 24 100.00% 14 100.00% 11 100.00% 8 100.00% 7 100.00% 64 100.00%

Appendix Appendix 12 : Candida spp. infection among patients according to previous antibiotic use.

previous antibiotic use Yes No No. 13 11 C.albicans % 54.17% 45.83% No. 10 4 C. glabrata % 71.43% 28.57% No. 6 5 C. parapsilosis % 54.55% 45.45% No. 7 1 C. krusei % 87.50% 12.50% No. 5 2 C. tropicalis % 71.43% 28.57% Total No. 38 26 Df=4;P-value= 0.425 non sig.(P> 0.05) Candida spp.

Total 24 100.00% 14 100.00% 11 100.00% 8 100.00% 7 100.00% 64

Appendix 13 : Candida spp . infection among patients according to age of the mothers. Age of mothera 0.05) Candida spp.

- 123 -

Total 24 100.00% 14 100.00% 11 100.00% 8 100.00% 7 100.00% 64 100.00%

Appendix Appendix 14: Candida spp . infection among patients according to education of the mothers. Mother education level Total illiteracy read and write primary secondary post-secondary No. 0 14 4 4 2 24 C.albicans % 0.00% 58.30% 16.70% 16.70% 8.30% 100.00% No. 0 8 5 0 1 14 C. glabrata % 0.00% 57.10% 35.70% 0.00% 7.10% 100.00% No. 1 7 2 0 1 11 C. parapsilosis % 9.10% 63.60% 18.20% 0.00% 9.10% 100.00% No. 1 3 1 0 3 8 C. krusei % 12.50% 37.50% 12.50% 0.00% 37.50% 100.00% No. 1 4 1 0 1 7 C. tropicalis % 14.30% 57.10% 14.30% 0.00% 14.30% 100.00% No. 3 36 13 4 8 64 Total % 4.70% 56.30% 20.30% 6.30% 12.50% 100.00% Df=16;P-value= 0.967 non sig.(P>0.05) Candida spp.

- 124 -

‫(‪ٔ ,)63.2% vs. %:1.1‬صذ اٌ االطفال انًشضٗ انز‪ٚ ٍٚ‬ؼخًذٌٔ ػهٗ انغزاء انخاو اكزش ػشضت‬ ‫نإلصابت بٕالغ (‪ )97.0%‬يٍ انز‪ٚ ٍٚ‬ؼخًذٌٔ ػهٗ انشضاػت انًخخهطت أ انز‪ٚ ٍٚ‬ؼخًذٌٔ ػهٗ انشضاػت‬ ‫االصطُاػ‪ٛ‬ت فمظ أ ‪ٚ‬ؼخًذٌٔ ػهٗ انشضاػت انطب‪ٛ‬ؼ‪ٛ‬ت فمظ )‪ ,(68.4%, 63.1%, 50.0%‬كًا ٔصذ‬ ‫اٌ االطفال انًشضٗ انز‪ٚ ٍٚ‬ؼ‪ٛ‬شٌٕ ف‪ ٙ‬يُاطك يفخٕحت شبكاث انًضاس٘ ٔبذٌٔ حصش‪ٚ‬ف اكزش ػشضت‬ ‫نإلصابت )‪ (67.3%‬يٍ انز‪ٚ ٍٚ‬ؼ‪ٛ‬شٌٕ ف‪ ٙ‬يُاطك يفخٕحت ٔراث حصش‪ٚ‬ف )‪ (52.6%‬أ يغهمّ )‪(35.4%‬‬ ‫‪ٔ ٔ,‬صذ اٌ انًضًٕػت انؼًش‪ٚ‬ت نأليٓاث الم يٍ (‪ )61‬سُت اطفانٍٓ اكزش ػشضت نإلصابت يٍ‬ ‫انًضًٕػاث انؼًش‪ٚ‬ت االخشٖ بٕالغ )‪ ,(:;.6%‬كًا ٔصذ اٌ يسخٕٖ انخؼه‪ٛ‬ى االبخذائ‪ ٙ‬نأليٓاث اطفانٍٓ‬ ‫اكزش ػشضت نإلصابت بٕالغ (‪.(76.5%‬‬

‫الخالصة ‪:‬‬ ‫شًهج ْزِ انذساست (‪ )011‬ػ‪ُٛ‬ت خشٔس صًؼج يٍ االطفال بأػًاس الم يٍ رالرت سُٕاث ‪ٚ‬ؼإٌَ يٍ‬ ‫االسٓال ف‪ ٙ‬يسخشفٗ انبخٕل انخؼه‪ ًٙٛ‬ف‪ ٙ‬يحافظت د‪ٚ‬انٗ يٍ بذا‪ٚ‬ت شٓش حشش‪ ٍٚ‬االٔل‪ٔ 6108‬نغا‪ٚ‬ت ان‪ٕٛ‬و‬ ‫انزانذ يٍ شٓش كإٌَ االٔل ‪.6108‬‬ ‫ٔشخصج ‪ )86%(86‬ػ‪ُٛ‬ت خشٔس ػهٗ آَا ححخٕ٘ اصابت فطش‪ٚ‬ت نــ ‪Candida species‬‬ ‫بٕاسطت ػًه‪ٛ‬اث حشخص‪ٛ‬ت حأك‪ٛ‬ذ‪ٚ‬ت انًخًزهت بانفحص انًضٓش٘ انًباشش نؼ‪ُٛ‬ت انخشٔس ٔ انزسع ػهٗ‬ ‫انٕسظ انزسػ‪ٔ Sabouraud Dextrose Agar (SDA) ٙ‬يٍ رى انفحص انًضٓش٘ نهًسخؼًشة ‪.‬‬ ‫بؼذ انؼزل االٔن‪ ٙ‬ألَٕاع ‪ Candida spp.‬اظٓشث انُخائش اٌ‪ )59.7%(66‬يٍ انؼزالث كاَج‬ ‫‪ )60.;%(06 , Candida albicans‬كاَج ‪glabrata‬‬

‫‪ )17.2%(10 , Candida‬كاَج‬

‫‪ )12.5%( 8, Candida parapsilosis‬كاَج ‪ )10.9%(7 ,Candida krusei‬كاَج ‪Candida‬‬ ‫‪ٔ tropicalis‬يٍ رى صُفج ‪ Candida albicans‬ص‪ُٛٛ‬ا باالػخًاد‪, PCR, rDNA 25S‬اظٓشث‬ ‫انُخائش اٌ ‪ )83.3%)20‬يٍ انؼزالث كاَج ححج انصُف انض‪ )8.3%(2, A ُٙٛ‬نكم يٍ انصُف‬ ‫انض‪ Cٔ Bُٙٛ‬يٍ ح‪ٛ‬ذ انحضى انًُخش نخفاػم انبهًشة ‪.‬‬ ‫ٔاظٓشث انُخائش اٌ ُْانك اًْ‪ٛ‬ت أ فشٔق يؼُٕ‪ٚ‬ت نألطفال )‪ (P0.05‬‬

‫ح‪ٛ‬ذ ٔصذ اٌ انزكٕس اكزش ػشضت نإلصابت بانـ ‪ Candida‬يٍ االَاد )‪ ,(66.7% vs. 60.5%‬كزنك‬ ‫فاٌ االطفال انًشضٗ يٍ انًضًٕػت انؼًش‪ٚ‬ت شٓش‪ ٍٚ‬أ الم حضًُج اػهٗ َسبت نإلصابت‬ ‫بٕالغ‪ ,(77.8%)7‬كزنك ٔصذ اٌ االطفال انًشضٗ انز‪ٚ ٍٚ‬ؼ‪ٛ‬شٌٕ ف‪ ٙ‬االس‪ٚ‬ا‬

‫اكزش ػشضت نإلصابت يٍ‬

‫انز‪ٚ ٍٚ‬ؼشٌٕ ف‪ ٙ‬انًذ‪ُٚ‬ت )‪,(65.6% vs. 61.1%‬كًا ٔصذ اٌ االطفال انًشضٗ انز‪ٚ ٍٚ‬ؼإٌَ يٍ اسٓال‬ ‫يسبك اكزش ػشضت نإلصابت يٍ انز‪ ٍٚ‬نى ‪ٚ‬ؼإٌَ يٍ اسٓال يسبك )‪ٔ ,(64.5% vs. 62.5%‬صذ اٌ‬ ‫االطفال انًشضٗ انز‪ٚ ٍٚ‬ؼإٌَ يٍ اسٓال يزيٍ اكزش ػشضت نإلصابت يٍ انز‪ٚ ٍٚ‬ؼإٌَ يٍ اسٓال حاد‬

                َ ّ‫ذ‬ َ َۡ َ ُ ٓ ‫َ ََ ذ َ ُ ُ ْ ۡ ۡ َ ذ‬ ‫ك‬ ‫ب‬ ‫ر‬ ‫ِو‬ ‫ن‬ ‫ك‬ ‫َل‬ ‫إ‬ ‫ل‬ ‫ىز‬ ‫أ‬ ‫ِي‬ ‫ٱَّل‬ ‫م‬ ‫ل‬ ‫ع‬ ‫ٱل‬ ‫وا‬ ‫وت‬ ‫أ‬ ‫ِيو‬ ‫ٱَّل‬ ‫ى‬ ‫ر‬ ‫ي‬ ‫و‬ ِ ِ ِ ِ   ۡ ۡ َ ٓ ‫ٱۡل ذق َو َي ۡه ِد‬ َ ‫ص َر ٰ ِط ۡٱل َعزيز‬ َ ‫ُه َو‬   ٰ ِ‫ي إ‬ ٦ ‫ٱۡل ِهي ِد‬ ‫َل‬ ِ ِ ِ           ‫صدق اهلل العظيم‬     )٦( ‫ اآليت‬-‫سورة سبأ‬                            

‫جمهورية العراق‬ ‫وزارة التعليم العالي و البحث العلمي‬ ‫جامعة ديالى‬ ‫كلية الطـــب‬

‫الفطريات المسببة لإلسهال بين االطفال في محافظة ديالى‬ ‫رسالة مقدمة الى‬

‫مجلش كلٍت الطب – جامعت دٌالى‬ ‫وهً جزء من متطلباث نٍل شهادة الماجستٍر‬ ‫فً االحٍاء المجهرٌت الطبٍت‬

‫قدمها‬ ‫علي رياض حميد مجيد‬ ‫بكالىرٌىس طب وجراحت بٍطرٌت ‪ /‬كلٍت الطب البٍطري‪ -‬جامعت دٌالى‬ ‫)‪)2014‬‬

‫بإشراف‬ ‫ا‪.‬م‪.‬د‪ .‬لمى طه احمد‬ ‫كلٍت الطب ‪ /‬جامعت دٌالى‬

‫‪ 1028‬م‬

‫ا‪.‬م‪.‬د‪ .‬صباح محسن علي‬ ‫كلٍت الطب ‪ /‬جامعت المستنصرٌت‬

‫‪2419‬هـ‬