University of Ghana http://ugspace.ug.edu.gh CHARACTERIZATION OF ENVIRONMENTAL CRYPTOCOCCUS FROM SELECTED SITES IN THE GREATER ACCRA REGION By NANA EGHELE ADADE (10551867) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON, IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER OF PHILOSOPHY DEGREE IN MEDICAL MICROBIOLOGY JULY, 2017 University of Ghana http://ugspace.ug.edu.gh DECLARATION The work in this thesis is original and was carried out by me at the Department of Medical Microbiology, School of Biomedical and Allied Health Sciences and supervised by the supervisors below. Works from other authors that were used were duly acknowledged in the text or by references cited. This work has not been submitted to any institution for the award of any degree. Student: Signature:………………………...Date:…………… NANA EGHELE ADADE Supervisors: Signature:…………………………Date:…………… Dr. Japheth A. Opintan Department of Microbiology, School of Biomedical and Allied health Sciences, College of Health Sciences, University of Ghana Signature:…………………………Date:……………. Prof. Mercy J. Newman Department of Microbiology, School of Biomedical and Allied health Sciences, College of Health Sciences, University of Ghana ii University of Ghana http://ugspace.ug.edu.gh DEDICATION This work is dedicated to God, who is always gracious and kind, to the memory of my Dad, who sacrificed his comfort to give me the best education he could afford and my family, who always have my back. iii University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT I profusely thank the Lord God Almighty for the strength and guidance throughout my work and for bringing me this far. The Lord is indeed gracious and kind. My heartfelt appreciation goes to my supervisors Dr. Japheth A. Opintan and Prof. Mercy J. Newman for the finance, other logistics and directions in carrying out this work. I also wish to thank other lecturers for their encouragement. Special thanks go to Pastor Earnest Omoleme, Late Prof. Kingsley Twun-Danso, Mr Prince Pappoe, Dr Noah Obeng and Dr Kwaku Labi, for the various roles played to ensure the success of this work. I say a big thank you to Mary Magdalene Osei, Georgina Tetteh-Ocloo, Wright Amesimeku, Esther Gyinae and Seth Agyemang for their help with sample collection. My appreciation also goes to all staff of the Medical Microbiology Department, Central Laboratory, Korle-Bu Teaching Hospital for their support. Special thanks go to the staff especially Evelyn Omane, Emelia Aryeetey and colleague students of the Department of Microbiology, School of Biomedical and Allied Health Sciences. Finally, I wish to thank my family, friends and numerous others whose names could not be mentioned here, for their prayers and support. iv University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS DECLARATION ................................................................................................................................................... II DEDICATION ..................................................................................................................................................... III ACKNOWLEDGEMENT .................................................................................................................................... IV TABLE OF CONTENTS ...................................................................................................................................... V LIST OF TABLES.............................................................................................................................................. VII LIST OF FIGURE .............................................................................................................................................. VII LIST OF ABBREVIATIONS ............................................................................................................................ VIII ABSTRACT ........................................................................................................................................................... 1 CHAPTER ONE ..................................................................................................................................................... 3 INTRODUCTION .................................................................................................................................................. 3 1.0 BACKGROUND ................................................................................................................................................. 3 1.1 PROBLEM STATEMENT .................................................................................................................................. 4 1.2 JUSTIFICATION ................................................................................................................................................ 5 1.3 AIM ..................................................................................................................................................................... 6 1.3.1SPECIFIC OBJECTIVES .............................................................................................................................. 6 CHAPTER TWO .................................................................................................................................................... 7 LITERATURE REVIEW ....................................................................................................................................... 7 2.0 BIOLOGY OF CRYPTOCOCCUS ...................................................................................................................... 7 2.1 CRYPTOCOCCUS VIRULENCE FACTORS .................................................................................................... 8 2.2 CHARACTERIZATION OF CRYPTOCOCCUS ................................................................................................ 9 2.2.1 HOST INFECTIVITY ................................................................................................................................... 9 2.2.2 BIOCHEMICAL CHARACTERIZATION .................................................................................................. 10 2.2.3 MOLECULAR AND SEROLOGICAL CHARACTERIZATION ................................................................. 11 2.3 ECOLOGICAL NICHES OF CRYPTOCOCCUS ............................................................................................. 13 2.4 DISTRIUTION OF CRYPTOCOCCUS ............................................................................................................. 15 2.5 ANTIFUNGAL SUSCEPTIBILITY OF ENVIRONMENTAL CRYPTOCOCCUS ......................................... 16 2.6 MODE OF ACTION AND RESISTANCE PATTERN OF ANTIFUNGAL AGENTS .................................... 18 2.6.1 POLYENES ................................................................................................................................................ 18 2.6.2 AZOLES ..................................................................................................................................................... 19 2.6.3 ALLYLAMINES AND THIOCARBAMATES .............................................................................................. 19 2.6.4 5-FLUOROCYTOSINE ........................................................................................................................... 19 CHAPTER THREE .............................................................................................................................................. 21 MATERIALS AND METHODS ............................................................................................................................ 21 3.0 STUDY SITE, DESIGN AND SAMPLE .......................................................................................................... 21 3.1 ENVIRONMENTAL SAMPLE COLLECTION .............................................................................................. 23 3.2 ISOLATION AND IDENTIFICATION OF ENVIRONMENTAL CRYPTOCOCCUS ................................... 23 3.3 ANTIFUNGAL SUSCEPTIBILITY TESTING OF ENVIRONMENTAL CRYPTOCOCCUS........................ 24 3.4 MOLECULAR ANALYSES ............................................................................................................................. 25 3.5 DATA ANALYSIS ........................................................................................................................................... 27 v University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR ................................................................................................................................................ 28 RESULTS ............................................................................................................................................................... 28 4.1 ISOLATION AND IDENTIFICATION OF ENVIRONMENTAL CRYPTOCOCCUS SPECIES ................... 28 4.2 SAMPLE SOURCE DISTRIBUTION OF CRYPTOCOCCUS ......................................................................... 28 4.3 LOCATION DISTRIBUTION OF CRYPTOCOCCUS ..................................................................................... 28 4.4 CRYPTOCOCCUS GENUS-SPECIFIC AND SPECIES-SPECIFIC PCR ........................................................ 31 4.4.1 DISTRIBUTION OF ENVIRONMENTAL CRYPTOCOCCUS BY LOCATIONS ...................................... 33 CHAPTER FIVE .................................................................................................................................................. 38 DISCUSSION ......................................................................................................................................................... 38 5.1 ISOLATION OF ENVIRONMENTAL CRYPTOCOCCUS SPECIES ............................................................. 38 5.1.1 NATURE OF POSITIVE ENVIRONMENTAL SAMPLES AND SAMPLING SITES ................................. 40 5.2 HEALTH IMPLICATIONS OF ENVIRONMENTAL CRYPTOCOCCUS ...................................................... 42 5.3 LIMITATIONS OF THE STUDY ..................................................................................................................... 43 CHAPTER SIX .................................................................................................................................................... 44 CONCLUSIONS AND RECOMMENDATIONS .................................................................................................. 44 6.1 CONCLUSIONS ............................................................................................................................................... 44 6.2 RECOMMENDATIONS .................................................................................................................................. 44 APPENDICES......................................................................................................................................................... 65 APPENDIX I ........................................................................................................................................................... 65 PREPARATION OF AGAR MEDIA AND STERILIZATION OF CONSUMABLES .......................................... 65 APPENDIX II: RESULTS FROM PHENOTYPIC ANALYSES ........................................................................... 67 vi University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES TABLE 4.1 Distribution of Cryptococcus by sample locations…………………………………30 TABLE 4.2: Distribution of Cryptococcus species by sample location…………………………34 TABLE 4.3: Antifungal Susceptibility of environmental Cryptococcus………………………...37 LIST OF FIGURE FIGURE 3.1: Map showing Sampling sites……………………………………………………..22 vii University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS % - Percentage ˚C - Degree Celsius µl –Micro liter µg-Micrograms AFLP - Amplified Fragment Length Polymorphisms AIDS - Acquired Immunodeficiency Syndrome BBB- Blood Brain Barrier CAP- Capsule CGB- Canavanine Glycine bromothymol Blue C. gattii - Cryptococcus gattii C. neoformans -Cryptococcus neoformans CLSI- Clinical Laboratory Standard Institute CNS - Central Nervous System DNA-Deoxyribonucleic Acid EDTA- Ethylene Diamine Tetraacetic Acid GM-CSF-Granulocyte-Macrophage Colony-Stimulating-Factor GMB- Glucose Methylene Blue HIV-Human Immunodeficiency Virus ITS- Internal Transcribed Spacer NACP- National AIDS/STI Control Programme viii University of Ghana http://ugspace.ug.edu.gh SDA- Sabouraud Dextrose Agar TAE- Tris Acetate EDTA PBS- Phosphate Buffered Saline PCR-Polymerase Chain Reaction var- variety VG- Variety gattii VN- Varietry neoformans YPD-Yeast Potato Dextrose ix University of Ghana http://ugspace.ug.edu.gh ABSTRACT Background: Cryptococcus neoformans and C. gattii affect both immunocompromised and immunocompetent individuals. Cryptococcal infections are acquired when cryptococcal spores or dehydrated yeast cells from contaminated environmental samples such as pigeon and bat droppings are inhaled. Immunocompromised individuals especially HIV/AIDS patients are however at a higher risk of death due to cryptococcosis. Sub-Saharan Africa records the greatest mortality due to cryptococcal meningitis, estimated to be as high as 70%. This study characterized Cryptococcus from pigeon and bat droppings from selected sites in the Greater Accra region. Methods: Droppings from pigeons (n=643) and bats (n=55) were collected from 4 sources (markets, church, residential quarters and a public park) from November 2016 to April, 2017. Droppings were inoculated on Sabouraud dextrose agar (SDA) supplemented with chloramphenicol (0.05 g/L). Creamy yeast colonies suggestive of Cryptococcus were purified and identified using standard microbiological methods. Microbiologically identified cryptococcal isolates were confirmed by PCR using yeast genus specific primers (ITSI and ITS4). Additionally, species specific primers (CN70 and CN49) were used to characterize the isolates. All characterized Cryptococcus were subjected to susceptibility test using two antifungal agents (fluconazole and voriconazole), referencing the Clinical Laboratory Standards Institute (CLSI), 2015 guidelines for yeast. Results: 49 of 698 (7.0%) samples were confirmed to be positive for Cryptococcus. The distributions of the isolates were as follows; church =26.3% (21/80), markets =4.9% (12/ 243) residential quarters =5.0% (16/ 320). No Cryptococcus was isolated from the droppings collected from the public park. Of the 49 Cryptococcus isolates, 1 (2.0 %) was characterized as C. gattii, 4 1 University of Ghana http://ugspace.ug.edu.gh (8.2 %) as C. neoformans, 20 (40.8 %) as C. neoformans /C.gatti, while 24(49.0%) of them could not be characterized with the species-specific primers used. Most of the isolates 36(73.5%) showed resistance to fluconazole. Conclusion: This study reported the presence of the pathogenic C. neoformans and C. gatti species complex from environmental sources for the first time in Accra, Ghana. A greater proportion of the species were C. neoformans with many of them resistant to fluconazole. Further studies, comparing the genetic relatedness of environmental and clinical Cryptococcus isolates is recommended. 2 University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE INTRODUCTION 1.0 BACKGROUND The yeast genus Cryptococcus contains numerous species with only Cryptococcus neoformans and Cryptococcus gattii considered as the pathogenic species (Nester et al., 2009). They are saprophytic in nature, yet are able to infect and cause disease (Casadevall and Perfect, 1998). Immunocompromised individuals suffer mainly from diseases caused by C .neoformans and C. gatti cause disease mainly in apparently healthy individuals (Chowdary et al., 2012, Idnurm A and Lin X, 2015). The yeasts invade the pulmonary alveoli and cause an initial pulmonary infection. In healthy individuals, the pulmonary infection will most often be cleared or remain latent, whereas in individuals with compromised immune systems the organism can become pathogenic with dissemination from the pulmonary alveoli to the central nervous system (CNS), then crosses the blood brain barrier (BBB) with subsequent inflammation of the meninges, eventually culminating in fatal cryptococcal meningitis or meningoencephalitis (Kwon-Chung et al., 2014; Park et al., 2009; Lin X, 2009). Immunocompromised individuals, especially HIV/ AIDS patients are therefore at a higher risk of death from the acquisition of this organism. C. gattii infections are thought to be more severe and are often more associated with the formation of cryptococcomas compared to infections caused by C. neoformans. Cryptococcoma formation is 3 University of Ghana http://ugspace.ug.edu.gh thought to confer a variable response to antifungal therapy, hence C. gattii is reportedly not as susceptible to antifungal agents compared to C. neoformans, although some studies do not agree with this (Chen et al., 2000; Gutch et al., 2015). Studies on antifungal susceptibility patterns of cryptococcal isolates recovered from the environment in different geographical regions, reported variable susceptibility patterns to same antifungal agents such as flucytocine, Amphotericin B, fluconazole, itraconazole, voriconazole (Soares et al., 2005; Matos et al., 2012; Nnadi et al., 2016). In the environment, Cryptococcus is ubiquitous; hence there is constant exposure to the organism. An examination of the niches of C. neoformans species complex show that avian, especially pigeon roosting areas and pigeon droppings, bat droppings and arboreal habitats are the main niches of this important yeast (Dongmo et al.,2016; Nester et al., 2009; Mitchell et al., 2011, Chowdary et al., 2012). 1.1 PROBLEM STATEMENT Cryptococcal meningitis is reported every year in over one million individuals suffering from HIV, resulting in over 625,000 deaths (Park et al., 2009). The AIDS-defining condition cryptococcal meningitis is the main meningitis form among adult HIV/AIDS patients in Africa and also the leading cause of community-acquired meningitis with prevalence between 20-45% (Hakim et al., 2000). The greatest mortality due to cryptococcal meningitis is also recorded by the continent (Holmes et al., 2003; Desalermos et al., 2012). 4 University of Ghana http://ugspace.ug.edu.gh Cryptococccal infections are acquired when cryptococcal spores or their dehydrated cells are inhaled (Park et al., 2009; Lin X, 2009; Kwon-Chung et al., 2014). The genetic profiles of environmental and clinical cryptococcal isolates from same geographical regions are reportedly identical (Alves et al., 2016; Litvintseva et al., 2005). In the Greater Accra Region, bats and especially pigeons can usually be found roaming the environment, such as motor parks, playgrounds, markets, churches and residential areas where they are also kept as pets. Pigeons are also sold in different markets, exposing a large number of people to their droppings which may contain cryptococcal spores or dehydrated cells. 1.2 JUSTIFICATION Ghana has a national HIV prevalence of 1.6% with approximately 3000 deaths (NACP, 2016). Ghana also records a crypotococcal meningitis prevalence of 11.7%even among those whose HIV status were not known (Owusu et al., 2012). Cryptococcosis in HIV/ AIDS patients and other immunocompromised individuals have also been reported at the Korle Bu teaching hospital, Accra, Ghana (Akakpo et al., 2018; Unpublished data). This study will inform on the relevance of pigeon and bat droppings as important sources of cryptococcal infections in the Greater Accra Region. 5 University of Ghana http://ugspace.ug.edu.gh The antifungal susceptibility patterns and prevalent species type of these environmental cryptococcal isolates will be known and could inform on what to expect with clinical isolates. 1.3 AIM The study aim was to isolate and characterize environmental Cryptococcus from selected sites in the Greater Accra Region. 1.3.1SPECIFIC OBJECTIVES  To determine the distribution of Cryptococcus species in environmental samples.  To characterize (speciate) environmental Cryptococcus isolates by PCR.  To determine the susceptibility profiles of environmental cryptococcal isolates to antifungal agents. 6 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO LITERATURE REVIEW 2.0 BIOLOGY OF CRYPTOCOCCUS Cryptococcus neoformans and C. gatti species complex have distinguished themselves as important human and animal pathogens. Some other species which make up the genus, in rare cases may also cause infections in humans. Examples are Cryptococcus liquefaciens, C. laurentti, C. flavescens, C. uniguttulatus, C. macerans, C. magnur, C. humicolus, C. curvatus, C. adelensi, C luteolus and C. uzbekistanesis (Rosario et al., 2005; Takemura et al., 2015). Cryptococcus reproduces by budding. It is a round to oval yeast-like cell and usually measures between 3 to 10µm in diameter. It is facultatively intracellular and the Cryptococcus species complex differs from most of the non-pathogenic cryptococcal species by their ability to grow at 37oC and in their synthesis of important virulent factors which includes the capsule (CAP59) and laccase genes responsible for melanin production (Nester et al., 2009; Bicanic et al., 2007; Kubowski and Heitman,2011). C. gatti and C. neoformans are dimorphic fungi (existing in both yeast and hyphal phases).Their primary morphology is the budding yeast. The hyphal phase is primarily responsible for the formation of spores small enough to be inhaled into the lungs. Their ability to exist as yeast in infected tissues and natural substrates differentiates them from other dimorphic fungi (Wickes, 2002). 7 University of Ghana http://ugspace.ug.edu.gh 2.1 CRYPTOCOCCUS VIRULENCE FACTORS The C. neoformans species complex exhibit virulence factors with dual purposes. They are useful in both their environmental survival and pathogenesis (Casadevall et al., 2003). The capsule protects the organism against dehydration and desiccation in the environment, while in pathogenesis; it is antiphagocytic (Park et al., 2014; Casadevall et al., 2003; Butel et al., 2007). Melanin protects the organism from heavy metals temperature extremes, ultraviolet radiation, antimicrobial peptides and oxidants, conferring a survival advantage on Cryptococcus strains in the environment (Wang and Cassadevall, 1994; Eisenman et al., 2007). In pathogenesis melanin protects the organism from the host’s immune defense mechanisms and aids its resistance to antifungal agents (Ikeda et al., 2003;Van Duin et al.,2002 ;Nosanchuk et al., 1999), preventing the drug from reaching its active site. The ability of the Cryptococcus species to grow at 37°C enables its survival in mammals. There is also increased transcription of heat shock proteins at this temperature (Steen et al., 2002). A number of secreted extracellular enzymes also serve dual purposes like virulence factors during human infection and also in the environment. Phospholipase is necessary to initiate pulmonary infection as well as dissemination from the lung to the blood stream. This enzyme destroys the host cell membrane enabling easy penetration and establishment of the fungal infection. It maintains the fungal cell’s integrity ensuring its survival. Several virulence phenotypes such as melanin production and growth at 37oC are also dependent on phospholipase (Siafakas et al., 2007). In the environment, phospholipase protects Cryptococcus from amoeboid predators (Ghannoum, 2000). 8 University of Ghana http://ugspace.ug.edu.gh Urease aids the yeasts dissemination from the central nervous system (CNS) and the crossing of the blood brain barrier (BBB). Ammonia, the usable form of nitrogen by environmental cryptococcal isolates such as those found in pigeon guano is made available by the conversion of urea to ammonia by Urease (Almeida et al., 2015; Wozniak et al., 2015; Santangelo et al., 2004). 2.2 CHARACTERIZATION OF CRYPTOCOCCUS Cryptococcus gattii and C. neoformans were once considered a homogenous anamorphic species, but the two were separated based on numerous differences which included; host infectivity, geographical distribution, ecological niches, epidemiology, molecular and biochemical characteristic as well as genetics (Kavanaugh et al., 2006, Chowdaryet al., 2012; Chan and Tay, 2010). 2.2.1 HOST INFECTIVITY Cryptococcus gattii usually presents as a primary pathogen and C. neoformans as a secondary pathogen. C. gattii infections are more often than not, associated with pulmonary disease and C .neoformans with meningoencephalitis. This may be because, C. gatti’s ability to cross the blood brain barrier (BBB) to infect the brain is less efficient in comparison to C. neoformans’ (Ngamskulrungroj et al., 2012). C. gattii infections occur in individuals who have subtle defects in phagocytic function. Studies indicate that individuals with anti-granulocyte-macrophage colony-stimulating-factor (GM-CSF) 9 University of Ghana http://ugspace.ug.edu.gh autoantibodies are predisposed to C. gattii infections and not to C. neoformans infections. The reason for this selective predisposition is still unclear (Saijo et al., 2014). These autoantibodies make GM-CSF dysfunctional hence Th1-cell responses, innate immunity and phagocytic activity are impaired. C. gattii also induces a more powerful immune response compared to C. neoformans (Rolston, 2013) and this is indicated by the formation of larger cerebral and pulmonary cryptococcomas. Cryptococcomas are also more frequently seen in C .gattii infections (Ngamskulrungroj et al., 2012; Chen et al., 2014). 2.2.2 BIOCHEMICAL CHARACTERIZATION C. gatti and C. neoformans are morphologically indistinguishable with both producing a characteristic brown pigmentation on Bird seed/ Niger seed agar (Kwon-Chung and Bennett, 1992).They are biochemically differentiated by their growth on media incorporated with canavanine and glycine with bromothymol blue (CGB medium) as the colour indicator for growth. This is because, C. gattii utilizes glycine as carbon and nitrogen sources so, a majority of C. gattii isolates are resistant to canavanine while the majority of C. neoformans strains can utilize glycine only as a nitrogen source but not as a carbon source and are susceptible to L- canavanine. Hence C. gattii will grow on CGB agar, producing a blue colour, indicating the assimilation of glycine, while C. neoformans fails to cause a colour change (Kwon-Chung and Bennett, 1992; Ngamskulrungroj et al., 2012). 10 University of Ghana http://ugspace.ug.edu.gh Several studies though have shown that some strains of C. neoformans obtained from both clinical and environmental sources may produce the blue colour change characteristic of C. gattii on CGB agar (Nakamua et al., 1998; Khan ZU et al., 2013), implying that CGB alone may not adequately discriminate between the two species. 2.2.3 MOLECULAR AND SEROLOGICAL CHARACTERIZATION Multiplex PCR and other molecular techniques such as PCR fingerprinting, AFLP among others have been shown to discriminate between these two species using primers specific for both species (Meyer et al., 2009; Cogliati, 2013; Leal et al., 2008). C. gattii and C. neoformans can be characterized further into serotypes using their mucopolysaccharide capsule as antigenic determinants. For C. neoformans the divisions are; Serotypes A and D. These can recombine to produce, diploid or aneuploid inter-varietal AD hybrids. C. gattii species has serotypes, B and C without a variety status (Cogliati, 2013, Xu and Mitchell, 2003). Based on multiple genetic analyses carried out globally using different molecular methods,C.neoformans and C.gatti are divided into nine molecular types. C. neoformans is broadly divided into five molecular types; AFLP1/VNI, AFLP1A/ VNII, AFLP1B/VNB, AFLP3/VNIII and AFLP2/VNIV. AFLP1/VNI and AFLP1A/VNII are globally distributed whilst the third, AFLP1B/VNB is aminly found in Southern Africa (Chen et al., 2015). Also AFLP1/VNI, AFLP1A/VNII and AFLP1B/VNB correspond with C. neoformans var. grubii (serotype A), AFLP2/VNIV corresponds to C. neoformans var. neoformans (serotype D) and 11 University of Ghana http://ugspace.ug.edu.gh AFLP3/VNIII corresponds to AD hybrids (Meyer et al., 2009; Litvintseva et al., 2006). AFLP1/VNI, AFLP1A/VNII and AFLP1B/VNB (Serotype A) has a predilection for HIV/AIDS patients and is the most commonly implicated serotype in meningitis worldwide (Park et al., 2009) C. gattii (serotype B and C) has four molecular types; AFLP4/VGI, AFLP6/VGII, VGIII/AFLP5, and AFLP10/AFLP7/VGIV (Cogialti, 2013; Chen et al., 2015).VGI/AFLP4 and VGIII/AFLP5 are the more virulent molecular types. AFLP4/VGI, AFLP6/VGII and AFLP10/VGIV correspond to serotype B isolates, while genotypes .AFLP5/VGIII and AFLP4/VGIV are serotype C isolates (Meyer et al., 2009). AFLP4/VG1 and AFLP6/VGII cause infection primarily in healthy individuals, while AFLP5/VGIII and AFLP7/VGIV in immunocompromised individuals (Hagen et al., 2010). The species complex also form hybrids between species. C. neoformans var. neoformans AFLP2/VNIV and C. gattii VGI/AFLP4 form serotype BD/AFLP8 (Bovers et al., 2006), C. neoformans var. grubii VNI/AFLP1 and C. gattii AFLP4/VGI form serotype AB/AFLP9 (Bovers et al., 2008b) and C. neoformans var. grubii VNI AFLP1 combines with C. gattii AFLP6/VGII to form AFLP11/ serotype AB (Aminnejad et al., 2012). In 2015, a new taxonomy for C. neoformans complex was proposed. This new taxonomy is based on phylogenetic analysis of several genetic loci of 115 globally collected isolates where a significant genetic diversity was reported. Based on this genetic diversity, they proposed the division of C. gatti into a total of five species (C. bacillisporus, C. deuterogattii, C. gattii, C. tetragattii and C .decagattii) and C. neoformans into two species (C. neoformans and C. deneoformans) (Hagen et al., 2015). This taxonomy is still under discussion (Cogialti et al., 2016). 12 University of Ghana http://ugspace.ug.edu.gh 2.3 ECOLOGICAL NICHES OF CRYPTOCOCCUS Cryptococcus species was first isolated in 1894 by Abraham Buschke and Otto Busse from the bone marrow of a lady (Busse, 1894) and in that same year by Sanfelice from fruit juices (Sanfelice, 1894).Since then Cryptococcus species have been discovered to occupy diverse environment and is therefore rightly referenced as ubiquitous. The discovered niches of the species complex include avian (particularly pigeon droppings and pigeon roosting areas) and arboreal habitats (including bark, tree-base soil, tree hollows and flowers of a variety of tree species), solid materials (soil, sand, rock, dust, roads, tires, shoe soles), liquid environments (fresh water, sea water, brackish water, swamp debris, mud, tree sap) and from both domestic and wild animals (Mitchell et al., 2011; Cogialti, 2013). A global survey of the environmental niches of Cryptococcus species complex indicates that their niches also differ from one geographical location to another (Cogliati, 2013). In the Oceania, environmental C. neoformans isolates were found associated with trees such as Pine needles and Eucalyptus species and C. gattii with Eucalyptus species, Olive seedlings and with plant debris (Boekhout et al., 2001). In Asia, environmental sampling showed C. gattii was mainly isolated from tree samples such as Eucalyptus species, Tamarindus species, Mangifera species amongst others and C. neoformans, birds’ excreta mainly those of pigeons (Chowdary et al., 2011; Gugnani et al., 2005; Gokulshankar et al., 2004). Europe’s environmental C. neoformans isolates were from bat guano, red fox faeces, birds excreta notably those from pigeons, as well as Eucalyptus species and C. gatti was isolated from Eucalyptus species ,Oak tree, Douglas tree, Carob tree, and Stone pine (Frasés et al.,2009; Montagna et al., 2003). 13 University of Ghana http://ugspace.ug.edu.gh Environmental C. neoformans isolated from Central and South America were from pigeon droppings , soil, dust, and contaminated dwellings, Eucalyptus species and C. gattii was isolated from psittaciformes bird excreta, dust, soil and Eucalyptus species and several other tree species (Escandon et al., 2010; Leite et al., 2012). North America isolated environmental C .neoformans mainly from pigeon droppings and C. gatti from soil, air and trees (Litvintseva et al., 2005; Licea et al., 1999). Reports of environmental cryptococcal survey from Africa showed that C. neoformans were isolated from bat and pigeon droppings, other birds’ droppings, soil, house dust and several tree species such as Mopane trees, Eucalyptus species, and Baobab and C. gattii isolates were from Eucalyptus species, Olive trees, almond trees and bat droppings (Dongmo et al., 2016; Mseddi et al., 2011; Litvinseva et al., 2011; Ellabib et al., 2016; Nweze et al., 2015; Nandi et al., 2016) Globally, C. neoformans is associated mainly with pigeon droppings and so pigeon droppings are a primary source of infection especially in urban communities. C. gattii is primarily isolated from a variety of tree species (Cogliati, 2013). Certain conditions favour the presence of these organisms in different environmental niches. These conditions include; dry and desiccated niches, exposure to direct sunlight and varying climatic conditions. Cryptococcal isolates were mostly isolated from dry/desiccated pigeon droppings compared to moist droppings and also from protected areas that favour desiccation compared to unprotected areas (Granados and Castañeda, 2005; Montenegro and Paula, 2000). High temperature inhibits the growth of C. neoformans. Temperatures above 40 ºC does not support the growth of Cryptococcus and there’s reportedly a higher density and frequency of C. neoformans during the rainy season than in the dry season and there’s an inverse relationship 14 University of Ghana http://ugspace.ug.edu.gh between C. gattii and humidity, temperature, evaporation and solar radiation (Mak et al., 2015; Granados and Castañeda, 2006). 2.4 DISTRIUTION OF CRYPTOCOCCUS C. neoformans is distributed worldwide and is the most prevalent species (Cogialti, 2013; Chen 2000), while C. gattii was mainly associated with tropical and subtropical regions, until the outbreak in North West Pacific of America and Vancouver Island, showed that it could be found in temperate regions as well (Cogialti, 2013; Bartlett et al., 2012). A global survey of the distribution of the molecular and species types of the C. neoformans species complex indicated that C. neoformans species is more frequently isolated than C. gatti (Cogialti, 2013).The variability in C. neoformans: C. gattii isolation based on geographical location showed a ratio of 33: 1 for Africa, 68: 1 for Europe, 7.6: 1 for Asia, 3.5: 1 for North America, 4.5: 1 for Central and South America and1: 1.5 for Oceania. The molecular types for each species are also variably encountered. VNI is most prevalent, except in Papua New Guinea and Australia, where VGI is recorded to be most prevalent (Litvintseva et al. 2006; Cogliati, 2013). Briefly, the variability in global molecular type distribution is as follows. In the Oceania, the prevalent molecular types are VGI, VNI and VGII. No VGIV isolates were reported. In Asia, VNI and VGI were the prevalent molecular types. VNI was the main molecular type in Europe and North America and all molecular types except VNB were also isolated from North America. Central and South America recorded VNI, VGI and VG II as the prevalent molecular types. Africa records a molecular type prevalence of VNI.VNB, thought to be limited 15 University of Ghana http://ugspace.ug.edu.gh to Botswana was also isolated from South Africa, Rwanda, and Democratic Republic of Congo. VNIV was not reported (Cogliati, 2013). 2.5 ANTIFUNGAL SUSCEPTIBILITY OF ENVIRONMENTAL CRYPTOCOCCUS The acquisition of Cryptococcus species by inhaling cryptococcal spores or dehydrated yeast cells from environmental samples necessitates their antifungal susceptibility testing (Kwon-Chung et al., 2014; Park et al., 2009; Lin X, 2009; Souza et al., 2008). Cryptococcosis is treated effectively by combination therapy. This combination therapy using Amphotericin B and flucytocine combination and azoles like fluconazole is the gold standard for treating cryptococcal meningitis infection (Govender et al., 2013; Perfect et al., 2010). This however has some limitations especially in settings where resources are limited because Amphotericin B and flucytocine may not always be available in these settings. Also Amphotericin B is administered intravenously and notably toxic (Loyse et al., 2013). Fluconazole is therefore commonly employed in treatment as it is orally administered and has the ability to easily penetrate the central nervous system (CNS) and its side effects are minimal (Brandt et al., 2001). Cryptococcosis that is limited to the respiratory system can be treated orally with 400 mg fluconazole taken daily for 6 to 12 months in both immunocompromised and immunocompetent hosts while for severe pulmonary Cryptococcosis, the dose is increased to 1200 mg of fluconazole, once daily for 14 days, then decreased to 600-800 mg once daily for 8 weeks, and 200 mg fluconazole orally once daily for 6-12weeks is recommended as maintenance dose (Perfect et al., 2010; Chaya and Perfect, 2006). 16 University of Ghana http://ugspace.ug.edu.gh The antifungal susceptibility profiles of environmental cryptococcal isolates to the same antifungal agents vary from one location to another (Soares et al., 2005 ; Nnadi et al., 2016; Gutch et al., 2015; Matos et al., 2012; ). Souza et al., in 2008 did not report any resistance in their environmental cryptococcal isolates to the antifungal agents tested. They tested the isolates against itraconazole, Amphotericin B, voriconazole and fluconazole. In India an analysis of the susceptibility pattern of environmental cryptococcal isolates to fluconazole, ketoconazole, itraconazole showed that some of the isolates were resistant to the three azoles (Gutch et al., 2015). Susceptibility testing of environmental Cryptococcus species to antifungal agents from the West Region of Cameroon showed all isolates tested were resistant to Amphotericin B ketoconazole and fluconazole (Dongmo et al., 2016). There are also reports that there seem to be no difference between antifungal resistance patterns of environmental and clinical cryptococcal isolates indicating that, antifungal susceptibility pattern of Cryptococcus is independent on their environmental or clinical origins ( Moraes et al., 2003; Trilles et al., 2004). C .gattii is reportedly more resistant to antifungal agents compared to C. neoformans in some studies (Trilles et al., 2004; Chowdhary et al. 2011) while some other studies report the reverse (Thompson et al., 2009; Gutch et al., 2015).However C. gatti’s reportedly delayed response to antifungal therapy is corroborated by the formation of larger cryptococcomas in the brain and/ or lungs compared to C. neoformans. These cryptococcomas are not easy to treat and usually require an extended antifungal therapy (Perfect et al., 2010; Chen et al., 2000). 17 University of Ghana http://ugspace.ug.edu.gh In both species the age of the cryptococcal cell has an influence on their antifungal susceptibility pattern with older cells demonstrating an increased resistance to antifungal drugs compared to younger cells (Bouklas and Fries, 2015). 2.6 MODE OF ACTION AND RESISTANCE PATTERN OF ANTIFUNGAL AGENTS The systemic antifungal compounds currently in clinical use are; the polyene antibiotics, the azole derivatives, the allylamines/thiocarbamates, and the fluoropyrimidines. 2.6.1 POLYENES The polyene antibiotics produced by Streptomyces species are fungicidal and have the broadest activity spectrum of all the clinically useful antifungal. They are able to complex with ergosterol in the fungal plasma membrane, thereby compromising its barrier function. They are also known to cause oxidative damage which may contribute to their fungicidal action (Georgopapadakou and Walsh, 1994). Amphotericin B which is the only systemic polyene in clinical use, has a higher affinity for ergosterol than the cholesterol, and is thus less toxic to mammalian cells. Its side effects notably nephrotoxicity, are significantly reduced when used in lipid formulations, such as liposomes, lipid complexes, and colloidal dispersions (HartseJ and Bolard, 1994). Fungal resistance to polyenes is associated with altered membrane lipids, particularly sterols. Other resistance mechanisms may involve altered phospholipids and increased catalase activity with decreased susceptibility to oxidative damage (Kelly et al., 1997). 18 University of Ghana http://ugspace.ug.edu.gh 2.6.2 AZOLES The azole antifungals (fluconazole, voriconazole e.t.c.) are totally synthetic and have fungistatic, broad-spectrum activity. Azoles act on ergosterol biosynthesis at the C-14 demethylation stage. Ergosterol is depleted and lanosterol and other 14-methylated sterols are accumulated and they interfer with the functions of ergosterol as a membrane component. There’s damage to the plasma membrane, and this alters the activity of several membrane-bound enzymes, such as those associated with nutrient transport and chitin synthesis (Vanden Bossche and Marichal, 1994). Resistance is due to decreased membrane permeability resulting from changes in membrane sterols and active efflux (Kelly et al., 1995). 2.6.3 ALLYLAMINES AND THIOCARBAMATES These are also totally synthetic. The only systemic allylamine antifungalin clinical use is terbinafine. Resistance has not been reported for human pathogens (Orth et al., 1990). 2.6.4 5-FLUOROCYTOSINE The fluoropyrimidine, 5-fluorocytosine (5-FC) though fungicidal, has a limited activity spectrum. It is mainly used in combination with amphotericin B in cryptococcal meningitis and in cases of disseminated candidiasis. 5-FC is taken up into fungal cells by a cytosine permease, deaminated to 5-fluorouracil (5-FU), converted to the nucleoside triphosphate, and incorporated into RNA where it causes miscoding. In addition, 5-FU is converted to deoxynucleoside, which inhibits thymidylate svnthase and thereby DNA biosynthesis. 19 University of Ghana http://ugspace.ug.edu.gh Primary resistance to 5-FC is usually due to impaired cytosine deaminase (Francis and Walsh, 1992; Vanden Bossche and Marichal, 1994). 20 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE MATERIALS AND METHODS 3.0 STUDY SITE, DESIGN AND SAMPLE The study sites were locations within the Greater Accra region where pigeons and bats were found. The region has a population estimate of over 4million people with more than 3.5million of this population dwelling in urban settlements (Ghana Statistical Service, 2012). The region is relatively dry and exhibits two rainy seasons, with the first beginning in March and peaks notably in May/June and the second in September/October. Sampling was done from November 2016 to April 2017, a relatively dry time in the region. 643 droppings from pigeons and 55 from bats were collected. The sources of pigeon droppings included markets (Agblogbloshie market, Kaneshie main market, Abossey Okai spare parts market, Kantamanto market, Nima market, Ashaiman main market and Malata market) residential quarters (Ashaiman- Lebanon, Agege-last stop, Korle-Bu Tropics, Teshie Greda estates, Mamprobi, Dansoman), a church (Agege) and bat droppings were collected from a park (37 Military hospital bus parks). Three residential quarters (Budumbura, Kasoa and Ofaakor) were located in the Central region. A total of 18 locations from 4 sources were therefore the sampling locations (Figure 3.1). The study was a cross sectional study. . 21 University of Ghana http://ugspace.ug.edu.gh Courtesy: Google map Figure 3.1: Map showing sampling sites 22 University of Ghana http://ugspace.ug.edu.gh 3.1 ENVIRONMENTAL SAMPLE COLLECTION Pigeon and bat droppings were collected into sterile plastic bags using spatula and brushes and then sealed. They were then transported to the Laboratory of the Medical Microbiology Department, SBAHS for processing and microbiological analyses. The natures of the specimens were noted, to be either moist or dry. The numbers of droppings collected from the different sources and locations were also recorded. Droppings were either immediately processed or kept at room temperature for not more than two days before processing. 3.2 ISOLATION AND IDENTIFICATION OF ENVIRONMENTAL CRYPTOCOCCUS Approximately 1 g of sample was suspended in 20 ml of sterile distilled water and vortexed for two minutes. The mixture was allowed to settle for thirty minutes, after which 100μl of the supernatant was inoculated onto Sabouraud dextrose agar (SDA) plates supplemented with chloramphenicol at a concentration of 0.05 g/L. The inoculated plates were then incubated for 7 days at 37oC with daily observation from day 2 for creamy colonies suggestive of Cryptococcus. These colonies were streaked onto fresh SDA plates to isolate pure colonies. They were then identified using standard microbiological methods which included; wet preparation, Gram stain, Urea test, Thermo-tolerance test and Germ tube test. Wet preparations were done by emulsifying pure creamy yeast colonies on clean grease-free glass slides, Cover slips were placed on them and viewed under x40 objective lens. Smears of heat fixed creamy yeast colonies were subjected to Gram stain technique. Slide was flooded for one minute with Crystal violet, after which it was washed under running tap water and then flooded with iodine for one minute, followed by rinsing under running tap water. A decolourizer, 95% ethanol was subsequently added for a few seconds, rinsed and finally, a counter 23 University of Ghana http://ugspace.ug.edu.gh stain, neutral red was added for two minutes and also rinsed. Slides were air-dried and examined under the microscope using the oil immersion objective lens. Pure creamy yeast colonies were also streaked onto the surfaces of urea agar slants and agar incubated for 2-5days at 250C. Thermo-tolerance tests were carried out by inoculating two sets of plates of Sabouraud dextrose agar (SDA) with pure colonies of the cryptococcal isolates. One plate was incubated at 37oC and the other at 25oC.The plates were examined every day for up to 7days. For germ tube test, the colonies were emulsified in tubes containing 0.5ml serum and incubated at 37o C for 2-4hours. Using a Pasteur pipette, drops of the suspension were placed on a clean grease- free slide, cover slips were placed on them and thereafter examined microscopically using the x40 objective lens. All microbiologically identified environmental cryptococcal isolates were subsequently coded with the type of sample and the sampling location and then inoculated on Sabouraud dextrose agar (SDA) slants and stored at -4oC, prior to further analysis. 3.3 ANTIFUNGAL SUSCEPTIBILITY TESTING OF ENVIRONMENTAL CRYPTOCOCCUS Antifungal susceptibility testing was done for all 49 microbiologically identified cryptococcal isolates. A known clinical Cryptococcus species isolate was used as control. Isolates were tested against voriconazole and fluconazole. Susceptibility testing to two antifungal agents (voriconazole and fluconazole) was performed on all cryptococcal isolates referencing Clinical Laboratory Standards Institute (CLSI), 2015 guidelines for yeast. Briefly, distinct colonies of the isolates from 24 University of Ghana http://ugspace.ug.edu.gh a 24-hour old culture were suspended in 5ml of sterile saline, and turbidity was adjusted to produce a 0.5McFarland standard. Sterile cotton swabs were dipped into the inoculums and were evenly streaked over the entire surface of plates containing Mueller-Hinton Agar supplemented with 2% Glucose and 0.5μg/ml Methylene Blue Dye (GMB) Medium. Disks were then dispensed onto the surface of the inoculated agar plates and incubated at 37oC. Plates were examined after 20-48hours of incubation and zone sizes were interpreted and recorded. 3.4 MOLECULAR ANALYSES DNA was extracted from the microbiologically identified environmental cryptococcal isolates and was subsequently amplified by PCR. DNA extraction was done using the Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit following the manufacturer’s instructions. Cryptococcus grown on YPD (Yeast potato dextrose) broth were centrifuged for 5 minutes at 15000 x g. 1000μl phosphate buffered saline (PBS) was then added to the sediment and centrifuged for 5minutes. 750μl lysis solution was added to the sample, vortexed for 30 seconds, aspirated and transferred to a bead beater fitted with a 2ml tube holder assembly and centrifuged for one minute at 10,000 x g. Supernatant was aspirated, centrifuged at 7,000 x g for 1minute and 1,200μl of Genomic lysis buffer and 200μlof DNA Pre- wash buffer were added and centrifuged at 10,000 x g for 1minute. 500μlof g-DNA Wash Buffer was also added and centrifuged at 10,000 x g for 1minutes. DNA elution buffer was added, centrifuged at 10,000x g for 30seconds to elute the DNA. DNA was then stored at -20oC for subsequent use. 25 University of Ghana http://ugspace.ug.edu.gh Two sets of primers were used to perform PCR. The first sets of primers were yeast genus specific primers. The primers were ITS1 (5’-TCC GTA GGT GAA CCT GCGG-3’) and ITS4 (5’-TCC TCC GCT TAT TGA TATGC-3’) (Mitchell et al., 1994). The second sets of primers were C. gattii and C. neoformans species specific primers and this was done by Multiplex PCR. The primers were CNa-70S(5’- ATTGCGTCCACCAAGGAGCTC -3’) and CNa-70A (5’ATTGCGTCCATGTTACGTGGC -3’) primer pairs which amplifies a specific DNA fragment from C. neoformans and primer pairs CNb-49S (5’- ATTGCGTCCAAGGTGTTGTTG-3’)and CNb-49A (5’- ATT GCG TCC ATC CAA CCG TTA TC-3’) which amplifies a specific DNA fragment from C. gattii (Aoki et al., 1999). PCR were performed using PCR Master Mix, 2x (Promega Corporations, Madison, USA) in a volume of 25μl containing; 1-5μl of DNA, 50units/ml of Taq DNA polymerase supplied in a proprietary reaction buffer (50mM Tris-HCl PH 9.0; 50mM Nacl; 200μM each of deoxynucleotide triphosphates and 5mM MgCl2)and 0.6μl of each primer. The thermo cycling conditions included an initial denaturation at 94oC for 5 minutes and 35 cycles at 94oC for 1 minute, annealing at 50oC for 1 minute, extension at 72oC for 2 minutes, and a final extension at 72oC for 10 minutes. The PCR amplicons were electrophoresed on 2% agarose gels in 1X Tris-acetate-EDTA (TAE) buffer containing 10μl ethidium bromide at 120V for 50 minutes. Cryptococcus species isolate from a clinical sample was used as control. All Cryptococcus were characterized into species using the species specific primers CN49 and CN70 primer and recorded. Those that the species specific primers did not characterize were recorded as Unidentified. 26 University of Ghana http://ugspace.ug.edu.gh 3.5 DATA ANALYSIS Sampling and Laboratory data were recorded manually and then transferred to an Excel file template. Analysis of the data collected was done using SPSS VER. 22. Descriptive statistics such as percentages and tables were used to determine isolation prevalence and distribution of species from sampling sites and the antifungal susceptibility pattern of species type 27 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR RESULTS 4.1 ISOLATION AND IDENTIFICATION OF ENVIRONMENTAL CRYPTOCOCCUS SPECIES Forty-nine of the 698 droppings were positive for environmental Cryptococcus species. This gave a prevalence of 7%. 234 droppings were also positive for Candida species. Germ tube tests to presumptively identify pathogenic Candida albicans were negative. All environmental Cryptococcus species were positive for urea test, Gram stain and thermo- tolerance test and cells were round/oval cells when viewed on wet preparation slide (Appendix III). 4.2 SAMPLE SOURCE DISTRIBUTION OF CRYPTOCOCCUS The distribution of environmental Cryptococcus recovered from the location sampled ranged between 0.0 and 26.3%. Cryptococcus was obtained from 12(4.9%) of 243 samples from markets, 16(5%) of 320 samples from residential quarters, while 20(26.3%) of 80 samples from church. Cryptococcus was not isolated from the public park, where bat droppings were collected 4.3 LOCATION DISTRIBUTION OF CRYPTOCOCCUS Cryptococcus was recovered from 6 of the 18 locations sampled. Five of the positive locations were in Accra (Ashaiman, Ashaiman-Lebanon, Agege-Last stop and Makola) and one from the central region (Budumbura). (Table 4.2), shows the distribution of Cryptococcus from the various 28 University of Ghana http://ugspace.ug.edu.gh locations sampled. Cryptococcus was mostly (30%) isolated from Ashaiman-Lebanon All environmental Cryptococcus, irrespective of location or source were from dry pigeon droppings. 29 University of Ghana http://ugspace.ug.edu.gh TABLE 4.1: DISTRIBUTION OF CRYPTOCOCCUS BY SAMPLE LOCATIONS Location No. of samples Nature Positives (%) positive Dansoman 15 Dry 0 0 Agege 80 Dry 21 26.3 Makola 40 Dry 9 22.5 Kasoa 40 Moist 0 0 Kaneshie -spare parts 10 Dry 0 0 Mamprobi plaza 15 Moist 0 0 Korle-Bu Tropics 40 Dry 0 0 Agege-Last stop 45 Dry 4 8.89 Budumbura 50 Dry 5 10 Ashaiman 65 Dry 4 6.2 Agblogbloshie 27 Dry 0 0 Ofaakor 80 Dry 0 0 Kaneshie 43 Dry 0 0 Aishaman-Lebanon 20 Dry 6 30 Nima 23 Dry 0 0 Malata 35 Dry 0 0 Teshie Greda estate 15 Dry 0 0 37-Public park 55 Moist 0 0 Total 698 49 100 30 University of Ghana http://ugspace.ug.edu.gh 4.4 CRYPTOCOCCUS GENUS-SPECIFIC AND SPECIES-SPECIFIC PCR ITS1 and ITS4, the yeast genus specific primers were expected to amplify Cryptococcus DNA fragments between 600 and 650-bp. According to the size of the fragments all 49 microbiologically identified Cryptococcus species were molecularly confirmed as such. Figure 4.1 shows DNA bands from gel electrophoresis of the PCR products. Multiplex PCR with species-specific primers pairs, CNa-70S and CNa-70A for C. neoformans and CNb-49S and CNb-49A for C. gatti, amplified DNA fragments of 695-bp for C. neoformans and 448-bp for C. gattii as expected (Figure 4.2). According to the size of the fragments, all environmental Cryptococcus were characterized. One (2%) isolate was characterized as C. gattii, 4(8%) were C. neoformans, and 20(40.8%) were a mixture of C. gatti and C. neoformans (C. neoforman/ C. gatti) 24 (49%) did not show any of the expected band sizes and were referred to as Unidentified (Table 4.2) 31 University of Ghana http://ugspace.ug.edu.gh Figure 4.1: Gel Image showing DNA bands from environmental Cryptococcus species isolates amplified withITS1 AND ITS4 primers. Marker (100bp DNA ladder, New England Biolab); Cryptococcus species are lanes 1-4 and 7-8. Lane 5. Control; Lane 6.Negative control for the amplification. Figure 4.2: Gel image showing DNA bands from environmental Cryptococcus species identified by Multiplex PCR amplified with primer pairs CNa70A and CNa70S and primer pairs CNb49A and CNb49S. Marker (100bp DNA ladder, New England Biolab); Lane 1. Control; Lanes 2-4 and 7 C. neoformans; Lane 5.Unidentified; Lane 6: C. gattii, Lane 8. C. neoformans/ C. gattii; Lane 9.Negative control for the amplification. 32 University of Ghana http://ugspace.ug.edu.gh 4.4.1 DISTRIBUTION OF ENVIRONMENTAL CRYPTOCOCCUS BY LOCATIONS Table 4.5 shows the distribution of environmental Cryptococcus species isolated from the different locations. All isolates from Ashaiman and Ashaiman-Lebanon were Unidentified species even though the sources were different. The other locations had both Unidentified and known species. The only C. gatti isolate was from Agege and the four C. neoformans were distributed between Makola (n=2), Agege-last stop (n=1) and Agege (n=1) (Table 4.3). 33 University of Ghana http://ugspace.ug.edu.gh Table: 4.2: DISTRIBUTION OF CRYPTOCOCCUS SPECIES BY SAMPLE LOCATIONS Code Location Source Multiplex PCR 1CR Ashaiman-Lebanon Residence Unidentified 2CR Ashaiman-Lebanon Residence Unidentified 3CR Ashaiman-Lebanon Residence Unidentified 4CR Ashaiman-Lebanon Residence Unidentified 5CR Ashaiman-Lebanon Residence Unidentified 6CR Ashaiman-Lebanon Residence Unidentified 7CR Ashaiman Market Unidentified 8CR Ashaiman Market Unidentified 9CR Ashaiman Market Unidentified 10CR Agege –Last stop Residence C. neoformans/ C. gattii 11CR Agege –Last stop Residence C. neoformans 12CR Agege –Last stop Residence C. neoformans/ C. gattii 13CR Agege –Last stop Residence C. neoformans/ C. gattii 14CR Agege –Last stop Residence C. neoformans/ C. gattii 15CR Makola Market C. neoformans 16CR Makola Market Unidentified 17CR Makola Market Unidentified 18CR Makola Market C. neoformans/ C. gattii 19CR Makola Market Unidentified 20CR Makola Market C. neoformans/ C. gattii 21CR Makola Market Unidentified 22CR Makola Market C. neoformans/ C. gattii 23CR Makola Market C. neoformans 24CR Budumbura Residence C. neoformans/ C. gattii 25CR Budumbura Residence Unidentified 26CR Budumbura Residence C. neoformans/ C. gattii 27CR Budumbura Residence C. neoformans/ C. gattii 28CR Budumbura Residence Unidentified 29CR Agege Church C. neoformans/ C. gattii 30CR Agege Church C. gattii 31CR Agege Church Unidentified 32CR Agege Church C. neoformans/ C .gattii 33CR Agege Church C. neoformans/ C. gattii 34CR Agege Church C. neoformans/ C. gattii 34 University of Ghana http://ugspace.ug.edu.gh 35CR Agege Church C. neoformans/ C. gattii 36CR Agege Church Unidentified 37CR Agege Church Unidentified 38CR Agege Church C. neoformans/ C. gattii 39CR Agege Church Unidentified 40CR Agege Church Unidentified 41CR Agege Church Unidentified 42CR Agege Church C. neoformans/ C. gattii 43CR Agege Church C. neoformans/ C. gattii 44CR Agege Church C. neoformans 45CR Agege Church Unidentified 46CR Agege Church Unidentified 47CR Agege Church C. neoformans/ C. gattii 48CR Agege Church Unidentified 49CR Agege Church C. neoformans/ C. gattii 35 University of Ghana http://ugspace.ug.edu.gh 4.5: ANTIFUNGAL SUSCEPTIBILITY PROFILES OF ISOLATED ENVIRONMENTAL CRYPTOCOCCUS SPECIES The antifungal susceptibility profiles of isolates are shown in Table 4.4. The zones of inhibition for the control isolate were within the recommended ranges. All C. neoformans (n=4) and C. gatti (n=1) were resistant to fluconazole and C. neoformans/C. gatti (n=24) were all but one, resistant to fluconazole. A total of 36 (73.5%) of all isolated environmental Cryptococcus species were resistant to fluconazole. 38 (77.6%) of the isolates were susceptible to voriconazole. The highest percentage resistance recorded for voriconazole was for the Unidentified isolates (20.8%). 36 University of Ghana http://ugspace.ug.edu.gh TABLE: 4.3 ANTIFUNGAL SUSCEPTIBILITY PROFILES OF ENVIRONMENTAL CRYPTOCOCCUS Antifungal agent Species Susceptibility profiles (%) S S-DD R Fluconazole(25μg) C. neoformans(n=4) 0.0 0.0 100 C. gatti(n=1) 0.0 0.0 100 C. neoformans/C. gatti(n=20) 25.0 30.0 45.0 Unidentified(n=24) 4.2 0.0 95.8 Voriconazole(1μg) C. neoformans(n=4) 50.0 25.0 25.0 C. gatti(n=1) 100 0.0 0.0 C. neoformans/C. gatti(n=20) 100 0.0 0.0 Unidentified(n=24) 66.7 12.5 20.8 S= Susceptible S-DD=Susceptible dose dependent R= Resistant 37 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE DISCUSSION 5.1 ISOLATION OF ENVIRONMENTAL CRYPTOCOCCUS SPECIES There is currently no literature on the isolation of environmental Cryptococcus in Ghana. This study is therefore the first to provide, information on the availability as well as antifungal susceptibility testing of environmental Cryptococcus in Ghana. The reported environmental Cryptococcus prevalence of 7% was from pigeon droppings and none were recovered from bat droppings also analyzed in this study. It is noteworthy that the pigeon themselves do not serve as hosts to the organism, as their high body temperatures (about 42o) does not support the growth of these yeasts (Littman et al., 1968).They are reservoirs through which the organism can be disseminated. Isolation prevalence differs globally, but Cryptococcus is largely associated with pigeon droppings (Cogialti, 2013). Environmental Cryptococcus prevalence from some other African countries were between 3% and 28.5% (Nnadi et al., 2016; Mahmoud et al., 1999; Nweze et al, 2015; Mseddi et al., 2011; Dongmo et al., 2016). Reasons for this varying isolation rates are not exactly clear, but could be due to local environmental conditions at the different locations. In any case, studies which seek to isolate Cryptococcus from environmental samples usually record more negatives than positives (Mitchell et al, 2011). Worldwide, C. neoformans is predominantly isolated from pigeon droppings and C. gattii from a variety of tree species (Cogialti, 2013). From the current study, both C. neoformans and C. gattii were isolated from pigeon droppings. This is quite interesting as many studies around the world 38 University of Ghana http://ugspace.ug.edu.gh that have isolated Cryptococcus from pigeons droppings only associated the droppings with C. neoformans, but not C. gattii (Cogialti 2013; Mseddi et al., 2010; Cogialti et al., 2016). Pigeon droppings are reportedly not a favourable niche for C. gatti’s long term survival and its sexual reproduction is ineffective in this niche (Nielsen et al., 2007). However, C. gattii can thrive in droppings of some other birds. (Abegg et al., 2006). The isolation of C. neoformans and C. gattii from same niche as reported in this study is also an interesting finding although not novel , as both species have been isolated from other birds faeces and Eucalyptus species (Abegg et al., 2006) and more importantly in comparison to this study, from pigeon droppings in Cameroon (Dongmo et al., 2016). This could mean that C. gattii is evolving; therefore adapting to a new habitat or that the environmental conditions in these countries favour its survival. It could also mean that in these countries, the ecological niches for both species are the same. This current study’s isolation of C. neoformans and C. gattii (C. gattii/ C. neoformans) from same DNA sample could imply the formation of inter-species hybrid. Hybrid strains have been described in some studies with C. neoformans (serotype D) combining with C. gattii (serotype B) to form serotype BD (Bovers et al., 2006), C. neoformans var. grubii (serotype A) and C. gattii ( serotype B) forming serotype AB/AFLP9 (Bovers et al., 2008b ;Aminnejad et al., 2012). These hybrids have two genome copies in one gene which may give them an opportunity to possibly evolve new functions. These hybrids could also combine the characteristics of both species and this may have graver implications for human health. For instance, the BD hybrid above may combine the cosmopolitan distribution of serotype D with serotype B’s ability to infect mainly 39 University of Ghana http://ugspace.ug.edu.gh immunocompetent individuals (Bovers et al., 2006).Further characterization technique such as sequencing is needed to clarify if indeed the current study isolated hybrid strains of environmental Cryptococcus. The sizeable number (24/49) of Unidentified species observed in the current study was unexpected. But this could mean they were neither C. neoformans nor C. gattii and this does not exclude them from being potential pathogens (Takemura et al., 2015). 5.1.1 NATURE OF POSITIVE ENVIRONMENTAL SAMPLES AND SAMPLING SITES All cryptococcal isolates from this study were from dry pigeon droppings, none was recovered from moist droppings, indicating that the viability of the organism is intact even in dry excrement (Cassadel and Perfect, 1998; Nweze et al., 2015; Mseddi et al, 2010). The presence of fewer bacteria in the dry pigeon droppings to compete with the yeast may have also accounted for this (Ruiz et al., 1981). An exposure to direct sunlight will decontaminate sites that would otherwise support the growth of Cryptococcus. Hence frequency of isolation is increased in areas protected from direct sunlight and that favour desiccation (Granados and Castaneda, 2005; Montenegro and Paula, 2000). Also, accumulation of faeces and poor aeration can increase the frequency of isolation of Cryptococcus (Emmons et al., 1955).The current study agrees with above accounts. It was observed that all cryptococcal isolates were from dry pigeon droppings and indeed all pigeon droppings were collected from dovecotes and house- tops protected from direct sunlight and because of lack of 40 University of Ghana http://ugspace.ug.edu.gh aeration, these sites favour desiccation. Specifically, isolates from residential quarters were from dry pigeon droppings collected from house-tops that were protected from direct sunlight, with accumulated faeces and poorly aerated. Isolates from markets and church were also from dry pigeon droppings in dovecotes protected from direct sunlight and large accumulations of faeces were observed. The highest numbers of isolates were recovered from Agege (21/49). This is possibly due to the larger accumulation of dry pigeon droppings in the dovecote, where the droppings were collected compared to the other sites. The presence of only Unidentified isolates from Ashaiman and Ashaiman-Lebanon likely indicates that these isolates are related due to the proximity of the two locations. The extent of their relatedness can only be verified with further characterization. All positive samples were collected from dovecotes and house-tops. We can imply that dovecotes and rooftops with accumulated dry pigeon droppings are important in human infection. This study did not recover any Cryptococcus species from bat droppings as reported by Dongmo et al. in 2016, where they isolated Cryptococcus from both pigeons and bat droppings. This does not suggest that bat droppings in Ghana do not harbor the yeast, but probably due to the small sampled size, since a large number of environmental samples are usually needed to get positive results (Mitchell et al., 2011). 41 University of Ghana http://ugspace.ug.edu.gh Varying climatic conditions such as temperature and rainfall may influence the isolation rate of Cryptococcus. Samples used in this study, were collected during a relatively dry time in the sampling locations (December, 2016 t0 April, 2017). The influence, this may have had on the prevalence of environmental Cryptococcus reported in this study is uncertain, but there is thought to be a higher isolation rate and density of C. neoformans and vice versa for C. gattii during the rainy season than in the dry season (Mak et al., 2015; Granados and Castañeda, 2006). 5.2 HEALTH IMPLICATIONS OF ENVIRONMENTAL CRYPTOCOCCUS The account of both C. neoformans and C. gattii species in pigeon droppings indicates these droppings are a relevant reservoir for the acquisition of cryptococcal infections. This is an indication that both immunocompetent and immunocompromised individuals are at risk, since it has been established that C. neoformans is mainly a secondary pathogen and C. gattii a primary pathogen. Cryptococcus is airborne and can be widely disseminated in the environment with a large population exposure. In over 80% cases of pulmonary cryptococcosis, a history of exposure to pigeon droppings is reported (Gunda et al., 2015). An investigation of the outbreak of cryptococcosis among healthy individuals in Vancouver (1999) indicated that the infection was caused by an environmental strain of C. gattii. Environmental molecular types were found to be the same as clinical molecular types (Kidd et al., 2004) The high resistance pattern of the environmental cryptococcal isolates to fluconazole recorded in this study could be worrisome. This is because fluconazole is commonly employed in treatment of 42 University of Ghana http://ugspace.ug.edu.gh cryptococcosis and this resistance pattern in environmental isolates could translate to treatment failure of human cryptococcal infections (Brandt et al., 2001). 5.3 LIMITATIONS OF THE STUDY 1. CLSI breakpoints used were not specific for Cryptococcus species. 2. Minimum inhibitory concentrations (MIC) for Cryptococcus would have given more accurate resistance patterns, but were not done. 43 University of Ghana http://ugspace.ug.edu.gh CHAPTER SIX CONCLUSIONS AND RECOMMENDATIONS 6.1 CONCLUSIONS This study adds to the information on the ecology of Cryptococcus neoformans and C. gatti in Africa. Cryptococcus neoformans and C. gatti were discovered to be present in pigeon droppings in the Greater Accra Region. Pigeon droppings are therefore possible reservoirs for sources of cryptococcal infections in both healthy and immunocompromised individuals in the Region. Isolates were mainly resistant to fluconazole, suggestive that the drug may not be effective in treating cryptococcal infections. 6.2 RECOMMENDATIONS Public health authorities should strategize and educate the general population especially pigeon handlers on the risks of cryptococcal infections. 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Comparative gene genealogical analyses of strains of serotype AD identify recombination in populations of serotypes A and D in the human pathogenic yeast Cryptococcus neoformans. Microbiology, 149(8), 2147-2154. 64 University of Ghana http://ugspace.ug.edu.gh APPENDICES APPENDIX I PREPARATION OF AGAR MEDIA AND STERILIZATION OF CONSUMABLES a) Sabouraud dextrose agar (per litre) i. Composition 65 g Sabouraud dextrose agar powder 10.0 g Mycological peptone 40.0 g Glucose 15.0 g Agar PH 5.6+0.2 at 25˚C ii. Preparation According to the manufacturers’ protocol, 500 ml of Sabouraud’s dextrose agar (SDA) (OXOID LTD, Lot no. 1419386) was prepared by dissolving 32.5 g of SDA into 500 ml of distilled water. The solution was brought to boil to dissolve powder completely. Solution was then sterilized by autoclaving at 121˚C for 15 minutes. About 25 ml of molten agar was dispensed into each one of disposable Petri dishes. The molten agars in the petri dishes were allowed to solidify at room temperature. The plates were packaged and stored at 2˚C to 8˚C in a fridge. For every batch of SDA plates prepared, one plate was incubated at 37˚C and another plate was streaked with a known Cryptococcus spp. for sterility and quality control respectively. 65 University of Ghana http://ugspace.ug.edu.gh b) Sterilization of consumables Eppendolf tubes were fully opened and placed in a clean conical flask and the mouth of it sealed with a foil. They were then autoclaved at 121˚C for 15 minutes. They were then transferred into the hot air oven to dry. Micropipette tips were arranged in their racks and were autoclaved at 121˚C for 15 minutes. They were then transferred into the hot air oven to dry. 66 University of Ghana http://ugspace.ug.edu.gh APPENDIX II: RESULTS FROM PHENOTYPIC ANALYSES Wet preparation Gram stain Round/oval cells Urea test 67 University of Ghana http://ugspace.ug.edu.gh 68