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FUNGAL DISEASES: A SERIOUS THREAT TO HUMAN EXISTENCE IN

RECENT TIMES BY

PROF. JOSEPHINE IFEYINWA OKAFOR 1.1 INTRODUCTION It is with joy and in thanksgiving to God and also to the University Administration for giving me this opportunity to deliver my inaugural lecture today to this distinguished audience. My area of specialization is Medical Microbiology with special interest in Medical Mycology which is the study of fungi that cause diseases in man. For about thirty years, I have carried out extensive research on fungi and also taught my students all that I learnt and discovered about fungi which are responsible for many diseases of man. During all these years, I have also learnt that fungi have shifted their position from being agents of non-significant human diseases to agents of serious and life-threatening human diseases. Unfortunately, many people including those in medical profession, particularly in the developing countries like Nigeria, are yet to be convinced that fungi are major causes of morbidity and mortality in the world today. I have, therefore, decided to give this public lecture in order to inform people of Nigeria in particular that fungi are indeed responsible for many fatal diseases of man.

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1.1.1 What are fungi? They are a group of non-photosynthetic micro-organisms which live as saprophytes in the soil and on dead organic matter or as parasites of plants and animals including man. Fungi are found everywhere: in the soil, in decaying vegetation, in the air and in the waters. They are seen to grow on foods (bread, cooked food), damp clothes, wet walls, house roofs, damp leather materials (shoes, bags, boxes) and even in harsh environments like hot desert soil, hot spring waters, snow fields, hot compost pits and bottom of deep waters (oceans, seas, great lakes) with very low temperatures and high pressures and also in the refrigerators. They also form part of the microbial flora of both outside and inside of living organisms including man, animals and plants. Most fungi produce spores in large numbers which are useful for their survival and distribution. The spores are usually light and so can remain floating in the air for long periods and often blown for a distance of about 500km by strong winds. From the air, they are forced into houses through suction mechanism or into the exposed human tissues (cuts in the skin) and into the nose during breathing. Most fungi are microscopic multi-cellular filaments, consisting of septate or non-septate hyphae while a few are unicellular in form (yeasts). A few are macroscopic in size (mushrooms).

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1.1.2 Nutrition in fungi Every living organism requires energy for its metabolic activities (growth, movement, reproduction). Fungi, being non-photosynthetic organisms, obtain their energy by feeding saprophytically on dead organic matters which contain high energy compounds (carbohydrates) or parasitically on living organisms which contain stored energy compounds (sugars, proteins, fats) in their tissues. Their parasitic activities cause a lot of damage to their hosts and these actions often give rise to different types of diseases of plants and animals including man. 1.1.3 The beneficial roles of fungi Fungi, as saprophytes, are very useful to man in many ways: i) They are important in the decomposition of huge

organic matters that are regularly formed within the environment and which after decomposition, serve as food for the living plants.

ii) They are important in the recycling of inorganic substances (potassium, sodium, calcium etc.) which are trapped in the dead organic matter and then make them available to living plants.

iii) They are important in the production of fermented foods, wines, alcohols, and acids in homes and industries.

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iv) Some of them produce antibiotics (penicillin) as part of the products of their metabolic activities, which are useful in the treatment of some diseases of man.

1.1.4 The defence mechanisms against fungal invasion of the human tissue The human body is constantly putting up strong resistance against any organism including fungi that tries to gain entry into the tissue to feed and live in it. This body defence is a combined action of many components of the body known as “immune system.” The first line of defence consists of the protection by the skin and mucous membrane. The skin which covers the outer body of man and the mucous membrane which covers the mouth, nose and exposed part of the female genital organ, as long as they are intact, provide protection against most pathogenic fungi that constantly attempt to enter the human tissue. There are also as part of this defence, a group of micro-organisms that include fungi, bacteria and protozoa which are known as “microflora”, “microbiota” or “commensal organisms,” which colonize the skin and mucous membrane. They feed on substances that are excreted from the body without causing any harm to the host and remain there throughout the life of the host, preventing the establishment of other organisms including the pathogenic ones. The second line of defence consists of two major components of the immune system, namely B-lymphocytes

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and the T-lymphocytes which play key roles in destroying these fungi. Any cut on the skin and membrane (knife cut, surgery, gun shot, nail or thorn prick), will allow fungi (and other micro-organisms) to enter the tissue. However, the normal immune system, particularly in healthy individuals, will prevent the establishment of these fungi. The B-lymphocytes produce different types of soluble substance called “antibodies” (IgA, IgG, IgM, IgE, IgD) which are capable of destroying many micro-organisms including fungi. The T-lymphocytes, also known as T-cells, are of different types: T helper (CD4), T cytotoxic, T suppressor, and T delayed cells; and they have various functions in the elimination of the invading organisms. T helper cells are the most important of the T cells for they have multiple functions including helping in the maturity of B-lymphocytes and stimulating T cytotoxic cells to destroy micro-organisms and tumour cells. There are also a group of other immune cells called “phagocytes” (white blood cells, macrophages) or “cell eaters” which singly or in combination with some T cells destroy large numbers of organisms in the tissue in a process called “phagocytosis.” 1.1.5 Establishment of fungi in the human tissue Despite the immune defence that operates within the human tissue, some pathogenic fungi are still able to enter the tissue, establish there, and cause various diseases in man due to the following:

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i) They are capable of producing various substances which include enzymes and toxins that they use in destroying human tissues.

ii) Some of them possess protective coats (capsules) which prevent their being engulfed and destroyed by phagocytes.

iii) Regular and prolonged use of some drugs, including antibiotics often reduce the effect of the immune system and so allow the establishment of some pathogenic fungi.

iv) Some micro-organisms (viruses) are able to attack some of the immune cells and so prevent them from playing their role in tissue defence.

1.1.6 Classification of fungal diseases (Mycoses) Fungal diseases, also called “mycoses,” are classified according to the degree of tissue involvement and mode of entry into the tissue. These are: � Superficial or cutaneous mycoses � Subcutaneous mycoses � Deep or systemic mycoses � Opportunistic mycoses 1.2 SUPERFICIAL MYCOSES These are fungal diseases which are limited to the outermost part of the body (skin, mucous membrane, nails and hair). Some important superficial mycoses are: i) Dermatophytosis: A group of fungi collectively called

dermatophytes are known to attack the outer layer of

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the skin including the hair and nails to cause superficial lesions on the skin known as ringworm or tinea. Ringworm disease was initially thought to be caused by worms and that was how the disease got the name. It was only after some researchers identified some fungi as being the cause of this disease that fungi were added to the list of agents of diseases in man. Ringworm disease is common in children and it is usually seen as scaly lesions on their head. In most cases, children overgrow this disease since it often heals spontaneously without treatment. In adults the lesions are usually on the skin, feet (athlete’s foot), groin, beard, or nails. The disease may be chronic but not serious and also does not go beyond the upper part of the skin. This disease is very common in this country. We have published some of our work on dermatophytosis, particularly among children in Nigeria.

ii) Superficial candidiasis: Candida, a species of the

group of unicellular fungi which are called yeasts, normally form part of the microflora of the human body and live on waste products of the body without causing any harm to the host tissue. However, under certain conditions in the human body, this “commensal organism” may become an “opportunistic pathogen” sometimes growing out of proportion to produce superficial lesions on the skin and mucous membranes particularly in infants, old people, pregnant women, diabetics and women on oral contraceptives. Many of you are familiar with some common diseases such as: � diaper rash in infants

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� oral thrush (membranous white growth) on the tongue of infants, diabetics and old people

� white growth or discharges often from the genital organs of diabetics, pregnant women or those on oral contraceptives.

These diseases are usually caused by Candida. In most cases the lesions remain at the superficial part of the body, not serious but may be chronic, causing a lot of discomfort if not treated. In some of our studies, we identified Candida as a normal flora of the skin of healthy individuals. We also found them to be responsible for skin rashes and oral thrush in infants as well as vaginal candidiasis which is very common among women in this country.

iii) Tinea versicolor (“ngwo” in Igbo): This is also caused by a species of the yeast called Pityrosporum. The lesions appear as patches of discolouration of the skin particularly on young adults. This is a common disease which we are familiar with. In some of our studies we were able to isolate this fungus from some patients and also identified some of the factors that aid its pathogenicity.

Other superficial mycoses are caused by spores of fungi found floating in the air which manage to enter the superficial area of the body. Some of them can cause superficial lesions in the external part of the ear or on the cornea of the eye but these diseases are not serious mycoses and can be easily treated.

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1.3 SUBCUTANEOUS MYCOSES These are fungal diseases that are limited mostly to the tissue just below the skin which is the dermis but they rarely affect the deeper tissues. Some important ones are: i) Basidiobolomycoses. This disease is caused by a

fungus called Basidiobolus. The organism exists naturally in the soil, decaying vegetation and also as part of the microflora in the intestine of reptiles (lizards, wall gecko) and amphibians (toads, frogs). The spore enters the tissue of man through cuts in the skin, grows slowly to produce a hard and enlarged lump under the skin, often in the legs and arms. The disease, if not treated may spread to the deeper tissue sometimes affecting vital organs. In some serious cases amputation of the affected limb is carried out to avoid the spread to vital organs like brain which will result in death of the patient.

ii) Conidiobolomycosis. This disease is caused by a

fungus called Conidiobolus and it is in the same class of fungus as the Basidiobolus. It exists naturally in the soil and decaying plant vegetation. The spore of this fungus is believed to gain entry through a cut likely in the area of the nose of man where it grows slowly to produce a hard and enlarged lump under the skin. As the disease progresses, it causes excessive inflammation of the nasal area including the nose, upper lip and part of the upper face so that the patient

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eventually looks like a monster. The inflammation may eventually cause blockage of the nasal passage to prevent breathing through the nose. Death may occur if the disease is not well treated.

My M.Sc. and Ph.D. research work were mainly on these two fungi. In the first part of this work, I identified the prevalence of Basidiobolus in soil (including river sand), plant vegetation and in the intestinal tracts of animals (lizards, toads, turtle, wall gecko) both here in Nigeria and also in Florida in the United States of America where I did part of my work. The high prevalence of this fungus within these different environments, shows a high possibility of the spore entering the tissue of any body with a cut in the skin. This also explains why we have many cases of basidiobolomycosis especially in children in the tropical areas of the world (where we have the majority of the reported cases) who often play outside (with cuts not usually taken care of). This disease occurs mostly in children in rural areas where it is often treated successfully by native doctors who simply remove the fungal lump under the skin. However, in few cases especially where the lump is in the neck area, the organism may spread to the brain to cause death of the patient even when the patient is undergoing medical treatment.

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Figure 1: Right picture is a plate culture of Basidiobolus sp., which is cream coloured and shows radial furrows. It has been isolated from African lizard (Agama agama). Left picture shows conidia from a young culture of Basidiobolus sp. as seen under the microscope, X375. Figure 2: Scanning electron micrograph of Basidiobolus haptosporus showing primary globose conidium and a slender conidiophore, X1200.

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Figure 3: A typical clinical picture of

Basidiobolus infection causing massive subcutaneous swelling of the thigh.

Conidiobolomycosis, although it is a more serious disease than basidiobolomycosis and is also prevalent in the soil (including river sand), appears to be rare even in Nigeria which is a tropical country. However, we were able to identify one case recently at the University of Nigeria Teaching Hospital (UNTH), Enugu. I can also recall that over thirty years ago while I was still in the college, I saw a man with features of this disease who was consulting a medicine dispenser in one of the villages in Enugu State. We have tried to make inquiries on the existence of this disease in some villages in Enugu, Abia and Ebonyi States but so far we have not identified any. This paucity of identified cases may not necessarily mean that the disease does not exist in Nigeria, but may be due to poor diagnosis and poor record keeping in hospitals. It is also possible that patients with this disease often consult native doctors since

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such weird diseases are usually attributed to poison by enemies or evil forces. Figure 4: Conidia of Conidiobolus coronatus isolated from Nigerian soil.

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Figure 5: Typical clinical pictures of rhinoentomophthoromycosis caused by Conidiobolus coronatus. Note the subcutaneous swelling on the face. 1.4 DEEP OR SYSTEMIC MYCOSES These are fungal diseases that affect deeper tissues including the muscles, bones, and body organs (lungs, liver, kidney, heart, and brain). Some important ones are: i) Maduromycosis (mycetoma). This is caused by a

variety of soil-borne fungi. The disease occurs when the spore of any of these fungi enters the tissue through a cut especially in the leg or hand, germinates and develops into a typical form. A typical form of the disease is characterized by excessive enlargement of the affected part which is followed by massive destruction of the affected tissue including the muscles and in severe cases, the bones. As the disease progresses, a large amount of pus is produced which contains the fungus in the form of coloured granules (“sulphur granules”) oozing out through openings in the cracked skin. This disease is common among outdoor workers, particularly in developing countries (including Nigeria) where protective shoes and gloves are not worn especially during farming. In severe cases, other organisms including bacteria may also be involved and this often leads to rapid spread of the organisms towards vital organs which may sometimes require amputation of the affected limb so as to prevent death of the patient. Cases of mycetoma are very common in Nigeria and we recently identified a case at UNTH, Enugu. In the course my teaching of

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mycology I have continued to isolate various species of fungi responsible for mycetoma from the soil with ease and these are what we use for the students’ mycology practical lessons.

ii) Dimorphic fungi involved in respiratory diseases.

Dimorphic fungi are a group of fungi that can exist in two morphological forms (unicellular forms or filamentous forms) which often help them to live as parasites in one form and as saprophytes in another form. As parasites, they often cause serious respiratory diseases similar to tuberculosis. These fungi occur as saprophytes in the soil where they grow as filaments and produce numerous spores that can be inhaled by man. The spores, if not destroyed in the human tissue, will germinate to develop into the parasitic form which may be a yeast form (as is the case in Histoplasma and Blastomyces) or in the form of endospores (as is the case in Coccidioides). In healthy individuals who have active immune system (“immunocompetent” individuals), the inhaled fungal cells are often destroyed by the immune cells and so the disease fails to occur. However, in individuals with defective immune system (“immunocompromised”), the fungal cells will grow to attack the lung tissue and from there may spread to other vital organs. Whenever these fungi are left to spread uncontrollably, as many as 90% of the affected patients often die of the fungal invasion of the vital organs.

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Figure 6: Maduromycosis (mycetoma) of the right leg. Note the swelling with induration of the subcutaneous tissue. (Courtesy of Dr. E. R. Ezeome, Department of Surgery, UNTH, Enugu) Figure 7: Maduromycosis. This is the surgical excision specimen from the patient in Figure 6. Note the whitish fibrotic lesion destroying the subcutis and skeletal muscle and which contains the characteristic yellow granules (“sulphur

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granules”) of the fungal elements. (Courtesy of Dr. O.C. Okafor, Department of Morbid Anatomy, UNTH, Enugu) 1.5 OPPORTUNISTIC MYCOSES Healthy individuals with active immune system which helps them to resist fungal invasion of their tissues are said to be “immunocompetent individuals.” However, individuals whose immune system is defective, such that they are not able to resist fungal spread are said to be “immunocompromised individuals”. Some important factors that lead to the development of immunocompromised state in some individuals include: i) immature or genetically inherited defective immune

system ii) long therapy (use) of antimicrobial antibiotics iii) prolonged use of immunosuppressive drugs

particularly in patients who received organ transplant (to prevent rejection of transplanted organ)

iv) debilitated disease conditions including diseases like diabetes mellitus, tuberculosis, cancer, and acquired immunodeficiency syndrome (AIDS).

As a result of a significant increase in the number of immunocompromised individuals in the past one or two decades, fungal diseases which earlier on did not constitute any serious problem to man have now become important causes of morbidity and mortality in man. The death rate due to fungal diseases is alarming and so needs to be addressed. A number of fungal diseases are now in the list of life threatening mycoses and they include: i) Systemic candidiasis

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ii) Cryptococcosis iii) Trichosporonosis iv) Invasive aspergillosis v) Invasive mucormycosis vi) Histoplasmosis vii) Coccidioidomycosis viii) Blastomycosis ix) Mycotoxicosis 2 SYSTEMIC INFECTIONS 2.1 CANDIDIASIS Candida is a unicellular fungus and it is normally a member of the microflora of the body including the gut and intestine. In these parts of the body they are known to co-exist with other micro-organisms which include other fungi and some “beneficial bacteria” in a manner that the growth of each group is under control so that none is allowed to grow out of proportion. Various studies on the microflora of the body have shown that the growth of Candida is controlled by other micro-organisms that constitute the microflora (which is the case under normal conditions). However, certain conditions may cause a drastic change that can allow Candida in the body to grow out of control and this will result in a disease condition generally called candidiasis. There are a number of conditions that can predispose a healthy individual to have candidiasis and one of them is prolonged use of antibiotics especially antibacterial antibiotics. In recent times, development of resistance of some pathogenic

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bacteria to the conventional antibiotics has forced physicians to prolong the period of treatment and also use different antibiotics to cure their patients. Unfortunately, this long antibacterial therapy as well as the use of different types of antibiotics have increased the chances of eliminating both the pathogen as well as the beneficial bacteria that co-exist with Candida and other yeasts in the human body. This often results in an excessive proliferation of Candida cells. In developed countries, antibiotics are administered only through doctor’s prescription but in developing countries like Nigeria, there is no restriction to their purchase and use. Hence, there is a serious abuse of the use of antibiotics which allows untrained medicine sellers to administer antibiotics to any one who requests for them. Also many sick people easily self-administer antibiotics without doctor’s prescription or laboratory tests. A very common example of this uncontrolled use of antibiotics in Nigeria is seen in the treatment of “typhoid fever” where patients consume all kinds of antibiotics for weeks often without a confirmatory test to prove that the patient actually has the disease. Many such patients continue to feel sick despite the prolonged antibacterial therapy and quite often they develop serious complications. This gross abuse and overuse of antibiotics has been found to cause a serious elimination of most beneficial bacteria in the gut and intestine and this is often followed by an excessive growth of Candida. This uncontrolled growth of Candida has given rise to a newly discovered and challenging unhealthy condition called “chronic

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candidiasis syndrome” or “leaky gut syndrome” (LGS). It was discovered that Candida, during this excessive growth in the gut, changes from its unicellular form to a mycelial form. In this mycelial form, Candida appears as a “plant-like structure” and develops “root-like structures” called “rhizoids” (long burrowing legs). The rhizoids hook into and penetrate the mucous membranes into the gut to cause serious bowel pains and distress. Later, the rhizoids burrow right through the intestinal walls to produce “microscopic holes” which will allow toxins, undigested food particles, particularly digested proteins, yeasts cells and some other micro-organisms to enter the bloodstream. 2.1.1 Leaky gut syndrome (LGS) LGS is a common problem of modern society partly due to abuse of antibiotics and other non-inflammatory drugs. The gut, which is made up of both small and large intestines, hosts many hundreds of different kinds of beneficial bacteria as well as fungi and viruses. These bacteria are important for healthy metabolism and immune response within the human intestines. They use their enzymes to transform the human and microbial wastes (bile, toxins, chemicals etc.) before they are finally discharged from the body. “Bile” which is found in the liver plays an important role of elimination of many toxins that accumulate in the liver. Bile is extremely caustic to large intestine epithelium but the beneficial bacteria in the small intestine are able to breakdown the bile salts into less caustic compounds before they enter the large intestine. However, elimination of these bacteria due to prolonged use of antibiotics will allow free passage of bile from the small intestine into the large

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intestine which will eventually cause serious damage to the large intestine. Damage to the large intestine has been found to be one of the causes of “colon cancer.” Beneficial bacteria in the intestines also metabolise hormones like “oestrogen” which are discharged from the liver into the small intestine. Absence of these bacteria and presence of leaky gut, allows the hormones to be reabsorbed and carried to different parts of the body. Excess oestrogen is known to bind to sensitive areas such as the breast, uterus, and ovaries and this has been found to contribute to fibrosis and tumours in these organs. These tumours may sometimes become malignant (cancerous). The damage to intestinal barrier due to LGS allows absorption of partially digested food particles, toxins, chemicals and micro-organisms especially Candida cells into the bloodstream to different parts of the body. Candida is known to produce 79 distinct toxins. Increased absorption of “toxins” by the liver will place great stress on this vital organ which may lead to liver inflammation and toxic hepatitis. Some of the toxins will move to the brain cells where they can be mistaken for neurotransmitters to cause brain fog. Some will move to the skin to cause rashes. In immunocompromised individuals, Candida cells which enter the bloodstream through the leaky gut will grow profusely and eventually develop into systemic candidiasis and so travel through the blood stream to other parts of the body including the mouth, sinuses, throat, reproductive organs, lungs, and heart. In most severe cases of systemic

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candidiasis, the organism causes a type of blood poisoning called “candidal septicaemia” or “candidaemia”. This will cause the organism to invade every organ in the body which in turn may result in death due to shock and renal shut down. A high mortality rate of up to 40% has been reported. The body defence system also attacks the undigested food particles and produces antibodies against them for future attack. This will result in the development of allergic reactions to these food particles. Some antibodies can give rise to autoimmune diseases in an attempt to destroy any foreign matter. What started as a gut barrier problem can escalate to a serious problem of tissue toxicity and with time can cause pains in the muscles and joints, brain fog, drop in energy production and body temperature. This will result in conditions such as: � Opportunistic infections � Arthritis � Brain fog, inability to concentrate � Chronic fatigue � Cancer (particularly of the liver, breast, ovary and

uterus) � Stressed immune system Other causes of LGS include: � Consumption of allergic food � Radiation therapy � Alcohol (depletes N-acetyl glucoseamine) and caffeine � Excess sugar (leads to excess growth of yeast) � HIV infection

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� Excess stress LGS is hardly tested for or diagnosed by doctors in general practice especially in the developing countries. But its effects are seen in many unrelated symptoms. Researchers give it different names; for example Gibere calls it “intestinal terrorist” while Gellend calls it “vicious cycle”. Crook (1986; 1998) has written two popular books: “The Yeast Connection” and “The Yeast Connection and the Woman” in which he discussed the disturbing role of yeasts in human diseases. Figure 8: Pseudohyphae and a few yeast cells of Candida albicans from growth in corn meal agar, X825. 2.1.2 Candida - Cause of diarrhoea in immunocompetent individuals

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Excessive growth of Candida in the guts of immunocompetent individuals even in the absence of LGS has also been found to cause chronic persistent diarrhoea sometimes associated with abdominal cramps. It was found that the symptom continued in most patients until antifungal treatment was administered which cured them of the disease. Some doctors in Georgetown University Hospital, Washington DC, who handled some of these cases, lamented that many hospitals even in developed countries rarely request for yeast identification in stool samples. Furthermore, even the well trained laboratory technologists often do not report the presence of yeasts they come across during their investigation even when the number seen are abnormally very high. Chronic diarrhoea if untreated, may lead to serious dehydration that can be fatal especially in children. 2.2 CRYPTOCOCCOSIS IN IMMUNOCOMPROMISED PATIENTS Cryptococcus neoformans is a capsulated yeast which lives as a saprophyte in the soil particularly one contaminated with faeces of birds (pigeons). This fungus can enter the human tissue through inhalation of soil containing the organism which is eventually carried into the lungs. In a healthy individual the organism will not be able to grow and to spread in the lungs because the immune cells (particularly T cells) will be able to destroy it. However, in immunocompromised individuals particularly those suffering from “acquired immunodeficiency syndrome” (AIDS), the immune cells are no longer effective, hence, the organism will grow slowly in the lung tissue to produce

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a disease called cryptococcosis. The disease initially presents as pneumonia but later the organism moves to the meninges and to the brain to cause cryptococcal meningitis. The disease is usually fatal in AIDS patients and patients with organ and bone marrow transplants. Such patients usually have low CD4 lymphocyte counts (low T cells) which are not enough to destroy the capsulated organism. 2.3 TRICHOSPORONOSIS IN IMMUNOCOMPROMISED PATIENTS Trichosporon beigelii is an ubiquitous yeast which inhabits the soil and also lives as part of the microflora of the skin, nose and the intestinal tract of man. Ordinarily, it causes a superficial infection of the hair called “white piedra” (loose white fungal ball attached to the hair shaft) which is common among farmers in the tropical and subtropical regions in the world. Recently, this organism has been found to cause fatal systemic mycoses called “trichosporonosis” in immunocompromised patients. Most of the patients affected are: � AIDS patients � Bone marrow and organ transplant recipients � Low birth weight and premature infants � Patients with in-dwelling devices (catheter) � Patients on prolonged broad spectrum antibiotics � Cancer patients. T. beigelii in these patients is known to grow uncontrollably all over the body and also invades the blood vessels to cause various disease conditions which include: � Peritonitis especially in AIDS patients

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� Chronic meningitis in cancer patients � Hepatitis (liver infection) � Fungaemia (wide spread of the yeast in the entire body

system) Most patients with this disease are known to die in spite of the antifungal therapy. 2.4 INVASIVE ASPERGILLOSIS IN IMMUNOCOMPROMISED PATIENTS Aspergillus is an ubiquitous filamentous fungus found naturally in the soil and produces numerous spores which are easily aerosolised. The spores are very light and so can remain suspended in the air in very large numbers and spread all over the environment and for long distances by wind. They are also found regularly on surfaces of most materials including furniture, water, vegetations, cooked and raw foods as well as on bodies of plants and animals. They can enter human tissues through inhalation or cuts on the skin. There are many species of Aspergillus which include A. niger, A. flavus, A. fumigatus, A. orizae and A. parasiticus. In healthy individuals, Aspergillus can cause superficial mycoses like keratitis (infection of the cornea) and external otitis (infection of the external ear) which are easily treated with antifungals. Constant inhalation of air-borne spores of Aspergillus occurs daily in every individual. In immunocompetent individuals, these inhaled spores are regularly destroyed by the immune cells to prevent their growth and spread. However, in immunocompromised patients inhaled spores germinate

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and grow to produce, in many cases, a fatal disease called “invasive aspergillosis.” Invasive aspergillosis has been on the increase in the last decade and death rate has been extremely high despite antifungal therapy. The disease occurs in severely immunocompromised patients such as: � Patients with acute leukaemia (cancer of the blood) � AIDS patients with very low CD4 lymphocyte count � Patients with heart, kidney, liver, and bone marrow

transplant. For organ recipient patients, the highest risk period is the first two to three months after transplant often due to high dosage of immunosuppressive therapy. These groups of patients are usually hospitalised and become infected through inhalation of Aspergillus spores which occur in large numbers in dust particles from construction work (demolished hospital buildings) that often occur during hospital expansion or renovations. In one hospital in the State of Pennsylvania in the United States of America, three patients who had undergone cardiothoracic surgery died due to invasive aspergillosis. These patients (through a thorough investigation by the Hospital Administration) were found to have died of invasive aspergillosis contacted as a result of the fungal spores inhaled. These spores were traced to the dusty air from a building construction in the hospital which was going on at the time of surgery. The organ most affected in invasive aspergillosis is the lung, with growth of the fungus in the lung tissue followed by severe damage to the pulmonary blood vessels which

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may eventually cause a collapse of the affected lung. The fungus may also invade other blood vessels to cause skin and gastrointestinal ulceration. It may also spread to other organs especially the kidneys, heart, and brain. These activities of the invading fungus often result in the formation of microabscesses within multiple organs, blockage of the blood vessels and stroke which often cause death of the patient. Despite adequate treatment, invasive aspergillosis carries a high mortality. Pillows can serve as reservoirs for Aspergillus spores. A research carried out at the University of Manchester in England which was funded by “Fungal Research Trust” revealed that millions of fungal spores are literally under our noses particularly in our pillows. They found that spores of A. fumigatus were the most isolated fungal spores from pillows. They dissected both feather and synthetic pillows and identified several thousands of spores of fungus per gram of used pillow. They also discovered that pillows used between 17 months and 20 years, contain more than a million spores per pillow. They, therefore, concluded that immunocompromised patients particularly those that have undergone organ transplant may develop invasive aspergillosis through inhalation of Aspergillus spores in their pillows. Most hospitals in developed countries provide patients with plastic covered pillows to protect them from direct contact with fungal spores in pillows. Patients may get infected when they go home and use unprotected pillows. In developing countries like Nigeria, the chances of immunocompromised patients contacting invasive

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aspergillosis are very high since most homes and many hospitals may not provide plastic pillows for them. Figure 9: Conidiophores of Aspergillus fumigatus with flask-shaped vesicles bearing sterigmata in single series and chains of conidia, X550. This is an isolate from Nigerian soil. Figure 10: These are autopsy specimens from a 39-year-old female who died from cancer (lymphoma). Histology confirmed the presence of circulating hyphae of Aspergillus within and

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around the wall of blood vessels in the lungs (top), kidneys (bottom left), spleen (bottom right), and brain. (Courtesy of Dr. O.C. Okafor, UNTH, Enugu). 2.5 INVASIVE MUCORMYCOSIS IN IMMUNOCOMPROMISED PATIENTS Many fungi in the order Mucorales, which include Mucor, Rhizopus and Absidia, are saprophytes in the soil. They produce large quantities of spores which are continually inhaled by man during normal breathing. In healthy people, the immune cells prevent the growth of these spores. But in immunocompromised patients, some of these spores especially those of Mucor can grow uncontrolled to cause a disease called mucormycosis. This disease may occur as a pulmonary infection with destruction of the lung tissue, a rhino-cerebral infection with the destruction of the brain cells or disseminated infection which spreads through the blood stream to destroy vital organs (liver, heart, kidney) and eventually causing death of the patient. Patients who are at risk of developing mucormycosis are: � AIDS patients � Patients with organ transplant � Poorly controlled diabetic patients It is believed that in diabetic patients with ketoacidosis, acidosis interferes with phagocytic processes of the phagocytes and thereby prevents them from destroying the fungal cells in the tissues.

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Figure 11: Mucormycosis diagnosed in the right eyeball and peri-orbital tissue from a 41-year-old HIV-positive male. He presented with right eye swelling, loss of vision, and copious yellowish-white discharge from the eye. (Courtesy of Dr. O.C. Okafor, UNTH, Enugu) 2.6 HISTOPLASMOSIS This is caused by Histoplasma capsulatum var. capsulatum (American histoplasmosis) and H. capsulatum var. dubuosii (African histoplasmosis). These fungi are usually found in black bird roosts, chicken houses (soil contaminated chicken droppings), as well as bat droppings.

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H. capsulatum var. capsulatum, the causative agent for American histoplasmosis, enters the human through inhalation of spores of the fungus. Many cases of the disease are common in people who spread chicken manure in their gardens. The spores are able to grow and cause disease only in immunocompromised patients especially in AIDS patients where there is dissemination to other vital organs (spleen, liver, and bone marrow) with fatality rate up to 90%. H. capsulatum var. dubuosii, (the causative agent for African histoplasmosis) is endemic in West, East, and Central Africa. The exact portal of entry of this fungus is unknown but direct inoculation into the skin and inhalation through the respiratory tract have been incriminated. It commonly affects the skin, subcutaneous tissue, and bone; occasionally it may cause disseminated disease involving the liver, spleen, lungs, and other vital organs. Figure 12: African histoplasmosis diagnosed in the axillary lymph nodes of a 21-year-old female who presented with

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enlarged axillary lymph, night sweat, and weight loss nodes of 6 months duration. (Courtesy of Dr. O.C. Okafor, UNTH, Enugu) 2.7 COCCIDIOIDOMYCOSIS This is also primarily a pulmonary disease caused by Coccidioides immitis. The fungus is usually found in hot desert soil, pottery, archaeological middens, cotton, as well as rodent burrows. Their spores are produced in large numbers and many are found floating in the air and spread to hundred of miles by dust storms. The fungus is common in desert areas of northern part of America where many cases have been identified particularly in immunocompromised patients. The disease is usually progressive, chronic, and fatal particularly in AIDS patients where the skin, lymph nodes, spleen, liver, bones, kidneys, meninges and brain are mostly affected. 2.8 BLASTOMYCOSIS This is again primarily a pulmonary disease caused by Blastomyces dermatitidis. The fungus is usually found in soil containing rotting wood, animal droppings, and plant materials. The fungus infects mostly people collecting firewood, tearing down old buildings or engaged in other outdoor activities which disrupt soil. The disease is also fatal in immunocompromised patients when the vital organs are seriously affected. The last three genera of fungi are not well known to cause disease in Nigeria. One of the reasons for this observation is that, with the exception of H. duboisii which we have

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isolated from soil here, there is so far no documented report on the presence of the other ones here. Secondly, many doctors here in Nigeria rarely request for the detection of these fungi in the clinical specimens (sputum) sent to the laboratory for investigation. Hence, the laboratory scientists rarely make report on the presence of yeast cells (characteristic of Histoplasma or Blastomyces) or spherules/ endospores (characteristic of Coccidioides) which they may encounter in the clinical specimens. 2.9 MYCOTOXICOSES Mycotoxins are natural by-products of some filamentous fungi which are toxic to human and other animal tissues when introduced (mostly by ingestion) in low concentrations. Mycotoxins are usually produced in plants such as maize, wheat, barley, rye, as well as groundnut while still growing in the field, after they have been harvested, during storage and also when they are processed into food or animal feed concentrates. There are about twenty mycotoxins which are known to occur naturally in foods and feeds at significant levels. Human intake of mycotoxins occurs mainly through ingestion of mycotoxin-contaminated foods as well as contaminated animal-derived foods like milk, cheese, and certain fermented meat products. Recently, mycotoxin contamination of food and feeds has become a world-wide problem. It is now believed that as much as 25% of the world food crops are affected annually by variable levels of mycotoxins. This has caused serious economic problems

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for individual crops, livestock producers, grain handlers, processors, as well as consumers. The effects of mycotoxins on human health are complex and not well understood. Human mycotoxicoses have been mainly associated with ingestion of contaminated foods and some scientists have been able to link some mycotoxins to certain human cancer. There is also some scientific proof that several mycotoxins can damage components of human immune system especially cellular immunity. The parts involved are the T lymphocytes as well as the non-specific humoral factors. Some important mycotoxins which are known to cause serious human diseases are: � Ergotamine and ergonomine produced by Claviceps

purpurea � Amatoxin produced by Amanita phalloides

(mushroom) � Aflatoxin produced mainly by Aspergillus flavus and A.

parasiticus. 2.9.1 Ergotamine and Ergonomine These two toxins produced by Claviceps purpurea were first discovered in Europe in the “middle ages” when rye, a staple food of the peasant population was somehow connected with a deadly disease called “ergotism.” A close study of the implication of rye in the many deaths revealed that a fungus called Claviceps purpurea grew in some of the developing seeds of rye and eventually replaced them with hard fungal mass called ergot. At maturity, both the ergot and the good rye seeds were all harvested, ground together and eaten as food. A detailed study of the ergot

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revealed that it releases a “Pandora’s box” of deadly poisons which include ergotamine and ergonovine which often led to death of consumers. Most medical historians noted that ergot mycotoxins exerted a major role in preventing population expansion in the middle ages in Europe. However an increase in the population of Europe was noted in the 16th and 17th centuries, and this has been attributed to the introduction of wheat and potatoes as staple food and also the reduction in the consumption of rye. 2.9.2 Mushroom Toxins (Amatoxins) Mushrooms which are composed of a group of macroscopic fungi that grow in the soil often serve as meat for the poor especially in developing countries. Unfortunately some species produce deadly toxins which kill human beings on consumption. The most deadly of these toxins are the amatoxins (known to be among the deadliest mushroom toxins in our planet) which are produced by Amanita phalloides (“death cap”) and has no known antidote. The toxins are found to destroy the liver and kidney and death usually occurs within 3 to 7 days following consumption. Fortunately, most people in the developing countries no longer eat mushroom for fear of consuming the deadly ones. Hence, only few cases of death from mushroom meals are now reported. However, in developed countries, mushrooms are eaten as special delicacies since mushroom farmers grow safe species for marketing.

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2.9.3 Aflatoxins Aflatoxins are toxic metabolites produced by certain fungi in or on foods and feeds. Aflatoxins were discovered accidentally in 1960 when over one hundred thousand (100,000) young turkeys on poultry farms in England died in the course of a few months from an apparently new disease which was then called “Turkey X Disease.” The disease was not limited to turkey but also affected ducklings and young pheasants (large birds with long tails). A detailed investigation of these outbreaks revealed that the birds were fed on groundnut feeds from Brazil which happened to contain aflatoxins that killed the turkeys. The toxins were later found to be produced by a fungus identified as Aspergillus flavus which influenced the naming of these toxins (aflatoxins). Other species of Aspergillus namely A. niger, A. parasiticus and A. nomius were also found to produce aflatoxins. I still recall that I first knew about aflatoxins in 1972 in my final year in the Department of Botany where I got my first degree. I presented two seminars as part of the requirements for the B.Sc. degree in Botany. I chose to talk on “Yeasts” in my first seminar. In the second one I talked on “Aflatoxins” which I found very interesting and revealing and my presentation was highly commended by staff and students in the department. After my graduation, my good results as well as these two seminars I presented helped me to earn a job and a University Scholarship as a

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“Junior Fellow” in the Department of Microbiology. The department wanted to train somebody in the area of Medical Mycology as part of the University Academic Staff Development. During the interview, the Vice Chancellor then, Prof. H. C. Kodilinye of blessed memory, who was the Chairman of the interview panel was so impressed with my discussion on aflatoxins that he wanted to know if I had travelled to England to get all the facts I presented on the toxins. I then told him that I did not go any where but read about them from books and journals. His reply was: “Well read, well read,” and soon after this I was offered the job and the scholarship. Aflatoxin-producing fungi infect plants while still growing in the farms and after harvest, especially during storage if the crops are not properly dried. Insects and rodent infestations facilitate their invasion into the crops in storage places. Favourable conditions for crop infection are high moisture content and high temperature. Crops with the highest risk of infestation are cereals (corn, sorghum, millet, rice and wheat), oil seeds (peanuts/groundnuts, soybean, sunflower, and cotton), spices (pepper and ginger) and tree nuts (almond, walnut, and coconut). Corn which is the staple food of many countries is of great concern for its aflatoxin contamination because it is grown in climates that are likely to have perennial contamination with aflatoxins. Again aflatoxins are stable or moderately stable in most food processes from where they enter the human tissues through food ingestion. The toxins can also be found in the milk of animals which are fed with aflatoxin-contaminated feed. The disease they cause in man is called “aflatoxicosis.”

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Acute aflatoxicosis has been reported in developing countries like Taiwan, Uganda, and India. The disease is characterised by vomiting, abdominal pain, convulsion, coma with serious involvement of the lungs, liver, kidneys, heart, brain and death may occur in severe cases. Conditions that favour development of acute aflatoxicosis in humans include limited availability of food, environmental conditions that favour aflatoxin-producing fungi in crops and commodities and lack of regulatory systems for aflatoxin monitoring and control. It is now accepted that aflatoxins are undoubtedly human carcinogens and not only of birds. A World Bank report has strongly incriminated mycotoxins especially aflatoxin as a significant modulating factor in childhood diseases and deaths. A number of epidemiological studies done in Africa have demonstrated a positive association between dietary aflatoxins and “primary liver cell cancer” (PLCC). Other investigations also revealed that aflatoxin ingestion through contaminated foodstuffs is one of the major etiological factors in PLCC in China and Sub-Saharan Africa. In certain regions of these countries, at least 250,000 deaths from PLCC are known to occur annually. For almost four decades, National Institute of Environmental Health Sciences (NIEHS) funded scientists who conducted research on the role of aflatoxins in the promotion of liver cancer. NIEHS-funded scientists at the Massachusetts Institute of Technology (MIT) in the U.S.A. were among the first to show that exposure to aflatoxins can lead to liver cancer. From their work it has been discovered that high

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level of aflatoxins exposure produces an acute necrosis, cirrhosis, and carcinoma of the liver. No animal species, man inclusive, is immune to acute effects of aflatoxins. However, humans have been found to have an extraordinarily high tolerance level to aflatoxin exposure and so, rarely succumb to acute aflatoxicosis. Children are particularly affected by aflatoxin exposure which usually leads to stunted growth and delayed development. Chronic exposure also leads to high risk of developing liver cancer through aflatoxin M1. In 2001, many reports appeared in the news media in South Africa on what was described as “poison in the peanuts”. These reports were based on the discovery of high levels of aflatoxins which caused serious health problems in school children fed on aflatoxin-containing peanut butter used in preparing their sandwich meal. Following this report, The South African Medical Research Council, an internationally recognised centre of excellence on mycotoxins, made some recommendations on the ways to control aflatoxins in peanut butter. It is difficult to avoid consumption of aflatoxin contaminated foods because fungal growth in foods is not easy to prevent. However, the ability of aflatoxins to cause cancer and related diseases in human given their unavoidable occurrence in foods and feeds, makes the prevention and detoxification of these mycotoxins one of the most challenging toxicology issues in recent times particularly in countries with high temperature and humidity. In developed countries like United States of America, foods with high levels of aflatoxins are not

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allowed to be sold in their food stores. Agencies like Food and Drug Administration (FDA) have specific guidelines on acceptable levels of aflatoxins in human foods and animal feeds. This step had to be taken because aflatoxins are considered unavoidable contaminants of food and feeds even where good manufacturing practices have been followed. Studies on human populations exposed to diets naturally contaminated with aflatoxins revealed a direct relationship between the high incidence of liver cancer in Africa and some other places and dietary intakes of aflatoxins. Following these findings, careful control of aflatoxins in foods has been recommended for each country. Effective control and monitoring of levels of aflatoxins in foods and feeds can, therefore, be handled by agencies such as: � National Agency for Food and Drug Administration

(NAFDAC) � Ministry of Health � Ministry of Agriculture � Natural Environment Committee Board � Food and Agricultural Organization I have a postgraduate student who is currently looking at some common staple foods consumed in Nigeria to determine the levels of aflatoxin contamination in them. The results will help to determine the safety of some of our foods with regards to aflatoxin contamination.

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3. CONCLUSION AND RECOMMENDATIONS I will not like any one to have the impression, after listening to this lecture, that fungi are a group of dangerous organisms which must be eliminated. Surely this is not true since I had earlier in this lecture, listed the beneficial aspects of fungi. Some of them are the decomposition of refuse in the environment which has helped to give us clean environment and subsequently provide food for plants and other organisms. The production of antibiotics and other useful metabolites by some species of fungi have helped to save humanity from serious harm and even death by some other pathogens. One must not fail to accept the fact that fungi are naturally saprophytic organisms and can live their lives by depending solely on organic matters within our environment. Living organisms including man are colonised by fungi only if they are able to gain entry into their tissues and also if they are able to overcome the defence mechanisms of their host tissues. The immune status of every individual is a key factor in the prevention or establishment of fungal diseases. Healthy individuals with effective immune system (“immunocompetent”) are able to overcome most fungal invasion. However, individuals with defective immune system (“immunocompromised”) are rarely able to resist the dissemination of fungal cells throughout the entire body even in the presence of appropriate drugs. This situation is a great concern for the medical group who are now faced

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with so many treatment problems including resistance of many pathogens to the best available drugs. The emergence of “human immunodeficiency virus” (HIV) which is destroying the CD4 lymphocytes (that are most effective in the destruction of fungal pathogens) has given rise to AIDS in man. The numbers of individuals with full blown AIDS are on the increase each year. Many fungi which were mere members of the body microflora, are now causing death (at a high percentage) in patients with advanced stage of AIDS. We are all aware that AIDS is real and that AIDS patients have the highest risk of being easily infected by opportunistic fungi. It is therefore, very necessary that everyone should take every adequate precaution to avoid infection by HIV. A word is enough for the wise. Prolonged use and abuse of antibiotics have elevated the status of some fungi like Candida found normally in the intestines of man, from being a harmless commensal organism to a serious pathogen that now causes systemic candidiasis and the subsequent “leaky gut syndrome.” This now calls for an effective control of both the sale and the administration of antibiotics in this country. The medical personnel particularly the Medical Doctors and the Pharmacists must, therefore, be more cautious in the administration of antibiotics to patients to avoid the elimination of the other important members of the microflora of the human body. Every Nigerian who treasures his or her life must know that self medication (especially of antibiotics) is very risky and can create

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serious complications that can lead to the death of the individual. In developed countries, there are effective laws preventing the purchase of antibiotics at pharmacy shops without the doctors’ prescription and everyone obeys because the people there are disciplined. Unfortunately, in Nigeria, I am not aware of any law preventing the purchase of any drug from any medicine store without doctors’ prescription. Even if there is one, no one is enforcing it. Moreover, most Nigerians are not disciplined enough to obey such laws. The only effective option left at the moment (before Nigerians realise the need to do things properly) is for every sensible Nigerian to avoid taking antibiotics at the slightest discomfort so as to prevent diseases like systemic candidiasis. Medical Doctors and Pharmacists are now challenged to champion an effective law that will stop the abuse of drugs particularly antibiotics in this blessed country. It is now clear from all we have learnt about aflatoxins that it is impossible to avoid contamination of foods and animal feeds by these toxins. We have also learnt that the crops most affected by the aflatoxin-producing fungi are corn and groundnut. Let us reflect for a moment, on the rate of consumption of corn in this country as a major staple food (corn flour, pap, roast and boiled corn, corn flakes, corn cakes and corn bread) and imagine the health implications if corn is highly contaminated with aflatoxins before it is eaten as food. We should also be concerned about the rate at which groundnut is being consumed in this country, as snacks in schools, work places and in homes as soup ingredient and the health hazard associated with aflatoxin-contaminated groundnuts. Groundnut is also popular as

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peanut butter (“ose oji” in Igbo) for kola nuts. Since it is now confirmed that aflatoxins are highly implicated in cases of liver cancer particularly in Africa, one may not be wrong in suggesting that consumption of corn and groundnut containing high levels of aflatoxins, may be partly responsible for some of the many cases of liver cancer being reported recently in our country. Nigeria, therefore, needs to established effective agencies (as is the case in most developed countries) which will monitor the levels of aflatoxins in our foods and animal feeds. The Director of NAFDAC, Prof. Dora Akunyili, can be appealed to, so that she can institute aflatoxin monitoring in foods and animal feeds to save lives regularly lost through aflatoxicosis. This is another challenge particularly to Pharmacists in this country. Part of the health problems created by fungal diseases is poor diagnosis of many diseases where fungi are the major cause. In many such cases especially in developing countries like Nigeria it is usually the culture methods that are used. In most developed countries the more advanced, faster, and more specific non-culture methods such as serological test, detection of fungal antigens in the infected tissue and detection of specific nucleic acid or distinctive fungal metabolites are used. Again many doctors do not request for search for fungi as possible aetiological agents in most of the clinical specimens. The doctors and laboratory scientists should therefore, look out for fungal cells (yeast cells, spherules and fungal balls) in clinical specimens which include sputum, faeces and urine collected for diagnosis. Very often the medical laboratory

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scientists rarely report the occurrence of fungal cells even when they are seen in an abnormally large numbers. There is now need for them to do so. Many doctors are yet to accept the fact that fungi have moved from being confined to the superficial part of the human body to a new position of penetrating into deeper tissues and organs, challenging the immune system effectively, and establishing fatal and systemic diseases. A number of other conditions such as AIDS have facilitated this development. Doctors should therefore, pay more attention to fungal diseases and not mostly on bacterial diseases so as to save lives of those facing death due to fungal diseases. One of the problems facing medical doctors recently in the successful treatment of fungal diseases is the emergence of fungal resistance to the available antifungal drugs. The Chemists are now challenged to discover new effective drugs that can destroy these invaders of the human territory. Unfortunately, fungi, being also eukaryotic organisms, share the same cellular structure as human beings with the result that many antifungal drugs produced are toxic to human tissues. Hence research into antifungal drugs is more challenging. Scientists working on medicinal plants (Pharmacists, Chemists and Microbiologists) are also challenged to identify medicinal plants that can serve as cheaper and safer alternative drugs that can be used for the treatment of fungal diseases especially for the large population of the masses in developing countries who will not be able to

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afford the expensive prescription drugs. I know that people are working on anti-microbial medicinal plants but more work is needed particularly on the antifungal ones. I have done some work on both antibacterial and antifungal plant extracts with some of my colleagues and students and the results obtained so far are promising. The Nigerian government needs to be more involved in tackling many health problems caused by fungi so that Nigerians can also enjoy high quality lives like other people in developed countries. There is need for modern and well equipped research and diagnostic laboratories for efficient and quick diagnosis of fungal diseases particularly the deadly systemic ones. Funds should, therefore, be provided by the government for these. There is also need to establish effective laws with the support of the national legislators, for the control of sale of drugs with doctors’ prescriptions to avoid the serious abuse of drugs in this country. Finally, the Government needs to establish agencies that will monitor the levels of mycotoxins, particularly aflatoxins in foods and animal feeds sold to the public. If the various effective arms of the government , the medical people, the scientists and the Nigerian populace can act effectively towards the fight against fungal attack on humanity, Nigerians can join the rest of the developed countries in the global plan to save humanity from extinction by these silent but energetic destroyers, the fungi.

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4. ACKNOWLEDGEMENTS I will not like to end this lecture without expressing my appreciation to all who have in one way or the other played key roles in my academic achievements so far. My highest honour and thanksgiving go to God, the Creator and Provider of all good things to humanity. I will for ever cherish the numerous blessings He has bestowed on me, the challenges He allowed me to experience and for making it possible (out of His mercy) for me to be alive today to deliver this lecture as a testimony of his goodness to me. I also thank my Spiritual Directors, Very Rev. Msgr. Prof. F. C. Okafor, Rev. Fr. Dr. A. O. Onyeneke, Rev. Fr. Prof. E. M. Nwabuisi and Rev. Fr. Prof. A. Ekwunife for their prayers and my spiritual guidance. I am most grateful to the Vice Chancellor, Ven. Prof. C. O. Nebo and the other members of the University Administration who made sure that assessment for my professorship was followed up and released after many years of undue delay just as he did for many others with similar experience. May God reward them for putting smiles on the faces of many who were unjustly treated in this University. I also wish to thank him and his Administration for financing these series of inaugural lectures which have given lecturers the opportunity to enlighten the general public on their research findings and experience in life.

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Next in my list are members of my family starting with my parents, late Mr. Joseph Igboko and late Mrs. Christiana Igboko who nurtured me right from birth with so much love and care. Despite the fact that they had many other children including two brilliant sons just behind me and also at a time when education for women was considered unprofitable, they were determined to give me the best education. My dad being a dedicated teacher coached me thoroughly for the highly competitive secondary school entrance examination particularly to good colleges. This was what made it possible for my name to appear top on the merit list published in one of the daily newspapers of thirty brilliant young girls who gained admission into one and only Federal Government Girls College, east of the River Niger which was then called “Queens School Enugu”. At the end of my first year in this college, I was awarded a college scholarship which I enjoyed all through the rest of my stay in the college (both for the secondary school and higher school education). Six months after completing my college education, my parents made sure that I got into the university before getting married because they believed that education is a major key to success in life. Unfortunately my mum died at a young age but God allowed her to see her first two grand children and she also witnessed my B.Sc. graduation. However, I thank God that my dad lived long enough to see the success of his labour for he saw my six children and also witnessed the graduation ceremonies for my B.Sc., M.Sc. and Ph.D. degrees before God took him at the ripe age of 84 years. Another member of my family whom I owe so much gratitude is my darling husband, Prof. Charles Okafor, a

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renowned academic and a great achiever to whom I got married at the end of my first year in the university. At the time we got married, he was a young lecturer, just back from the United States of America after his brilliant academic records, with First Class Honours Degree in Chemistry from the University of London and a Ph.D. degree also in Chemistry from Michigan State University, East Lansing, Michigan, U.S.A. Although he was very much involved in teaching, research and administration, he was still able to accommodate a student wife and babies who were born into the family during the many years I had to study for my B.Sc. degree in Botany, M.Sc. and Ph.D. degrees in Microbiology. Each time I reflect on the level of support and encouragement I received from him, I count myself as one of the happiest and luckiest wives in the whole universe. At a certain time during my undergraduate programme, he reminded me that spending too much time with our children, would prevent me from earning a good degree which he felt I could make easily on merit judging from my previous academic performance, by working hard. This message from him clicked in my brain and so I started working as hard as I used to and finally got the second best result in the B.Sc. degree examination. This was the result that gave me a job as a “Junior Fellow” in the Department of Microbiology and a university scholarship to pursue a higher degree in microbiology any where in the world although I opted to study here so that I would be able to take proper care of my family. My husband continued to give me strong support until I finally got my Ph.D. degree. Soon after my B.Sc. degree, he took me on holidays to Harvard University Cambridge, Massachusetts, U.S.A. where he did his Post-doctoral Fellowship following an

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invitation from a Nobel Prize winner in Chemistry, the late Prof. R. B. Woodword. Incidentally, my husband was nominated twice in 1985 and 1994 by the Royal Swedish Academy of Science for consideration for the award of Nobel Prize in Chemistry. You can now see that behind this lecturer who gave you an inaugural lecture is a loving and caring great man who happens to be my husband. I will also want to thank my lovely children for being very submissive and cooperative which made it possible for me to combine my studies with home management. The eldest among them is Dr. Okechukwu Charles Okafor, a Consultant Pathologist at the University of Nigeria Teaching Hospital and a Senior Lecturer in the Collage of Medicine UNTH, Enugu. His brilliant academic career made him a model for his siblings. Others are Mrs. Nnenna Ofordu, an Assistant Manager with Zenith Bank PLC, Ikeja, Lagos, Mrs. Ifeoma Ezuma - Ngwu, a Senior Benefits Analyst with Metropolitan Transit Authority, Houston, Texas, U.S.A., Dr. Chidi Okafor, M.D. and a Resident Doctor, specialising in Internal Medicine at St Vincent Charity Hospital, Cleveland, Ohio, U.S.A., Mrs. Amaka Awanye, an Assistant Lecturer in Pharmacy at the University of Port Harcourt and Mummy’s handbag, Obioma Okafor who, by the grace of God, will be graduating with Doctor of Pharmacy degree (Pharm. D.) from the University of Houston, Houston, Texas, U.S.A. early next year, 2009. I also want to extend my gratitude to my wonderful sons-in-law, Mr. Cyprain Ofordu, a Chartered Accountant in a Federal Ministry at Abuja, Mr. Nnanna Ezuma - Ngwu, a Real Estate Consultant, Houston, Texas and Mr. Emeka Awanye, a Chartered Accountant with Elf Petroleum Company as well as my cute

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grandchildren: Masters Kenechukwu, Ugochukwu and Ikechukwu Ofordu, Master Chikezie, Miss Chisom and Amaka Ezuma - Ngwu, Miss Ogochukwu, Ogechukwu and Master Obichukwu Awanye who have brought so much joy to the family. I will also like to express my gratitude to my siblings, Arc. Chris Igboko, Engr. Godwin Igboko, Dr. (Mrs.) Ijeoma Iloeje, Prof.(Mrs) Nnenna Kanno, Mrs. Tessy Ekwunife, Mrs. Cecilia Asika, Barrister Gab. Igboko, Mr. Patrick Igboko, Barrister (Mrs.) Angela Emesih, Dr. Charles Igboko, Mrs. Nkechi Nwafor, Pharm. Ikechukwu Igboko and Miss Ujunwa Igboko, who are always available for their big “Sister Joe”. I cannot fail to thank my nice parents–in-law, Mr. Thomas Okafor of blessed memory and Mrs. Agnes Okafor who accepted me and treated me as one of their daughters ever since I got married to their son. Many thanks also go to my sweet sisters–in-law, late Justice Monica Edozie, Barrister (Mrs.) Betty Madike, Mrs. Ngozi Nwosu, Prof. Chinyere Grace Okafor as well as my brothers-in-law, Dr. Emeka Okafor, Dr. Tony Illo and many others. I cannot forget the amicable support I continue to enjoy from other close relations particularly from my wonderful brother-in-law, Prof. O. C. Iloeje , a Commissioner in the Electricity Regulatory Commission, Abuja, Engr. Val Emesih of Houston Electricity Corporation, Texas, U.S.A, Mr. Rich Ekwunife, Engr. Ogbonnia Nwafor as well as my

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lovely sisters-in-law, Mrs. Nnenna Igboko, Engr. Ngozi Igboko, Barrister Chinelo Igboko and Dr. Ifeoma Igboko. I also wish to express my sincere gratitude to my dedicated teachers who taught me at different levels of my academic career. I remember, in particular, some of my teachers at Queens School, Enugu, namely, Mrs. Kirk Patrick, my Principal and Mathematics teacher, Mrs. Iwobi, my Chemistry teacher and Miss Penny, my Biology teacher at the Higher School. I am ever grateful to my lecturers in the Department of Botany, especially to Prof. J.N.C. Maduewesi who made me to develop keen interest in Mycology, late Prof. Eni Njoku, our one time Vice-Chacellor and great scholar, late Prof. S. N. C. Okonkwo and Akunne O. C. Mozie as well as late Prof. A. N. U. Njoku Obi and Prof. Nduka Okafor for their inspiring lectures in Microbiology. My special thanks go to my supervisor for my M.Sc. and Ph.D. degrees, Prof. H. C. Gugnani who guided me effectively and who also made it possible for me to study in Nigeria and also to take care of my family especially my young children who could have derailed in my absence. I am also indebted to my other co-supervisors in the United States of America, Prof. Diane. TeStrake of the Department of Biology and Dr. B. G. Yangco of the Department of Medicine, Division of Infectious and Tropical diseases, College of Medicine both of University of South Florida, Tampa, Florida, U.S.A. I will also like to acknowledge the contributions of my research collaborators, Prof. I. C. Ononogbu, Prof. O. U. Njoku both of the Department of Biochemistry and Prof. Maria O. Nwosu of the Department of Botany as well as

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my postgraduate students especially Mr. E. I. Nweze and Mr. E. A. Eze who are most likely to obtain their Ph.D. degrees in Microbiology before the end of the year. I also owe my gratitude to the staff in my faculty particularly to Prof. Jasen Obeta, Prof. Chris Iroegbu, late Dr. A. C. Emeruwa and late Dr. Joe Oguike who were very helpful to me during the period I was the Head of the Department of Microbiology. I cannot end without thanking Dr. (Mrs.) Ifeoma Ezeonu, my former student and now a professional colleague, for being a good ambassador of the department. I am also very grateful for the cooperation I received from other colleagues: Prof. L. C. Eze and Dr. Eddy Alumanah both of the Department of Biochemistry, Prof. J. C. Ogbonna, my current Head of department, Drs. Jerry Ugwuanyi, Chukwudi Anyanwu and many others. I also wish to thank Dr. O. C. Okafor (my son) and Dr. E. R. Ezeome both of UNTH, Enugu who provided the clinical pictures of some patients with fungal diseases. To all of you I say thank you and thank you and thank you and may you all be blessed in Jesus name.

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5. LIST OF PUBLICATIONS

(i) Chapters in Books

1. Anyanwu, B.N. and Okafor, J.I. (1981). Man and Microorganisms. In: Nwankiti, O.C. (ed). Man and His Environment. Longman Group Ltd., Essex, England, pp. 38-42.

2. Okafor, J.I. (1981). Terrestrial and Cosmic Life. In: Nwankiti, O.C. (ed). Man and His Environment. Longman Group Ltd., Essex, England, pp. 81-83.

3. Okafor, J.I. (1999). Morphology of Bacteria. In:

Maduewesi, J.N.C. and Iheagwam, E.U. (eds). College Laboratory Manual of Tropical Biology. Ihem Daris Press 8 Ltd, Owerri, Nigeria, pp 12-16.

(ii) Refereed Journal Articles/ International

Conference Papers

4. Njoku-Obi, A.N.U., Okafor, J.I. and Gugnani, H.C. (1976). Yeast-like fungi recovered from normal human skin in Nsukka (Nigeria). Anotnie Van Leeuwenhock Journal of Microbiology and Serology. 42: 101-105.

5. Gugnani, H.C and Okafor, J.I. (1980). Mycotic flora

of the intestine and other internal organs of certain reptiles and amphibians with special reference to

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characterization of Basidiobolus isolates. Mykosen. 23: 260-268.

6. Okafor, J.I. (1981). Bacterial and Fungal pathogens from Intestinal tracts of cockroaches. Journal of Communicable Diseases. 13: 128-131.

7. Okafor, J.I. and Gugnani, H.C. (1981).

Dermatophyte and other keratinophilic fungi associated with hairs of rodents in Nigeria. Mykosen. 24: 616-620.

8. Okafor, J.I., Testrake, D.I. and Mushinsky, H. R.

(1982). Ecological studies on Basidiobolus spp. Presented at the Florida Academy of Sciences 22nd-24th April at Stetson University, Deland, Florida, U. S. A.

9. Okafor, J.I., Testrake, D.I. and Yangco, B.C. (1982).

Physiological studies on Basidiobolus isolated from South Florida and Nigeria. Presented at 33rd Annual Meeting of the American Institute of Biological Sciences (AIBS) 6th-12th August at Pennsylvania, U.S.A.

10. Ibekwe, A.O. and Okafor, J.I. (1983). Pathogenic

organisms in chronic suppurative otitis media in Enugu, Nigeria. Tropical and Geographical Medicine. 35: 389-391.

11. Okafor, J.I., Yangco, B.C. and Testrake, D.I. (1983).

In Vitro antifungal susceptibility studies of human and wild isolates of Basidiobolus sp. and

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Conidiobolus sp. Presented at the 83rd Annual Meeting of the American Society for Microbiology 6th-12th March at New Orleans, Louisiana. U.S.A.

12. Testrake, D.I., Beker, V., Yangco, B.C. and Okafor,

J.I. (1983). The effect of nutrition on morphology and pigment production of Basidiobolus sp. Presented at the 3rd International Mycological Congress 28th August-3rd September at Tokyo, Japan.

13. Okafor, J.I., Testrake, D.I., Mushinsky, H.R. and

Yangco, B.G. (1984). A Basidiobolus sp. and its association with reptiles and amphibians in Southern Florida. Sabouraudia. 22: 47-51.

14. Yangco, B.G., Testrake, D.I. and Okafor, J.I. (1984).

Phialophora richardisae isolated from infected Human Bone: morphological, physiological and antifungal susceptibility studies. Mycopathologia. 86: 103-111.

15. Yangco, B.G. Okafor, J.I. and Testrake, D.I. (1984).

In vitro susceptibility of human and wild isolates of Basidiobolus and Conidiobolus sp. Antimicrobial Agents and Chemotherapy. 25: 413-416.

16. Yangco, B.G., Nettlow, A., Okafor, J.I. Park, J. and

Testrake, D.I. (1986). Comparative Antigenic studies of species of Basidiobolus and other medically important fungi. Journal of Clinical Microbiology. 23: 679-682.

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17. Okafor, J.I. Gugnani, H.C. Testrake, D.I. and Yangco, B.G. (1987.) Extracellular Enzymes of Basidiobolus and Conidiobolus isolates on solid media. Mykosen: 30: 404-407.

18. Okafor, J.I. and Gugnani, H.C. (1992). Lipase

activity of Basidiobolus and Conidiobolus species. Mycoses. 33: 81-85.

19. Okafor, J.I. (1994). Purification and characterization

of protease enzymes of Basidiobolus and Conidiobolus species. Mycoses. 37: 265-269.

20. Okafor, J.I. and Okunji, P.O. (1994).

Cryptosporidosis in patients with diarrhoea in five hospitals in Nigeria. Journal of Communicable Diseases. 26: 75-81.

21. Nwosu, M.O. and Okafor J.I. (1995). Preliminary

studies of the antifungal activities of some medicinal plants against Basidiobolus and some other pathogenic fungi. Mycoses. 38: 191-195.

22. Okafor, J.I. and Okunji, P.O. (1996). Prevalence of

Cryptosporidium oocysts in feacal samples of some school children in Enugu State, Nigeria. Journal of Communicable Disease. 28: 49-55.

23. Okafor, J.I. and Uka, N.A. (1996). Extracellular

Enzyme Activities by Pityrosporum ovale on solid media. Journal of Science, Engineering and Technology. (Accepted for Publication).

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24. Okafor, J.I. and Okonowo, N.O. (1997). Some

pathogenic fungi involved in serious cases of urinary tract infections in Nigeria. Journal of Communicable Diseases. 29: 101-107.

25. Njoku, O.U., Onongbu, I.C. and Okafor, J.I. (1997).

Constituents of fixed oils of Piper guinense and Xylopia aethiopica. Journal of Natural Products and Medicine. 1:41-43.

26. Okafor, J.I. and Agbugbaeruleke, A.K. (1998).

Dermatophytosis among school children in Aba, Abia State, Nigeria and some physiological studies on the isolated etiologic agents. Journal of Communicable Diseases. 30: 44-49.

27. Njoku, O.U., Okeke, U., Ezugwu, C.O. and Okafor,

J.I. (1999). Preliminary Investigation on some Nutritional and Toxicological properties of Caesalpinia crista seed and oil. Bollenttino Chimico Farmaceutico. 138: 552-554.

28. Njoku, O.U., Ezugwu, C.O. and Okafor, J.I. (1999).

Investigation of some important nutritional and phytochemical properties of Averrhoa carambola fruit. Journal of Pharmaceutical Research and Development. 4: 109-112.

29. Okafor, J.I., Eze, E.A. and Njoku O.U. (2001).

Antifungal activities of the leaves of Baphia nitida, Cassia alata, Ficus exasperata and Gossypium

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arboreum. Nigerian Journal of Natural Products and Medicine. 5: 59–60.

30. Okafor, J.I. and Ngwogu A. (2002). Keratinolytic

activities of five Human isolates of the dermatophytes. Journal of Communicable Diseases. 32: 300-305.

31. Okafor, J.I., Eze, E.A. and Njoku, O.U. (2002).

Antibacterial activities of the extracts of leaves of Baphia nitida, Lodd, Cassia alata Linn, Ficus exasperata Vahl and Gossyupium arboreum Linn. Applied Natural Sciences Research. 4: 1-5.

32. Okafor, J.I., Nweze, E. and Njoku, O.U. (2002).

Antimicrobial activities of the methanolic extracts of Zapotica portericensis Benth and Cissus quadrangularis Linn. Nigerian Journal of Pure and Applied Science. (Accepted for publication).

33. Okafor, J.I. and Ngwogu, A. (2002). In vitro effects

of three metallic salts and carbon black (soot) on the growth of five of dermatophytes. Journal of Medical Investigation and Practice. (Accepted for publication).

34. Nweze, E.I., Okafor, J.I. and Njoku, O. (2003).

Antimicrobial activities of methanolic extracts of Trema guineensis (Shumm and Thorn) and Morinda lucida Benth used in Nigerian Herbal Medicinal Practice. Bio-Research. 2: 39-46.

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35. Nweze, E.I. and Okafor, J.I. (2005). Prevalence of dermatophytic fungal infections in children: A recent study in Anambra State, Nigeria. Mycopathologia. 160: 239-243.

36. Nweze, E.I., Okafor, J.I. and Njoku, O.U. (2005).

Antifungal activities of pair combinations of extracts from Morinda lucida Benth by decimal additive assay. Bio- Research. 3: 99-104.

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OTHER REFERENCES 1. Crook, W.C. (1986). The Yeast Connection. Vintage

Book. New York. 2. Dismukes, W.E. (1988). Cryptococcal meningitis in

patients with AIDS. J. Infect. Dis. 157: 624-628. 3. Anon. (1989). Mycotoxins, Economic and Health

Risks. Council for Agricultural Science and Technology. Report No. 116, pp. 91.

4. Woods, G.L. and Goldsmith, J.C. (1990). Aspergillus

infection of the central nervous system in patients with acquired immunodeficiency syndrome. Arch. Neurol. 47: 181-184.

5. Denning, D.W., Follansbee, S.E., Scolaro, M., Norris,

S, et. al. (1991). Pulmonary aspergillosis in acquired immunodeficiency syndrome. N. Engl. J. Med. 324: 654-662.

6. Saral, R. (1991). Candida and Aspergillus infections

in immunocompromised patients: an overview. Rev. Infect. Dis. 13: 487-492.

7. Pappas, P.G., Pottage, J.C., Powderly, W.G., Fraser,

V.J., et al (1992). Blastomycosis in patients with acquired immunodeficiency syndrome. Ann. Intern. Med. 116: 847-853.

8. Lecciones, J.A., Lee, J.W., Navarro, E.E., et al (1992). Vascular catheter- associated fungemia in

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patients with cancer: analysis of 155 episodes. Clin. Infect. Dis. 14: 875-883.

9. Fraser, V.J., Jones, M., Dunkel, J., Strofer, S.,

Medoff, G., Dunagan W.C. (1992). Candidemia in a tertiary hospital: epidemiology, risk factors and predictors of mortality. Clin. Infect. Dis. 15: 414- 421.

10. Crook, W.C. (1994). The Yeast Connection and

Women. Jackson T.N. Professional Books. 11. Girmenia, C., Gastaldi, R. and Martino, P. (1995).

Catheter-related cutaneous aspergillosis complicated by fungemia and fatal pulmonary infection in an HIV- positive patient with acute lymphocyte leukaemia . Eur. J. Clin. Microbial. Infect. Dis. 14: 524- 526.

12. Chandenier, J., Goma, D., Moyen, G., Samba-

Lefebvre, M.C., et al (1995). African histoplamosis due to Hisloplasma capsulatum var. duboisii: relationship with AIDS in recent Congolese cases. Sante 5: 227- 234.

13. Jones, J.L.,Fleming, P.L., Ciesielski, C.A., Hu, D.J.,

et al (1995). Coccidioidomycosis among persons with AIDS in the United States. J. Infect. Dis. 171: 961-966.

14. Dunkin, M.M., Connolly, P.A., Wheat, L.J. (1997).

Comparism of radioimmunoassay and enzyme-linked immunoassay methods for detection of Histoplasma

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capsulatum var. capsulatum antigen. J. Clin. Microbiol. 35: 2252-2255.

15. Verweij, P.E., Poulain, D., Obayashi, T., Ptterson,

T.F., Denning, D.W., Ponton, J. (1998). Current trends in the detection of antigenaemia, metabolites and cell wall markers for the diagnosis and therapeutic monitoring of fungal infections. Med. Mycol. 36: 146- 155.

16. Berry, C.L. (1998). The Pathology of Mycotoxins. J.

Path. 154: 301- 311. 17. Erer, B., Galimberti, M., Lucarelli, G., Giardini, C.,

Polchi, P., Baronciani, D., Gaziev, D., Angelucci, E., and Izzi, G. (2000). Trichosporon beigelii: a life-threatening pathogen in immunocompromised hosts. Bone Marrow Transplant. 25: 745-749.

18. Ebright, J.R., Fairfax, M.R. and Vazquez, J.A. (2001).

Trichosporon asahii, a non-Candida yeast that caused fatal septic shock in a patient without cancer or neutropenia. Clin. Infict. Dis. 33: 28-30.

19. Girmensia, C.M., Nucci, M. and Martino, P. (2001).

Clinical significance of Aspergillus fungemia in patients with haematological malignancies and invasive aspergillosis. Br. J. Haematol. 114: 93-98.

20. Raad, I., Hanna, H., Huaringa, A., Sumoza, D.,

Hachem, R., Albitar, M. (2002). Diagnosis of invasive pulmonary aspergillus using polymerase

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chain reaction-based (PCR) detection of Aspergillus in BAL. Chest. 121: 1171-1176.

21. Williams, J.H., Phillip, T.D., Jolly, P.E., Stiles, J.K.,

Jolly, C.M., Aggarwal, D. (2004). Human aflatoxicosis in developing countries; a review of toxicology, exposure, potential health consequences and interventions. Am. J. Clin. Nutr. 80: 1106-

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Figure 13: Professor Okafor working in Professor TeStrake’s Laboratory in the Department of Biology at the University of South Florida, Tampa, FL. U.S.A. Figure 14: Professor Okafor working under the hood in Dr, Yangco’s Lab in the College of Medicine, Univ. of South Florida, Tampa, FL. U.S.A.

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Figures 15: Professor Okafor working with a scanning electron microscope in the Department of Biology, University of South Florida, Tampa, Florida, U.S.A.

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Figure 16: Professor Okafor preparing her paper for presentation at the Florida Academy of Sciences at Stetson University, Deland, Florida, U.S.A. Figure 17: Professor Okafor at work in the Microbiology Laboratory, University of South Florida, Tampa, Florida, U.S.A.