Chapters
- Introduction
- Fungal Morphology
- Fungal Taxonomy
- Immunity to Fungal Diseases
- Diagnosis of Fungal Diseases
- Antifungal Therapy
Medical Mycology is a newly established discipline of medical sciences, which has attained immense importance during the terminal two decades of 20th century, particularly after the onset of AIDS pandemic. In the past, fungi were believed to be merely non-pathogenic, commensals or contaminants, with exception of a very few pathogenic ones. But now these are recognized as medically significant organisms causing potentially life-threatening diseases invariably with fatal outcome. Therefore, this medical speciality is gradually gaining more and more importance in the present context, especially among the immunocompromised and debilitated patients with one or the other underlying risk factors.
This Section covers some of the fundamental issues pertaining to fungi, which are generally applied to the field of Medical Mycology. Their morphological features are entirely different from other causative agents like viruses, bacteria and parasites because they exist as yeast, mycelial or even both morphological forms. The understanding of these features is also essential to have high index of suspicion thereby timely detection of fungi as the causative agent in clinical setting of a particular disease.
The nomenclature, classification and taxonomy of many medically significant fungi are settled as and when teleomorphic state of a fungus is discovered, however, many of these agents are still included in phylum Deuteromycota i.e. ‘fungi imperfecti’ as no teleomorphic state is yet discovered. Other agents looking similar to fungi are now regarded as pseudofungal organisms like Prototheca, Rhinosporidium and Pythium species. Taxonomically such organisms are either classified as protistan parasites or oomycetes. These taxonomical intricacies are pertinent to understand the basic disease process adopted by a causative fungal agent.
How fungi overcome defense mechanisms of human body among immunocompetent as well as immunocompromised hosts is separately dealt in one chapter of this Section. By the time fungal infection is recognized, patient is found to be already immunocompromised and remedial modalities become quite cumbersome thereby such infections invariably prove to be fatal. From prophylactic point of view, role of vaccination in Medical Mycology is still in its rudimentary stage. Hence general hygienic measures are recommended for prevention of fungal diseases.
A high index of suspicion is essential in any clinical setting for diagnosing fungal diseases, which is often lacking among medical specialists. The diagnostic techniques are utilized to prove or disprove the suspicion thereby therapeutic modalities are resorted, accordingly. Therefore, an appropriate diagnostic approach to fungal diseases is also described briefly in separate Chapter of this Section.
After establishing the final diagnosis, appropriate antifungals are to be instituted the therapeutic modality. However, some of the fungi, yeasts as well as mycelia, are developing resistance to the commonly used antifungals. It may be because of the indiscriminate use of such drugs merely on an empirical basis, without specific indication and/or appropriate antifungal susceptibility testing. The understanding of antifungals is of paramount importance thus commonly used antifungals have been described.
The fungi are achlorophyllous eukaryotic organisms, which multiply sexually and asexually by production of spores. Their somatic structure is composed of either yeast or filament bordered by cell wall, which is mainly composed of chitin. The fungi, unlike plants, cannot produce their own food thus are called heterotrophs. It was generally believed in the past that fungi were plants and classified under kingdom Plantae. However, now these are classified under independent biological kingdom i.e. Fungi since 1969.
The fungus is essentially a Latin word that means mushroom. The branch of biological science, which deals with study of fungi, is known as Mycology (Myco+logy=- Fungus+study, Gr. Mykes, mushroom/fungus, logos, discourse/study). The term is basically derived from mykes, Greek word, which is also used for mushroom. Hence Medical Mycology is the study of epidemiology, ecology, pathogenesis, diagnosis and therapeutic modalities of fungal diseases found prevalent among human beings. This discipline of Medical Microbiology is emerging rapidly and has now attained full-fledged and independent status in biomedical fields. The subject of mycology is essentially contemplation of medical sciences in newer perspectives.
Analogous to the field of Medical Mycology, Veterinary Mycology is study of fungal infections among animals, which often presents in similar way as those found in human beings. Sometimes, these are transmitted from animal-to-human beings and such infections are called zoonotic diseases. In recent reports cats are recognised as source of human sporotrichosis in Brazil establishing animal-to-man transmission. Hence reference of animal mycoses in the Textbook of Medical Mycology is essential to understand the epidemiology of a fungal disease.
The diseases of warm-blooded animals caused by fungi are known as mycoses (singular = mycosis). The prefix ‘myco’ has been given to few bacterial organisms like Mycobacterium and Mycoplasma or diseases like Mycosis fungoides i.e. cutaneous T-cell lymphoma, etc. that were speculated in the past to have similarities with either fungi or diseases caused by them. But later on it was realized that these organisms are not at all related to fungi. However, their nomenclature is still popular as misnomer because these are now well-accepted terms of the organisms and/or diseases.
Historical Perspective
The clinical manifestations of some of fungal infections are known since antiquity, however, systematic study of fungi is hardly one and half century old. In 1835, Agostino Bassi (1773-1856) in Italy established that fungus (Beauveria bassiana) was the cause of disease (muscardine) in silkworms (Bombyx mori), which could be transferred from one silkworm to the other. This fungus has shown to be widespread geographically and is currently used as a biological agent for the control of important insect pests in agriculture. Bassi is rightly acknowledged to be the first to refer the etiology of an animal disease to a microbial infection and is universally regarded as “Father of Mycology”. On the basis of these findings he predicted that fungi could also cause infections in man.
In 1842, David Gruby demonstrated for the first time that infection of scalp (favus) was caused by fungus through its inoculation to the healthy skin. Gruby thereby fulfilled what later on became famous as Koch's postulates, comprising the criteria necessary for the acceptance of a microbe as the cause of a specific infectious disease. Since then knowledge of fungal infections, which was fragmentary to begin with, has increased exponentially over the years as an independent infectious disease speciality as Medical Mycology.
The discipline of Medical Mycology attained substantial recognition in the field of bioscience in 1910 when 4French dermatologist and microbiologist, Raymond Jacques Adrien Sabouraud (1864-1938), published his seminal treatise, ‘Les Teignes’. This monumental work was a comprehensive account of most of the known dermatophytes, which is still being referred by the mycologists. He laid down solid foundation of the field of Medical Mycology, which gained remarkable momentum. As a result official journal of ISHAM was initially named as Sabouraudia in his honour in 1961. The medium for cultivation of fungi still bears his name, which is universally used for primary isolation of pathogenic and non-pathogenic fungi from clinical as well as non-clinical specimens in the mycology laboratory. It is suitable for the growth of both yeasts as well as mycelial fungi. Therefore, Raymond Sabouraud has rightly been called “Father of Medical Mycology”.
Similarly, P A Saccardo has played significant role in the establishment of field of Medical Mycology in earlier days. The other scientists who have played an active role in development of mycology are J I Schoenlein, Norman Conant, Chester Emmons, David Gruby, H P R Seeliger, J W Rippon, L Ajello, K J Kwon-Chung and Arvind A Padhye.
The introduction of fungi to man came into existence through the findings of Agostino Bassi in 1835 and the field of Medical Mycology has now completed more than 1½ century (mid-19th and 20th) as it has now entered into the 21st century. There have been lot of developments during this period, specially after advent of modern molecular techniques, which are dealing with diagnostic as well as taxonomical matters. But many issues are still lying unresolved before the mycologists, which include classification of pseudofungal organism and successful cultivation of Lacazia loboi and Pneumocystis jirovecii on artificial culture media. It is expected that molecular techniques will be able to resolve most of the intricacies of pending issues in the field of medical mycology in the times to come.
There are significant developments in treatment modalities of fungal infections and we have now achieved new prospects. However, till 1950s there was no specific antifungal agent available. Nystatin was discovered in 1951 and subsequently amphotericin B was introduced in 1957 and was sanctioned for use for human beings. In 1970s field was dominated by azole derivatives. Now, this is the most active field of interest, where potential drugs are being developed to treat fungal infections. By the end of 20th century, fungi have been reported to be developing drug resistance, among yeasts and mycelia in hospital as well as community acquired infections.
Milestones in Medical Mycology
The discovery of relationship of certain fungi to disease precedes work of even Louis Pasteur and Robert Koch. Some of the important milestones in Medical Mycology are given below:
1835 | Bassi described fungal etiology of ‘muscardine of silkworms’. |
1839 | Schoenlein studied fungal infection of scalp i.e. tinea capitis (favus). Remak, succeeded in growing the fungus on apples and reproduced the disease in animals and on his own forearm. |
1839 | Lagenbeck described yeast-like organism of thrush. |
1842 | Gruby independently isolated fungus on artificial media responsible for tinea capitis and produced disease by inoculation of healthy scalp. |
1890 | Sabouraud began publishing large numbers of articles on fungal disorders of skin, which eventually culminated in an enormous contribution to the field of Medical Mycology. |
1928 | Penicillin was discovered by Alexander Fleming as product from Penicillium notatum (P.chrysogenum). |
1934 | According to Botanical Nomenclature, species concepts of dermatophytes was redefined by Chester Emmons. |
1969 | Separate Kingdom - Fungi, was created by Whittaker, in the five-kingdom classification. |
1976 | Organism causing human PCP i.e. Pneumocystis carinii was proposed as P.jirovecii by J K Frenkel, in honour of Czech Parasitologist Otto Jirovec, who described this microbe in humans but he is not widely recognised. |
1999 | Pneumocystis carinii strain infecting human was re-designated as P.jirovecii. |
2002 | Loboa loboi was re-designated as Lacazia loboi and C.posadasii was established as new species of genus Coccidioides. |
2017 | Penicilliosis Marneffei was renamed as Talaromycosis and Emmonsiosis as Emergomycosis. |
The observations of both Bassi, in 1835 and Gruby in 1842, demonstrating that fungi could be cause of diseases, was long before Pasteur's Germ Theory came into existence fulfilling Koch's Postulates, about four decades before Koch actually formulated them in 1884.
The start of the modern age of mycology begins with Pier Antonio Micheli's (1679-1737) publication of Nova Plantarum Genera. Published in Florence, this seminal work laid the foundation of the systematic classification of 5grasses, mosses and fungi. He is considered as the founding ‘Father of Scientific Mycology”.
Invasive fungal infections are becoming an increasingly important cause of morbidity and mortality, particularly for immunocompromised human populations. The fungal pathogens belonging to genera Candida, Cryptococcus and Aspergillus collectively contribute to over 1 million human deaths annually.
Epidemiology
The fungi are widely found in environment and most of them are harmless commensals, contaminants or non-pathogenic agents. Some of the fungi are even useful to mankind in several ways. However, small number of these organisms are causing disease among men, animals and plants. This has been estimated by Hawksworth that only 5% of total fungal species in world have been identified which constitute about 70,000 species out of an estimated 1,500,000 but hardly 600 are usually recognized as primary pathogens of man and other mammals. Out of these fungal species, less than 100 are frequently encountered in routine clinical practice. It is estimated that about 1,500 new species of fungal genera are being described every year.
By 1995, approximately 70,000 fungal species have been accepted as compared to 5,000 viruses and 3,100 known bacteria. Yet remarkably, very few of these fungi cause disease in humans. Moreover, most of fungi exist as molds but there are number of pathogenic yeasts and some of them are dimorphic as well. The dimorphic fungi exist either as yeast or spherule in their parasitic form when causing infection to host and assume mold form when growing as saprotrophic in nature. Out of 1.5 million species of fungi estimated to exist all over the world and one-third of global fungal diversity found in India. According to Maheshwari there are 27,000 species of fungi, which have been recorded from India.
As fungal infections are not notifiable like viral, bacterial or parasitic diseases hence these are not given much attention and usually diagnosis is established very late when the patient is terminally sick. The approach to identify fungi in developing countries is on gross morphological features whereas in developed countries it is molecular based approach that is important due to availability of requisite infrastructure. Moreover, most of serious fungal infections are more common in developed countries due to underlying immunocompromised situations as compared to developing countries where majority of disease are due to low hygienic standards and various environmental factors.
The overall incidence and prevalence of mycotic infections is increasing, particularly during the last three decades. A major contributor to this emergence is growing number of immunocompromised and more susceptible individuals. Previously the epidemiological features of these diseases were not well-documented but the understanding of epidemiology of fungal infections has increased considerably in recent years, largely because of studies that have mainly focused on specific patient groups, hospital series and autopsy surveys.
Candida species is the fourth most common organism recovered from blood cultures in hospitalized patients. Aspergillosis is common in selected populations, such as bone marrow transplant recipients and lung is the most common clinical site. Aspergillus species is isolated in substantial number of solid organ and bone marrow transplant recipients with clinical findings of pneumonia. In such patients, high mortality rates are observed in relation to Aspergillus species.
The diseases, which were not prevalent in a particular area, are now being reported very commonly due to frequent travel across the globe. In the times to come, diseases like paracoccidioidomycosis and coccidioidomycosis which are currently found in restricted zones of endemicity in the New World, may be reported from other parts of world. Some of fungal infections are only limited to African continent like Histoplasma capsulatum var. duboisii. Blastomycosis is also found in Africa in addition to its native prevalence in the southeastern parts of North America.
The liberalization and globalization of economy has increased the gap between rich and poor as well as urban and rural population. Consequently privatization of health services has targeted poorest of poor due to migration of laborers thereby affecting epidemiology of fungal diseases.
The fungi are now recognized as significant cause of morbidity and mortality among man and animal. They have emerged as important etiological agents of opportunistic infections as well as full-fledged diseases as true pathogens. The invasive fungal infections were regarded as very rare till half century ago. Since that time there has been steady increase in number of patients suffering from life-threatening fungal infections, specially in prevailing immunocompromised circumstances. In recent 6years opportunistic fungal infections have emerged as one of interesting areas due to number of contributory factors. This calls for increased awareness about fungal diseases and their definitive diagnosis. Care must be taken to exclude such type of infection in cases where fungus is grown in culture but not seen in lesion by smear preparations and histopathological examination of tissue sections.
Like other organisms, Koch's postulates are applicable to fungal infections as well despite the fact that some of agents like Lacazia loboi are yet to be successfully grown on artificial culture media. The present challenges in Medical Mycology are diagnosis, treatment, taxonomy, mycology training and changes in diseases patterns. However, new and more effective tumour chemotherapeutics, methods of surgery and irradiation, complex treatment procedures such as autologous and allogenic bone marrow and blood stem cell therapy as well as organ transplantation, have enabled treatment of various solid tumors and systemic hematologic diseases. The use of such chemotherapeutics and immunosuppressants as therapeutic modalities result in general increase in opportunistic bacterial, viral parasitic as well as fungal infections. The epidemiological features of important fungi are dealt with subsequent chapters of this Textbook.
As such, the arthropods are not acting as biological vectors of the fungal diseases however; sometimes these may act as mechanical carriers of fungi. In this regard, cockroaches have been reported to the source of transmission of fungi in hospital set-up.
As per the statement of G C Ainsworth in 1966, that in reality it is the distribution of the medical mycologists or active investigators in this field and not the true distribution of fungal disease, which is reflected as its epidemiology.
Ecology of Fungi
The fungi are capable of existing and flourishing in a wide variety of environment as parasites, saprotrophic or symbionts. In the past they were known to be cave dwellers but presently they are found existing on innumerable places. They may be parasites of organic substrate, such as wood and other decaying plant parts, paper, leather, cloth, keratinous and chitinous substrate, oils and fats, resins and even petroleum and draw their nutrition.
The definite ecological groups of fungi known to exist as soil fungi, aquatic fungi, coprophilous fungi (grow on animal excreta), entomogenous fungi (parasites of insects) predacious fungi (capture small animals and protozoa), marine fungi, osmophilic fungi (from saline soil) and thermophilic fungi in which organism show remarkable adaptations not only to exploit nutritionally rich substrate but also to reproduce themselves. As decomposers, fungi are essential because along with bacteria, they recycle vital elements, such as nitrogen and phosphorus, back to the ecosystem.
Although beneficial effects of fungi far outweigh their harmful aspects but there are some disease-causing and destructive species, which are clinically significant. The number of saprotrophic fungal species far outnumbers those that are parasitic and impact on the health of man, animal and plants. This is well-known that nearly one hundred saprotrophic fungi, which may have their existence in any of common habitats, mentioned above can adapt to infect man and animals and cause diseases. It is also essential to acquire sound knowledge of biology of these fungi and their role in ecological niche. The limited data is available about survival of fungi that commonly cause health-care associated infections in immunocompromised patients on typical hospital materials. This indicates that many of fungi like Candida, Aspergillus and Fusarium species, which are responsible for health-care associated infections, survive for at least one day and often longer on fabrics and plastics routinely used in hospitals. These survival results indicate fabrics and plastics to serve as potential reservoirs or vectors for fungi.
There have been three ecological categories of infectious mycological agents based on their natural habitat. Therefore, traditionally fungi have been described as geophilic, zoophilic and anthropophilic. Now, a fourth category has also come into existence that is called hydrophilic, which encompasses free living organisms that live in aquatic habitats on non-living organic matters or on aquatic plants. The diseases, caused by such fungal (lacaziosis) and pseudofungal organisms (rhinosporidiosis and pythiosis) are currently categorized as hydrophilic infections.
Magnitude of the Problem
Mycology is a subject matter of immense interest to botanists as most of fungi are plant pathogens. The veterinarians also take lot of interest as fungi are significantly affecting health of domestic animals as well. Now, those fungi, which were supposed to be non-pathogenic, are reported significantly infective agents. Hence microbiologists and histopathologists are taking more interest in this branch 7to render diagnostic services in hospitals. The field of Medical Mycology that was virtually limited to case reports in the 19th century had attained status of full-fledged subject, as an important branch of medical science by the turn of the 20th century. It is estimated that during the 21st century, magnitude of problem is going to be increased because of the immunocompromised nature of hosts entailing in secondary as well as primary fungal infections.
Another way to estimate that this branch has expanded enormously in recent past, is by increased number of publications on issues related to Medical Mycology. Now, not only case reports but detailed prospective and retrospective studies are being published regularly in national and international journals, more in number and quality to previous times and as such subject of Medical Mycology has entirely changed over the years. Therefore, the field of Medical Mycology has evolved to the present independent status as significant branch of medical sciences.
Although fungi were recognized as disease causing agents much earlier but their significance was overshadowed by bacteriology and even virology. The attitude towards Medical Mycology was ‘step-child’ of our doctrine and research. It was least bothered subject in medical institutions as compared to other branches of medical sciences. But now there is ‘obligatory’ attention to this subject because clinicians face challenges due to underlying immunocompromised situations leading to life-threatening secondary fungal infections taking lives of innumerable patients. The fungal infections had such an impact that now-a-days no health personnel can ignore mycology, no matter whatsoever may be their speciality.
In the past, Mycology Sections of medical institutions, used to get only few skin scrapings from the Department of Dermatology and Venereology. In due course of time this field has evolved to an extent that Mycology Sections are now receiving clinical specimens from almost all specialties whether medical or surgical. Moreover, their number and quality has enormously increased in recent past due to greater awareness about fungi as disease causing agents and microbiology laboratories are now receiving more number of clinical specimens for mycological investigations. Therefore, due to the circumstantial compulsions, the microbiologists and pathologists have left their myopic attitude towards the fungal infections.
As fungal diseases are not notifiable diseases hence data on morbidity and mortality attributable to these diseases undoubtedly is understated. But incidence and prevalence of fungal infections has increased enormously. It may be due to underlying predisposing factors such as immunocompromised situations like use of corticosteroids, antimicrobials, immunosuppressive and anticancer drugs, bone marrow or solid organ transplants, HIV-positivity, metabolic disorders i.e. diabetes mellitus, etc as narrated in Table 1.1. Out of all these factors two are most important and prominent namely therapy among cancer patients and pandemic of the AIDS. Moreover, elderly patients, whose life span has been extended by treatment of cancer or other debilitating diseases, are more susceptible to secondary fungal infections than younger individuals. In addition to that there are changing parameters of mycoses like host population, emerging drug resistance and reporting of newer fungal pathogens.
The rapid growth of fungi such as Candida albicans in debilitated patient with non-mycotic disease may be minor factor as cause of death or in contrast the other fungal infections. Similarly, lack of experience in diagnostic techniques, inadequate laboratory infrastructure and lack of pathological autopsy examination, may limit recognition and reporting of many deaths due to fungal infections. These days, it is feasible to detect about 30% of diagnosis of fungal infections are made antemortem and rest 70% as postmortem. This paradigm is going to be shifted depending on the awareness among the medical personnel.
The basic need of hour is to create awareness among clinicians so that they are motivated to an extent as to send samples to Mycology Section of Microbiology Department. In most of advanced medical institutions, it is estimated that about five percent autopsied patients turn out to have 8died of fungal infections. At present in most of the teaching hospitals every third clinicopathological conference (CPC) is reporting deep-seated fungal infection as secondary invader responsible for terminal events of deceased patients. The autopsy findings also substantiate an increasing prevalence of invasive fungal infections among hospitalized patients along with significant changes in aetiology and underlying disease process. Therefore, pathological autopsy is an essential feedback for clinicians in particular setting of severely immunocompromised population and emphasizes constant and urgent need for more efforts towards prevention, diagnosis and treatment of fungal infections. The autopsy besides being diagnostic method, is also a means of medical quality control because it acts as deterrent on the diagnostic procedures.
The prognosis among patients of fungal infections as such is very poor. Therefore application of early diagnostic and therapeutic modalities is essential. A fruitful outcome depends on a very close association between Medical Mycology laboratory and clinician incharge of concerned specialty. It has also been observed that increased awareness among medical personnel has contributed to an early diagnosis and commencement of prompt and timely treatment in life-threatening fungal infections. Despite all the available modalities, Medical Mycology is still labeled as tip of the iceberg as little is known about it to the medical fraternity.
The radiological investigations, like ultrasonography, computed tomography, magnetic resonance imaging, have contributed significantly, in addition to routine x-rays examinations. Now, there are effective chemotherapeutic agents available and further modalities are being developed for treatment of fungal infections, therefore, their application requires proper techniques. The opportunistic fungal infections vary greatly in their clinical and pathological manifestations due to underlying illness of patients at risk as well as treatment modalities undertaken.
The newly isolated fungi may be morphologically atypical because of partial treatment given to patient. Therefore, if fungus does not fit into any description provided, serial transfers on Sabouraud dextrose agar may be necessary to restore typical morphology. When fungal growth is observed from clinical specimen, three principal characteristics to be evaluated are texture, growth rate and pigmentation. Therefore, using these criteria, conventional fungal identity can be narrowed down quickly to a limited group of fungi having similar characteristics.
Burden of Fungal Diseases
In a large number of countries two organizations i.e. Leading International Fungal Education (LIFE) and Global Action Fund for Fungal Infections (GAFFI) have conducted extensive campaign to document the burden of fungal diseases in respect to that country. This data has been published in various medical journals and cited in Further Reading of this Chapter as well.
Fungal Taxonomy
During the 20th Century, until around 1950s, botanists used the term fungi to include all members of ‘plant kingdom’ that did not have stems, roots, leaves and chlorophyll. By this definition, even some bacteria were included with fungal category. As fungi were initially classified with plants hence lot of botanical influence is seen on this subject. This may probably be the reason that Medical Mycology has been mainly subject of interest to the botanists. These organisms were transferred to an independent kingdom, after five-kingdom classification by Whittaker in 1969, based on their cell morphology that subsequently substantiated by molecular studies. Now, after recent increase in prevalence of fungal infections, especially in immunocompromised patients, it has become subject matter of great interest to microbiologists, pathologists, internists and the allied medical specialists, who regularly encounter such type of infections.
The Medical Mycologists encounter many new fungal genera and species that are known to have clinical relevance and every year many species are being added in the list of significant fungal pathogens. Many fungi, formerly considered as insignificant commensals or merely laboratory contaminants, have now taken new roles as agents of infectious diseases in patients with AIDS and other immunodeficiency conditions, in drug abusers and in debilitated individuals with various underlying diseases. The subject has reached a level of understanding where any fungus recovered as pure culture from any of the body sites must be considered as potential human pathogen.
In recent times, the human genome has been fully described and this knowledge, based on molecular techniques, is applicable to Medical Mycology as well so that diagnostic dilemma and taxonomic confusions are now feasible to be resolved. In this very sequence a long-standing taxonomical controversy of Pneumocystis jirovecii has been substantially resolved. Microsporidium species had a similar fate. These atypical fungi are shifted from kingdom 9Protista to Fungi; thereby Parasitology to Mycology; hence being described in the Textbook of Medical Mycology.
The pseudofungal organisms like Pythium and Prototheca species are mycelial or unicellular microorganisms that are not classified in the kingdom Fungi and that produce infections with clinical and histopathological features resembling those caused by eumycetes. Taxonomically, on the basis of molecular studies, Rhinosporidium seeberi is also now considered as a protist, classified in Mesomycetozoea, recognized as DRIPs clade of protistan parasites.
The Medical Mycology is totally new and unfamiliar subject to medical personnel and presents difficulties, which are apparent while identifying newly isolated fungal agent. Even though procedures for direct examination and isolation have been mastered, mycologist is often left to confront fungal culture that may be totally unfamiliar both macroscopically and microscopically. This happens most frequently when person is new to this field or knows least what to do. In such a situation, even if the species identification is difficult, at least the isolate should be identified to the genus level and immediately conveyed to the clinician so that timely and definitive treatment is instituted then and there. In due course of time, further taxonomical studies can be performed for final identification of the clinical fungal strain.
Endemic Mycoses
The endemicity of some of the fungal diseases like histoplasmosis, blastomycosis, coccidioidomycosis, paracoccidioidomycosis as well as talaromycosis are restricted to limited geographical areas. This may be due to environmental and other factors, which favor growth of fungi in soil of these areas. Histoplasmosis and blastomycosis mostly afflict people in Mississippi and Ohio River Valleys, while coccidioidomycosis is found primarily in southwest desert regions of USA and paracoccidioidomycosis in certain parts of Latin America. However, talaromycosis is found prevalent in entirely different geographical zone away from Americas i.e. Southeast Asia. All these diseases are acquired through inhalation of air contaminated with conidia of mycelial phase and cause localized to diffuse pulmonary involvement entailing dissemination to distant body sites. Their clinical features are different but all of these fungi are dimorphic in nature.
There are now case reports of endemic mycoses from non-endemic areas also. In case of coccidioidomycosis, new area in northeast Brazil has been recognized. In this very context talaromycosis has been reported from Africa without patient's history of travel to its endemic zone. In India, it has been now significantly reported from Manipur, Mizoram, Nagaland and upper Assam. It may be because of its higher prevalence in adjoining countries like Myanmar and northern Thailand.
Imported Mycoses
Those fungal infections which are caused by endemically prevalent fungi, encountered in non-native locations are called imported mycoses. In this era of modern technology, the entire world has become a ‘global village’ and borders of diseases are being demolished day-by-day. Now, due to frequent traveling all over the world in this era of jet age and underlying immunocompromised situations, zones of endemicity are not limited to their original native areas of prevalence. Such types of endemic fungal infections are also on the rise.
Emerging and Re-emerging Fungal Diseases
In the past fungi were considered to be merely non-pathogenic or simply laboratory contaminants with very few exceptions with cutaneous manifestations. But due to circumstantial immunocompromised background among the patients, these very non-pathogenic and contaminants have now proved to be significant pathogens and are encountered as emerging agents of life-threatening fungal diseases. In addition, some of the clinical entities which were not of great magnitude have recently re-emerged as important diseases. Most of the dimorphic fungi have re-emerged after the advent of AIDS and other immunocompromised situations.
In Medical Mycology as such there is an emergence and re-emergence of different types of fungal diseases of paramount significance, which are caused by Saccharomyces cerevisiae, Candida auris, Scedosporium apiospermum, Talaromyces marneffei and Fusarium species, which have recently been reported in literature. Outbreaks due to fungal infections do occur but only in sporadic form and epidemics have not been reported. For example, incidence of cryptococcal meningitis has recently increased to 1000-fold in the New York City alone.
In addition, during late September 2012, a multistate outbreak of CNS fungal infection and septic arthritis was caused due to an emerging fungus, Exserohilum rostratum in the USA, which is otherwise a plant pathogen. There 10were over 700 patients with 64 deaths, who received epidural injections of methylprednisolone produced at a Massachusetts compounding center and subsequently developed meningitis with or without posterior circulation stroke and/or spinal or para-spinal infection and more than 30 patients who received intra-articular injections of the same drug developed osteoarticular infections.
Some of the fungi, which have been reported as merely environmental contaminants, have now emerged as significant human pathogens. Some of mucormycetes like Apophysomyces variabilis, Saksenaea erythrospora and Rhizopus homothallicus have now emerged as significant cause of various forms of mucormycosis and if not timely treated may lead to fatal consequences even in immunocompetent individuals.
Medical Mycology and AIDS
Mycology as such was an unknown field of medical sciences except in some of clinical specialties like Dermatology and allied subjects dealing predominantly with dermatophytosis and candidiasis. The real augment came three and half decades ago when new virus was brought to the human civilization probably a spillover of biological warfare laboratories, which created havoc by abrogating immune system of its targeted victims. Consequently the index case was recognized in the USA in 1981. The novel agent was subsequently designated as Human Immunodeficiency Virus (HIV) in 1986, the labeled cause of Acquired Immunodeficiency Syndrome (AIDS).
This type of clinical setting gave an open invitation to all the secondary invaders of viral, bacterial, parasitic and even fungal origin. The patients virtually became microbiological culture plates and all these organisms thrived upon them. The fungi were already well-known agents invading individuals with weaker immune system, made their abode among AIDS patients thereby leading to fatal consequences. This duo of HIV and fungi attained medical acceptability to an extent that some of the diseases like pneumocystosis, cryptococcosis, talaromycosis, etc. became AIDS-defining illnesses. Therefore, the fungal disease/parameters started defining the viral infections for the first time in the history of medical sciences.
Now, every year fungal pathogens cause more than two million infections leading to high morbidity as well as mortality. Out of this two fungal genera i.e. Candida and Cryptococcus are together accounting for about 1.4 million infections with higher mortality rates. The patients with disturbed immune system like HIV/AIDS, are mainly vulnerable to such infections. Although there is substantial control on this disease due to introduction of ART but fungal infections are still on the higher rates.
Hence advent of AIDS has altered the incidence as well as prevalence of fungal infections thereby the course of action of the field of medical mycology. During this period since 1981, epidemiological scenario about fungal infections has entirely changed. The fungal isolates, which used to be discarded considering as merely laboratory contaminants are now found to be playing significant role in pathogenicity of the variety of infections. This is true with fungi affecting not only immunocompromised patients but about those capable of infecting even healthy individuals as well. Therefore, the impact of syndrome is such that even true fungal pathogen like Histoplasma, Blastomyces, Coccidioides and Paracoccidioides species, infect immunocompetent individuals as well as now immunocompromised patients also and that too in a very large number.
This was probably warfare experiment to begin with but in due course of time it has attained the level of a full-fledged pandemic as it is now reality and not fiction-based story of a syndrome of ‘sealed’ origin. In the times to come, fungal infections will be encountered more in number, killing thousands of patients with underlying abrogated immune system.
Biological Warfare and Fungi
During the events of nine-eleven (September 11, 2001), so-called greatest imperialist power of the world was 'shaken' to the hilt. This was followed by incidents of spreading white powdery material pertaining to Bacillus anthracis. All these developments led to apprehension that fungi also might be used on similar pattern. Most of the governments secretly resort on to biological warfare acts for their survival in power through state organized terrorism thereby giving rise to origin of counterproductive terrorist groups, who may retaliate by indulging in such activities.
As such till date no fungal agent has been apparently used in biological warfare but reports are available about their secondary metabolic products. In the past, Yellow Rain in Laos is one of examples, where mycotoxins (Trichothecene) were used as agent of biological warfare. Similarly, Agent Orange, a herbicide and defoliant was also used during Vietnam War. The degradation of 11such material release dioxins, which cause serious health related maladies.
The danger of working with cultures of some of fungi like Coccidioides species, were even recognized as early as in 20th century. Now, in the changed international scenario, there is an apprehension that these fungi may be abused as biological warfare agents. Therefore, their interstate transport is restricted under provisions of US Anti-terrorism and Effective Death Penalty Act, 1996. These highly infectious agents require safe laboratory handling under Biosafety Level 3 facility.
Classification of Fungal Diseases
Most of the fungi are saprotrophic in nature as they use non-living organic material as source of their nutrition. They are significant scavengers in the ecosystems. Along with bacteria, they are important in recycling carbon, nitrogen and essential mineral nutrients. As far as their interaction with living world is concerned, they have various modes of existence like other microorganisms. The host-parasite relationship in fungi can be divided into three potential modes namely mutualism, commensalism and parasitism depending upon the basic interaction between fungus involved and the host. These are as follows:
(a) Commensalism: The fungus neither gets benefit nor harmed in the host-parasite relationship. There is usually no physiological interaction between two organisms. One organism uses other to get a better position in the environment.
(b) Mutualism: The fungus and host take mutual benefits from host-parasite relationship. For example in Mycorrhizae there is association of fungi with plant roots and in Lichens there is association with algae and cyanobacteria.
(c) Parasitism: The fungus gets benefits and host is harmed by host-parasite relationship. Most of the fungi causing disease in humans, animals and plants are placed in this category.
The basic mechanism of fungal pathogenicity is its ability to adapt to tissue environment, physiological barriers and temperature (37°C) thereby withstand lytic activity of hosts’ cellular defense mechanism. The same fungal agent can produce different manifestations depending upon underlying situations of the host. Based on their wide-spectrum of adaptability, various fungi causing human mycoses can be categorized into following groups:
(a) Pathogenic Fungi: These fungi have ability to adapt to the tissue environment, which is quite marked. This is expressed as thermal dimorphism and fungi are able to cause infection even in the immunocompetent individuals.
(b) Opportunist Fungi: These fungi cause infections only in patients with immunodeficiency or other debilitating conditions, which carry high morbidity and mortality. Some sort of defect in immune system of the host is prerequisite for establishment of such fungal infection and these do not exhibit thermal dimorphism.
(c) Toxigenic Fungi: These fungal species cause illness or even death of patients and/or animals after ingestion of contaminated food by fungi (mycetismus) or their mycotoxin metabolites (mycotoxicosis).
(d) Allergenic Fungi: These fungal species act as allergens and cause various allergic manifestations in human beings.
The disease process is established on the basis of net outcome of host-parasite relationship. In addition, the fungal diseases in man can be classified according to the anatomical site of primary involvement.
(a) Superficial Mycoses: The fungal infection is limited to outermost layers of skin and its appendages. The immune response in such infections is rarely induced or it is very mild.
(b) Cutaneous Mycoses: In this type the infection extends deeper into epidermis and it also invades hair and nails. The disease process may evoke high inflammatory response in the host.
(c) Subcutaneous Mycoses: The infection is due to organisms of low pathogenicity usually following traumatic injury. It involves dermis, subcutaneous tissues and sometimes muscles and fasciae. The causal agents of subcutaneous mycoses require mechanical introduction of organisms into tissues through traumatic implantation.
(d) Systemic Mycoses: The infection primarily involve a site like lungs and later on disseminates systemically to distant body sites. Systemic mycoses along with the opportunistic fungal infections, is called deep mycoses.
(e) Opportunistic Mycoses: Besides the above-mentioned four categories of fungal infections, fifth group has come into prominent focus because of increasing use of immunosuppressive therapy and pandemic of AIDS and is called opportunistic group. The infectious agents normally are of 12very low pathogenic potential, which produce full-fledged disease only under changed circumstances, mostly involving host debilitation.
The fungal infections are not transmitted sexually as commonly seen in viral, bacterial and even protozoal diseases. However, balanoposthitis caused by Candida species is supposed to be transmitted by the sexual contact. Moreover, piedra is also taken into this category as higher rates are reported among homosexuals in Denmark. In some of the fungal infections like histoplasmosis and mucormycosis, genital lesions have definitely been reported but their mode of infections is not considered to be through sexual route.
Significance of Fungi
The fungi have recently gained more importance due to increased incidence of various fungal diseases. Normally, most of these organisms are found as soil saprotrophic but whenever there is some underlying cause, the very same organism becomes significantly pathogenic. In addition to pathogenic potential, significance of fungi lies in the economy of nature is described below:
(a) Useful Properties of Fungi
The fungi are pathogenic to man, animals and plants causing various types of diseases. On the other hand it is also known that most of the fungi are beneficial to mankind. Their ability to break down complex organic substrate of almost every type thus contributing to recycling of carbon and other elements making fungi as an important component of our terrestrial ecosystem. The industrial uses of fungi as food, fermenter in food industry, producers of antibiotics, drugs, etc. are innumerable and may surpass damages, which these agents do as pathogens of man and animals. However, to date penicillin, lovastatin, cyclosporin, griseofulvin, cephalosporin and ergometrine are the most important pharmaceutical products that have been extracted from different fungal species.
The importance of fungi has been more emphasized towards beneficial effects, which are given below:
- The edible fungi are widely used in industry as source of food, particularly wild or domesticated mushrooms of all shapes, sizes and colors, which belong to basidiomycetes. They are also put in salads, pizza and burgers.
- The fungi are used to alter texture, improve flavor and increase palatability and digestibility of natural and processed foods. The yeast, Saccharomyces cerevisiae, is used in leavening of bread and other baked products. Hence it is known as Baker's yeast. The blue mold, Penicillium species, is used in ripening process to prepare speciality cheeses such as blue cheese.
- Some of yeasts, Candida fukuyamaensis, are used to prepare special type of tea, known as Russian or Manchurian tea, which is presumed to be having some therapeutic potential in certain medical ailments. Hence it is consumed by many people under this impression. Candida fukuyamaensis is believed to be asexual state of Pichia guilliermondii.
- At industrial level fungi are used to produce alcohol (Saccharomyces species), fat (Endomyces species) and proteins (Torulopsis species) through fermentation. Saccharomyces cerevisiae, also called Brewer's yeast, is used in the fermentation processes that result in production of beers, wines and spirits. All citric acid used in soft drinks, candies, artificial lemon juice, baked goods, etc. are produced industrially by fermentation by certain fungi like Aspergillus niger.
- The fungi are now therapeutically being used as probiotics along with bacterial species like Lactobacillus and Bifidobacterium. In peculiar circumstances when the bacterial probiotic agents do not withstand onslaught of antimicrobials in antibiotic-associated diarrhoea, yeasts such as Saccharomyces boulardii can bear this assault thereby prove to be very effective in ameliorating such common alimentary ailments, while acting as probiotics. Hence S.boulardii is shown to be useful in the treatment (biotherapeutic agent) of acute infectious diarrhoea, the prevention or treatment of diarrhoea associated with antibiotic use and as an adjunctive therapy for Helicobacter pylori infection.
- The discovery of popular antibiotic penicillin from Penicillium notatum (now called P.chrysogenum) by Alexander Fleming in 1928 was real breakthrough in history of medical sciences. In 1941, it revolutionized the basic approach in treating infectious diseases. P.chrysogenum is mold that is widely distributed in nature and is often found living on foods and indoor environments. Many fungi like P. chrysogenum are used as source of several β-lactam antibiotics, most significantly penicillin. Another fungus, Acremonium chrysogenum (also called Cephalosporium acremonium) is used in manufacturing cephalosporin. Cephalosporium chrysogenum was isolated for the first time by Thirumalachar and Sukapure from Pimpri, Pune, India.
- The antifungal agent produced from fungi (Penicillium griseofulvin) is griseofulvin, is narrow-spectrum fungistatic antibiotic used for treating dermatophytes.
- Fumagillin is produced from Aspergillus fumigatus and used in the treatment of microsporidiosis.
- In addition to β-lactam antibiotics (penicillins and cephalosporins), fungi are used to produce immunosuppressant like cyclosporin A, which is a primary metabolite of several fungi, including Trichoderma polysporum and Cylindrocarpon lucidum.
- The statin family of cholesterol-lowering drugs are derived from fungi. Simvastatin is prepared synthetically from fermentation product of Aspergillus terreus.
- The ergot produced by Claviceps purpurea, a fungal pathogen of rye, is used for inducing uterine contractions, controlling bleeding and alleviating localized vascular disorders e.g. migraine. The ergot alkaloids belong to two groups: (a) amine alkaloid - ergometrine and (b) aminoacid alkaloids - ergotamine. Ergometrine or its semisynthetic analogue methylergometrine is clinically used for oxytocic effects. As ergotamine has α-adrenergic blocking and vasoconstriction properties, it is clinically used in treating migraine.
- The fungi are now playing a vital role for producing natural products, most productive source of lead compounds in far reaching endeavor of new drug discovery. Epicoc-cum is known for its potential to produce diverse classes of biologically active secondary metabolites. Most of these fungal metabolites have cytotoxic, anticancer, antimicrobial and anti-diabetic activities.
- A few fungal species (Saccharomyces cerevisiae, Neurospora crassa and Ustilago maydis) are studied as model organisms. Such studies are useful to acquire more knowledge of the basic processes involved in biochemistry, physiology, genetics and molecular biology and these results are applicable to many organisms. The dung fungus, Sordaria fimicola has recently been exploited in genetics studies.
- The yeasts like Saccharomyces cerevisiae, Hansenula polymorpha and Pichia species have been used as heterogeneous host for preparation of recombinant vaccines for hepatitis B virus infection.
- Fungi like Culicinomyces clavosporus have been used as traps and pathogen to control mosquitoes in malaria eradication. The entomopathogenic genus, Coelomomyces is also known to be pathogenic to larva and develop within larval hemocoel of mosquitoes. The Coelomomyces species are obligate parasitic fungi that alternate their saprotrophic and gametophytic stages between mosquitoes and copepods, respectively.
- The pesticides of fungal origin have also been found to decrease malaria transmission in animal studies. After feeding on an infectious blood meal, mosquitoes have been exposed to surfaces coated with fungal biopesticides or entomopathogens, which have dramatically reduced their ability to transmit malaria.
- Some of biotechnological applications of fungi especially in the field of food, agriculture, etc. are very significant. The biological measures have been applied in agriculture in Havana where some of fungi are used e.g. Beauveria bassiana, to control banana root borer, sweet potato weevil, rice water weevil and sugarcane borer. Verticillium lecanii specifically is bred to control sweet potato white fly, one of the most damaging pests in Cuba. Trichoderma species is used to control soil-borne diseases that attack tobacco, tomatoes and pepper.
- Recently, one of the fungi (Gliocladium roseum), found prevalent in Ulmo tree in rain forests of Patagonia (South America), is tried to make biofuel. This digests the cellulose turning it into biofuel i.e. myco-diesel.
As an overall point of view, fungi have been used to cause man more satisfaction rather than trouble. They raised his bread, his wine and beer, carbonate his champagne, flavored his cheeses, furnished him edible mushrooms, produced drugs to stop bleeding, to reduce cholesterol, worked diligently to form antibiotics to help him fight bacterial as well as fungal infections and helped industry to produce many organics acids and other compounds. However, to a general mycologist they are even beautiful to behold and their unending variation is very fascinating, whereas to a medical mycologist they are now the dangerous and life-threatening organisms.
(b) Harmful Properties of Fungi
In addition to the useful properties, fungi are also important agents producing harmful effects, which are given below:
- The pathogenic fungi are responsible for superficial, subcutaneous, systemic and opportunistic infections in man and animals.
- Mycotoxicosis and mycetismus are becoming serious public health issues in most of the developing tropical and sub-tropical countries.
- The fungi can spoil improperly stored agricultural produce like grain, foodstuff, vegetables and fruits. These are commonly seen in eatables as ‘bread molds’. The fungal pathogens are responsible for damages amounting about 10% of world's crops. It is estimated that as agents of plant disease, fungi cause annual crop losses of billions of dollars.
- 14The fungi, especially molds, can cause decay of fabrics, timber, leather, electrical insulation and other synthetic materials. There may be extensive loss following failure to protect material from ravages of fungi in warm humid climates. It is also speculated that mutated fungi, which were breeding in space station Mir were responsible for the deterioration of its equipment.
- The fungal growth on plastic products, especially on plasticize components, is known since 1940s. Each plastic product must be examined for its suitability to colonizing fungi. Saprotrophic fungi, especially in humid tropical areas, can colonize diskettes, audiotapes, videotapes and computer disks. The fungi belonging to genera like Alternaria, Aspergillus, Epicoccum, Paecilomyces, Penicillium and Trichoderma are observed on diskettes in the tropical countries.
Aeromycology
As fungal agents are part and parcel of soil and the environment, especially atmospheric air, which acts as the most common source of opportunistic as well as true fungal infections. The fungi in atmospheric air may be regularly monitored to know the magnitude of problem under which the population is living in an area. The fungal species constitute major component of air-spores. Various studies have revealed that fungal spores in air mostly come from fungi on plants or large fungal fruiting bodies that rise above soil surface and not from the ground. The soil itself is sinking into which most airborne spores finally disappear rather than their being a source. Many organisms found outside atmosphere will also be found inside buildings where they are introduced by air currents but main sources of contamination arise from within buildings from animals, humans or plants.
The aeromycology is study of airborne fungi, which is usually done with following purposes: (a) to know variety of fungi prevalent in particular region and also (b) to know seasonal variations of allergenic fungal spores. There are two methods for studying airborne fungi that may be used individually or concurrently; (i) slide exposure method and (ii) petri-dish exposure method. The combination of both these methods is ideal to minimize drawback of each other.
The value of aerobiology lies in the study of fungi that cause diseases to man and animals. Generally three types of diseases based on fungal origin are recognized: allergies, mycotoxicosis and mycoses. Hence inhalation of fungal spores and perhaps their metabolites may cause respiratory system disease such as bronchopulmonary aspergillosis, pulmonary mycotoxicosis and hypersensitivity reactions.
Surveillance of Fungal Infections
A regular monitoring of fungal infections is also required as it is done in case of viral, bacterial and parasitic infections. This is more important when large number of infections are healthcare-associated in nature and found prevalent in hospital environment. Moreover, these are found to be resistant to the most commonly used antifungal agents.
Outside hospital setup, surveillance of fungal infections is mandatory as certain outbreaks are reported in particular geographical areas. Some of diseases are prevalent in certain geographical pockets but in addition to that diseases are found prevalent in certain communities as well, which may be detected in time with watch of surveillance system.
Now molecular techniques are playing an important role not only in fungal diagnosis but in taxonomy and epidemiological aspects as well. These techniques have resolved many long-standing controversies on these aspects. Therefore, in any hospital set-up, regular surveillance of fungal infections is the most important, especially in epidemiological aspects of the healthcare-associated ones.
Safety in Medical Mycology Laboratory
There are certain fungal pathogens, which cause systemic infections associated with work related exposure to the staff working in diagnostic as well as research mycology laboratory. Four of these genera are established pathogenic fungi in which greatest potential of exposure is involved in such laboratories. These are: Coccidioides immitis, C.posadasii, Histoplasma capsulatum, Sporothrix schenckii and Cryptococcus neoformans. Additional species are being documented as etiological agents of mycotic morbidity and may pose potential threat for such type of infections. These agents usually take entry through respiratory route and may establish infection due to inhalation.
There are general safety measures like never smell or examine fungal culture in the open petri dish. The handling of clinical material as well as the fungal isolates should be done under biosafety hood. It is essential for both medical as well as classical mycologists to adhere 15standard safety measures during their laboratory work. The basic priority is to recognize potential risk of infection by these fungi. Secondly, acknowledgments that investigators have primary responsibility for containment of any organism with which personnel are working in laboratory to avoid any occupational health hazards. In diagnostic laboratory every clinical sample should be taken as highly infectious unless it is proven otherwise. Biosafety issues are not only important from medical point of view but because of legal angle also. Therefore, essential safety precautions and techniques should be adhered to in all research and diagnostic laboratories handling infective microorganisms including fungi. The general guidelines on laboratory safety practices are briefly described in Chapter 36.
The specifically recommended safety procedures and equipment are set out according to four Biohazard Safety Levels or BioSafety Levels (BSL) of laboratory facilities, dealing with potentially hazardous pathogens, moving through basic laboratories handling routine sample of low-risk microorganisms (BSL 1) to maximum containment laboratories working with highly infectious pathogens (BSL 4). These are also called corresponding levels of Containment (CL1 to 4), Category (Cat1 to 4) or Protection (P1 to 4) and the counterpart microorganisms as Hazardous Group (HG1 to 4).
A classification of fungi into biosafety categories is proposed and various criteria for attribution to Biosafety Levels have been laid down. The list of fungi stressing on ecological criteria is derived from these agents rather than host factors and include all species accepted in recent medical literature. Each fungal species is assigned a defined biohazard categories i.e. Biosafety Levels.
There are three primary goals for biological safety in medical mycology laboratory, which are as follows: (a) to reduce potential level of exposure of laboratory personnel; (b) to protect diagnostic and research work from exogenous contamination and (c) to prevent inadvertent contamination of environment outside laboratory.
The intent of dealing with this issue is to cover some of important aspects of biosafety; (i) standards in handling dimorphic fungal pathogens; (ii) principles and criteria of biosafety levels and classification of known medically important as well as environmental fungi according to their biosafety levels; (iii) medically important fungal waste and its safe disposal and (iv) biosafety and regulatory considerations in handling and mailing medically important fungi to a Reference Laboratory or Culture Collection.
The following safety procedures are adopted while working with potential fungal pathogens. These should be mandatory for all personnel posted in clinical or diagnostic laboratory. There are two most common means of getting infections by fungi in laboratory i.e. inhalation of conidia that are aerosolized and accidental inoculation through sharp object e.g. needles, scalpel blades and broken glasses while handling specimens.
The Biosafety Levels include laboratory practices and techniques, safety equipment and appropriate laboratory facilities and have been defined to guide those working with infectious agents. Keeping all types of microorganism in view, Centers for Disease Control and Prevention and National Institutes of Health, USA have divided Biosafety Levels into following categories, which are described below:
(a) Generally Regarded As Safe (GRAS): The harmless industrial strains like domesticated mutants widely applied in food production. GRAS refers to individual strains or clones whereas BSL refers to species in taxonomic sense.
(b) Biosafety Level 1: This level requires basic laboratory facilities and use of standard microbiological practices. It applies to use of specific microorganisms not known to cause disease in healthy human adults. These saprotrophic agents or plant pathogens are occupying non-vertebrate ecological niches or these are fungi utilizing dead animal products. The infections in this category are coincidental, superficial and non-invasive or mild in nature. Saccharomyces cerevisiae, which was categorized under GRAS, is now considered under this category because of substantial number of case reports by this organism.
(c) Biosafety Level 2: This level requires basic BSL 1 practices plus aprons, decontamination of all infectious wastes, limited access to laboratory, protective gloves and pasting of biohazard warning signs. This level is used when handling microorganisms of moderate-risk, which are associated with human diseases. These species are principally occupying non-vertebrate ecological niche but with relatively low ability to survive in vertebrate tissue. In severely immunocompromised patients they may cause deep or opportunistic mycoses. The pathogens causing superficial infections also fall into this category. Therefore, majority of fungi infecting man and animals are categorized under BSL 2.
Moreover, BSL 2 practices and facilities are recommended for handling and processing clinical specimens and identifying isolates of Coccidioides species. BSL 2
16practices differ from BSL 1 practices in that (i) laboratory personnel have specific training in handling pathogenic agents and are directed by qualified scientists; (ii) access to laboratory is limited when work is being conducted; (iii) extreme precautions are taken with contaminated sharp items and (iv) procedures in which infectious aerosols or splashes may be created, are conducted in Class II biological safety cabinets.
(d) Biosafety Level 3: This level requires BSL 2 facilities and practices supplemented by controlled access to laboratory and use of special laboratory clothing and containment equipment e.g. biological safety cabinet. Biosafety level 3 facilities require a separate negative pressure room. This level is used when potentiality of infection from aerosols, autoinoculation or ingestion exists and when concerned personnel are handling microorganisms which may cause severe or lethal infections. These pathogens are potentially able to cause severe or deep mycoses in otherwise healthy patients. Earlier list of fungi included in this category was mycelial forms of dimorphic fungi but now it also includes additional other potentially pathogenic fungi like Cladophialophora bantiana.
(e) Biosafety Level 4: This is maximum containment level consisting of work with dangerous and exotic agents, which have high potential for causing life-threatening diseases. It does not include any of fungal pathogens as most of the agents covered under this level are mainly viral in origin.
All Mycology Laboratories are covered under purview of two levels i.e. BSL 2 and at the most BSL 3. The clinical specimens that may contain fungi pathogenic to man should be handled by using BSL 2 practices, containment equipment and other facilities. There is need for clinical laboratories to adhere to safety standards promulgated by CDC Office of Health and Safety in 5th edition of ‘Biosafety in Microbiological and Biomedical Laboratories’ published in 2007. This document summarizes four recommended biosafety levels for infectious agents and addresses which types of biological safety cabinets are appropriate for each biosafety level and which are available online (See Appendix F).
The standard precautions include use of Biological Safety Cabinet or hood when working with clinical material suspected of having fungi. These cabinets are most commonly used primary containment devices in laboratories working with infectious agents. The staff must be properly trained in use of these cabinets. Therefore, biological safety cabinets are essential protective element for investigators working with infectious or potentially infectious fungal pathogens. The Class II biological safety cabinets have vertical laminar airflow with high efficiency particulate air (HEPA) filtered supply and exhaust air, which are 99.99% efficient in removing particles of size 0.3 μm or larger. An additional advantage of using biological safety cabinets is that they also protect clinical specimens from extraneous and airborne contamination. Hence they protect worker, product as well as the environment. The types of biosafety cabinets and their proper use have been described in details in 3rd edition of CDC-NIH publication entitled ‘Primary Containment for Biohazard: Selection, Installation and Use of Biological Safety Cabinets’ issued in September 2007.
BSL 2 procedures are specifically recommended for personnel working with clinical specimens, which may contain Blastomyces dermatitidis, Coccidioides immitis, C.posadasii, Histoplasma capsulatum, Cryptococcus neoformans, Sporothrix schenckii and pathogenic members of genera like Trichophyton, Microsporum and Epidermophyton. If culture of dimorphic fungus like Blastomyces, Coccidioides or Histoplasma is grown in its mycelial form, BSL 3 containment procedures should be adopted since conidia of these fungi have greater potential to cause infection through inhalation. In other words yeast (tissue) forms of some of dimorphic fungi are covered under BSL 2 and their counterpart mycelial forms strictly under provision of BSL 3 due to their potentially higher infectious nature.
Risk Groups
The biological agents are also categorized in Risk Groups (RG) based on their relative risk. Therefore, depending on the country or organization, this classification system may take the following factors into consideration: (i) Pathogenicity of the organism; (ii) Mode of transmission and host range; (iii) Availability of effective preventive measures like vaccines; (iv) Availability of effective treatment like antimicrobials. It is important to understand that biological agents are classified in a graded manner such that the level of hazard associated with RG1 being the lowest and RG4 being the highest. EHS Biosafety follows the NIH Guidelines categorization of Risk Groups as follows:
RG1: Are not associated with disease in healthy adult humans or animals.
RG2: Are associated with disease, which is rarely serious and for which preventative or therapeutic is often available.
RG3: Are associated with serious or lethal human disease for which preventative or therapeutics may be available.
RG4: 17Are associated with lethal human disease for which preventative or therapeutics are not readily available.
Outbreaks of Fungal Infections
Outbreaks of fungal infections following natural disasters and accidental/hospital acquisition are increasingly recognised. They have important public health and infection control consequences. Infections after natural disasters typically result from inhalation or cutaneous inoculation of environmental fungi. Clusters result from shared exposures. There have been >13 major global outbreaks in the past decade from air-borne, near-drowning or near-burial events encompassing fungi from Coccidioides immitis (n=3) to mucormycetes. Hurricane Katrina in 2008, the Boxing Day tsunami in 2004 and Joplin Tornado in 2011 ‘stand out’ with horrendous soft tissue injuries and disseminated infection with a mortality of 30-80%. Healthcare infections after disasters can follow e.g. Aspergillus meningitis from contaminated syringes (Sri Lankan tsunami). Case clusters also occur through use of contaminated pharmaceutical products, fomites and environmental hospital exposure. Mucormycosis has resulted from contaminated bed linen and tongue depressors. Fungal endophthalmitis (Fusarium, Bipolaris) followed minor eye surgery after irrigation with contaminated triamcinolone and Brilliant Blue Green. Moreover, there were 752 cases (64 deaths) of Exserohilum CNS and joint infections from contaminated methylprednisolone vials used to treat back pain in the USA with a national crisis. Improving physician and public health awareness of mycoses is essential with an urgent need for infrastructure for case reporting and a network of alerts.
Index of Suspicion
In routine medical practice, the infectious diseases are usually considered to be caused by bacteria, viruses and seldom by the parasites. Fungus, as such the cause of infectious disease is rarely considered. Therefore, to tackle such infections, often antibacterial antibiotic are prescribed and underlying pathogenesis of fungal infection goes on increasing. By the time realization is there about the fungal infection, it is too late and situation becomes almost irreversible. Therefore, fungal infections should be considered as the differential diagnosis, wherever, a slightest doubt of fungal involvement is there. If the index of suspicion is kept very high then the additional diagnostic methodology is adopted along with the routine protocol and final diagnosis is reached without wastage of valuable time thereby proper antifungal treatment is instituted and life of the patient is saved. Hence a high index of suspicion is the key to an early diagnosis of fungal diseases, which is must to tackle such life-threatening infections.
Medical Mycology in India
In India, fungal infections are known since ancient civilization and have been mentioned in Aryan documents such as Atharva Veda, wherein mycetoma is described as Padavalmikam i.e. anthill foot. This finding was observed by John Gill in 1842 in Madurai district of a south Indian state, Tamil Nadu, which was subsequently designated as ‘madura foot’ by Henry Carter in 1860.
In the beginning of 20th century, School of Tropical Medicine and Hygiene was established during the British regimen in 1920s in Kolkata (then Calcutta), the eastern zone of country. There was separate Department of Medical Mycology to cater services in this field of medical sciences, where Prof. Anisetti Thammayya did his innovative work in this field. In the western zone, Prof. K R Joshi consolidated different aspects of medical mycology, especially in the field of mycetoma and opportunistic fungal infections. Similarly, in southern zone, Prof. A S Thambiah, Prof. A Kamalam, Prof. Pankajalakshmi V Venugopal and Prof. P A Thomas in Tamil Nadu and Prof. B M Hemashettar in Karnataka, have brought Medical Mycology in the country to the level of substantial recognition. In the central India, mycological services were consolidated by Prof. S M Singh and his colleagues.
In northern side, Department of Medical Mycology was established at Vallabhbhai Patel Chest Institute, Delhi in 1959 to provide research, diagnostic, therapeutic mycological services in the national capital and its surrounding areas. Prof. H S Randhawa and Prof. H C Gugnani further developed this Department and currently being looked after by Prof. Anuradha Chowdhary. During the same period Mycology Sections were developed in All India Institute of Medical Sciences (AIIMS) and National Institute of Communicable Diseases (NICD), both in New Delhi. Prof. L N Mohapatra did pioneering work in this field at AIIMS, which was continued by Prof. Uma Banerjee and her colleagues.
The Division of Mycology at Postgraduate Institute of Medical Sciences and Research, Chandigarh was started under enthusiastic leadership of Prof. Pushpa Talwar in 1964 and is at present being looked after by Prof. Arunaloke Chakrabarti. This is one of the leading centers of Medical Mycology and providing diagnostic services as well as 18conducting research activities. Some of Indian institutions, where active work is currently going on, have been mentioned in Appendix E of this Textbook.
A curriculum was designed by Prof. R. Sambasiva and colleagues in 1999, wherein it is emphasized that postgraduates in Microbiology must have basic knowledge of theory and practical aspects in Medical Mycology, along with its other branches. In India, this branch of Microbiology has now attained substantially sound footing. The following salient features are also relevant to describe on this issue:
- Previously there were only a few case reports from India about fungal infections but now many prospective studies are being conducted mainly based on epidemiology and diagnostic modalities in the field of Medical Mycology.
- The international leading organizations in this field like ISHAM are helping medical institutions in India especially for training of staff, who handle difficulties related to management of fungal infections.
- The first edition of Textbook of Medical Mycology was published on July 1, 1995 which has modified trends in understanding of Medical Mycology at the grass root level in India and its neighboring countries followed by second edition in April 2002 and third edition in October 2008. It is expected that this fourth edition (2017) will also be able to further consolidate its endeavor in the times to come.
- The national body of Indian scientists interested in field of medical mycology came into existence. The Society of Indian Human and Animal Mycologists (SIHAM) was founded in February 1996 in its First Conference held at Jabalpur, Madhya Pradesh. Actually idea of making national body of mycologists was conceived in 1994 at time of 12th ISHAM Congress held at Adelaide (Australia), which was materialized after two years under the dynamic leadership of Prof. S M Singh, in the form of SIHAM, which is actively working for enhancing the cause of Medical Mycology in India. The National Conferences of SIHAM are being regularly held after a gap of two years. The very 1st Conference was held in Jabalpur (1996), 2nd in Jodhpur (1998), 3rd in Chennai (2000), 4th in Annamalai Nagar (2002), 5th in Chandigarh (2004), 6th in Hyderabad (2006), 7th in Mumbai (2008), 8th in New Delhi (2010), 9th in Siliguri (2012), 10th in Coimbatore (2014) and 11th in Shimla (2016). Now, 12th National SIHAM Conference will be held under the leadership of Dr. Jayanthi Savio in February 2018 at Bangalore (Karnataka).
- Many Seminars, Symposia, Workshops, Continuing Medical Education (CME) and Professional Development Programs (PDP) are being held in this field from time to time in different parts of the country. The relevant documents of these events are referred in Appendix F as Bibliography. A number of monographs and proceedings of these meetings are now available dealing with various aspects of Medical Mycology to provide firm footing in India.
- During this course of development, Mycology Reference Laboratories (MRL) are supposed to be designated to provide basic facilities for identification of fungi, teaching of graduates and postgraduates, other academic and service-oriented infrastructure in this unique field of medical sciences. The Indian Council of Medical Research (ICMR) has recognized the Division of Mycology as the ‘Centre for Advanced Research in Medical Mycology’ in the year 2005. It is providing two ICMR-WHO-PGI Training Courses in Diagnostic Medical Mycology in a calendar year: (i) summer course for the young faculty and medical scientists (May-June) and (ii) winter course for technical staff (December). In addition, it is the national reference centre for the identification, national culture collection as well as antifungal susceptibility testing of medically significant pathogenic fungi.
- The quality control in diagnostic mycology is essential to maintain standard in imparting the services. This may be internal or external quality control. In India periodic exercise was started by VPCI, New Delhi in the form of Proficiency Testing as external quality control. This service is now being provided as EQAS by the PGIMER, Chandigarh and dealt briefly in Appendix E. Therefore, auditing of Medical Mycology services is mandatory to impart good quality laboratory practices.
Fungal Repositories
The Budapest Treaty on international recognition of deposit of microorganisms for purposes of patent procedure was done at Budapest on April 28, 1977 and was amended on September 26, 1980. In India, based on these guidelines, the Microbial Type Culture Collection Center and Gene Bank (MTCC), IMTECH, Chandigarh was established and is functioning since October 4, 2002. In addition to other microorganisms, it is repository for the fungi, mainly isolated from the environment. The National Culture Collection of Pathogenic Fungi (NCCPF) was set-up at the PGIMER, 19Chandigarh by the ICMR in 2010, where the fungal isolates can be deposited. The details of such issues are dealt in Appendix E.
Fungal Registries
In the recent times, lot of websites have been created which register data pertaining to any of the fungal issues, analyze it and periodically publish it from time to time. According to the feedback received, corrective measures are also taken. Some of the registries are: Mucormycetes (www.zygomyco.net), Emerging Fungal Infections (www.fungiscope.net), Trichosporon (www.trireg.com) and research collaboration focused on pediatric invasive fungal infections i.e. International Pediatric Fungal Network (www.ipfn.org), etc.
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