Đang tải... (xem toàn văn)
(BQ) Part 2 book Medical mycology - Current trends and guture prospects has contents: Classic histoplasmosis, paracoccidioidomycosis - An endemic mycosis in the americas, fungal allergens - recent trends and future prospects, fungalbiofilms - Formation, resistance and pathogenicity,... and other contents.
SECTION III Classic Mycoses Caused by Dimorphic Fungi © 2016 by Taylor & Francis Group, LLC CHAPTER Classic Histoplasmosis Ricardo Negroni Introduc on Classic histoplasmosis or histoplasmosis capsulati is a systemic endemic mycosis, caused by the thermally dimorphic fungus Histoplasma capsulatum var capsulatum This microorganism lives in the environment, especially in the soil, where it exists as mould In blood-agar medium at 37ºC and in tissues it grows as a budding yeast In the infected organs these yeasts are inside the cells of the reticuloendothelial system (Arenas 2011; Bonifaz 2012) Histoplasmosis has been registered in more than 60 countries, but it is more frequent in the middle east area of U.S.A and in Latin America (Borelli 1970) The infection is produced by inhalation of microconidia and the lungs are its portal of entry The majority of the infections in immunocompetent individuals are asymptomatic or mild and self-limited (Larsh 1970) The severity of the respiratory manifestations is related to the amount of conidia inhaled (Goodwin et al 1981) Chronic progressive pulmonary histoplasmosis is detected in males above 50 years of age with chronic obstructive pulmonary disease (Goodwin et al 1981; George and Penn 1993) Acute or chronic disseminated histoplasmosis occurs in patients with cell-mediated immunity failures and it is a life-threatening disease (Goodwin et al 1980; Alsip and Dismukes 1986) Amphotericin B and itraconazole have been successfully applied in the treatment of this mycosis (Wheat 2002) History In 1904, when Samuel Darling was working at the Ancon Canal zones Hospital, he observed the first fatal case of disseminated histoplasmosis He performed the autopsy Consultant MD, Mycology Unit, Hospital de Infecciosas Francisco J Miz, Buenos Aires, Argentina Director de la Maestría en Micología Médica de la Facultad de Medicina de la Universidad Nacional del Nordeste E-mail: ricnegroni@hotmail.com © 2016 by Taylor & Francis Group, LLC 208 Medical Mycology: Current Trends and Future Prospects of a black man from Martinique who had died of an infection resembling a severe tuberculosis, but microscopically he observed an intracellular parasite very similar to Leishmania Upon closer examination he noted that this microorganism lacked kinetoplasts Darling thought that this new infective agent was a protozoa and named it Histoplasma capsulatum (Negroni 1965) In 1906 Darling reported two new cases of disseminated histoplasmosis, one in a black man from Martinique and the other in a Chinese who had lived in Panama for 15 years (Kwon-Chung and Bennett 1992) In 1912 the eminent Brazilian parasitologist, Henrique Da Rocha Lima, who was working in Hamburg, suggested that the microorganism described by Darling was a budding yeast and not a protozoa (Negroni 1965) In 1934 Dodd and Tompkins reported a new case of histoplasmosis and studied the growth of H capsulatum in blood-agar at 37ºC In these cultures they obtained the growth of the yeast form of this dimorphic fungus and confirmed Da Rocha Lima’s hypothesis They also reproduced the disease in Macacus rhesus (Kwon-Chung and Bennett 1992) In the same year at Vanderbilt University De Mombreun discovered the dimorphism of Histoplasma capsulatum and thought that the mycelial form of this fungus probably existed in nature (Rippon 1988) Christie and Peterson in 1945 reported many patients with pulmonary calcifications who reacted negatively to tuberculin and positively to histoplasmin Palmer (1945– 1946) carried out a nation-wide project of histoplasmin skin testing and found a particular geographic distribution of histoplasmin hypersensitivity in U.S.A (George and Penn 1993) Furcolow (1945) made very important contributions to the knowledge of the epidemiology and ecology of histoplasmosis Emmons (1948) isolated H capsulatum from soil in a sample collected near a rat burrow under the edge of a poultry house (Negroni 1965) The first outbreak of histoplasmosis was detected in soldiers at Camp Grubber (Oklahoma, U.S.A.) (Rippon 1988) Kwon-Chung (1972) discovered the sexual reproduction of H capsulatum and identified the two mating types (+) and (–) She named the teleomorphic form Emmonsiella capsulata In 1979, McGinnis and Katz transferred E capsulata to the genus Ajellomyces, now named Ajellomyces capsulatus (Kwon-Chung and Bennett 1992; Deepe 2012) Pablo Negroni (1940–1941) published a mycological study of the first Argentinean case of histoplasmosis He performed a very careful study of the isolated fungus and was able to obtain the yeast form in vitro and employed a sterile extract of the mycelial form of H capsulatum (histoplasmin) for the first time (Negroni 1965) E ology Histoplasma capsulatum grows at 28ºC after a 15 day incubation in several culture media such as Sabouraud dextrose-agar, dextrose-potato-agar and Borelli lactrimel It presents a cottony aerial mycelia, white to tan in color The reverse is uncolored or brown Two types of colonies are found: white and brown White colonies grow © 2016 by Taylor & Francis Group, LLC Classic Histoplasmosis 209 faster and lose the capacity of producing spores after several subcultures The brown type is more virulent for mice and produces a great amount of conidia (Kwon-Chung and Bennett 1992; Kauffman 2011; Deepe 2012) Microscopically vegetative mycelia consist of septated, branched, hyaline hyphae of to μm in diameter Three types of asexual conidia are observed: (1) large, spherical or pear shape spores, 10 to 25 μm in diameter with a thick cell wall covered by tubercles, of which some are like a digital protuberance in shape, to μm in length This cell wall has a thin inner layer and a thick verrucous part These spores are named macroconidia, they are found in the aerial mycelia and are born on short sporophores (Fig 2); (2) conidia similar to the previous one, to 20 μm in diameter, spherical to oval with thin walls, usually born on short hyphae, and found in the submerged mycelia; (3) microconidia which are spherical or pyriform with thin walls, to μm in diameter, sessile or on short sporophores (Negroni 1965) H capsulatum yeast form develops in rich culture media such as brain heart infusion-agar with 5% of rabbit blood incubated at 37ºC Blood cisteine-agar is also an excellent culture medium for this purpose After to days of incubation colonies are visible as whitish, wrinkled or cerebriform, or mm in diameter, moist, glossy and of cream consistency growth Microscopically, small single budding yeasts, to μm in diameter, are observed They multiply by polar budding and the connection between the mother and daughter cells is narrow These yeast cells are uninucleated The transformation from the mycelial growth to yeast form is not easy and may require several attempts (Rippon 1988; Kwon-Chung and Bennett 1992) Mycelial form is also named saprophytic phase because it is found in the soil as well as in bat and bird droppings The yeast form is called tissues phase In animal tissues it is found in the form of small yeast-like elements of spherical to oval shape, single budding; they are 3–5 μm in diameter and have a thin cell wall which does not take aniline stains Due to this characteristic it was initially mistaken for a capsule The majority of these budding yeasts are found inside macrophages or giant cells in the granulomas and they are of the same shape and size (Salfelder et al 1990; Negroni 2004) In smears stained by Giemsa or Wright techniques the cell wall does not take up the stain and appears as a clear halo, the cytoplasm has a single distinct mass, half moon shaped, placed at the opposite side of the bud, which is darker than the rest of the cytoplasm (Fig 1) (Negroni 1965) H capsulatum is Gram positive, stains red with periodic acid Schiff (P.A.S.) and dark brown or black with Grocott methenamine-silver technique (G.M.S.) (Salfelder et al 1990) H capsulatum sexual reproduction follows the heterothallic conjunction of compatible mating types (+) and (–), paired in poor culture media as yeast extract-agar or soil extract-agar at 28ºC for several weeks The young cleistothecia are globose, 100 to 150 μm in diameter; they become irregularly stellate with age because of the radiated spinal peridial hyphae Asci are club to pear shape, 3–5 x 10–15 μm in diameter and contain oval ascospores This sexual (teleomorphic) form is called Ajellomyces capsulatus (Kwon-Chung and Bennett 1992; Kauffman 2011; Deepe 2012) H capsulatum has to chromosomes According to the number and characteristics of these chromosomes the strains of this fungus were initially divided into two chemo types, but new molecular biology techniques have allowed the © 2016 by Taylor & Francis Group, LLC 210 Medical Mycology: Current Trends and Future Prospects Figure Giemsa stained smear of a cutaneous lesion showing yeast-like elements of H capsulatum inside macrophages, X 1,000 Figure Mycelial form of H capsulatum, microscopic observation of macroconidia in a preparation with lacto phenol cotton blue, X 400 identification of clades: from North America, from Latin America and from each of these regions: Australia, Indonesia and Eurasia (Muniz et al 2001; Kauffman 2011; Deepe 2012) The genetic differences between H capsulatum strains are associated with different clinical manifestations; strains from South America produce more mucocutaneous lesions than those from North America This wide genetic variety of H capsulatum © 2016 by Taylor & Francis Group, LLC Classic Histoplasmosis 211 seems to have originated through a sexual recombination of the strains (ZancopéOliveira et al 1994; Negroni et al 2010a) The mating type (–) is isolated to times more frequently in patients than the (+) mating type (Kauffman 2008) Some Chrysosporium and Sepedonium species present mycelial growth with micromorphological findings very similar to the mycelial form of H capsulatum However, H capsulatum produces microconidia in addition to tubercular macroconidia; it is dimorphic and pathogenic for laboratory animals (Negroni 1965) Atypical H capsulatum strains can be identified by nucleic acid hybridization test, Gen-probe (San Diego, CA), kit or exoantigen testing; both have a good sensitivity and specificity (>90 %) (Wheat and Kauffman 2003; Negroni et al 2010) H capsulatum belongs to Ascomycotina sub-division, to Onygenaceae family and Ajellomyces capsulatus specie It is genetically related to Blastomyces dermatitidis (Ajellomyces dermatitis) and Paracoccidiodes brasiliensis (Deepe 2012) There are three varieties of Histoplasma capsulatum: H capsulatum var capsulatum (the one we have described), H capsulatum var duboisii, the etiologic agent of African histoplasmosis, which has a larger yeast form, and H capsulatum var farcinimosum, which produces epizootic lymphangitis in horses and mules in southern and central Europe, North of Africa, the Middle East and Southern Asia (Arenas 2011; Bonifaz 2012) Some minerals and vitamins are necessary for H capsulatum growth, such as thiamine, biotin and iron Amino acids containing sulfhydrilic groups are required for the growth and survival of the yeast form The mycelial to yeast transformation is related to the hyper expression of mRNA of calcium binding protein This transition is stimulated by a temperature of 37ºC, which gives way to an increase in cell membrane fluidity This process is very complex and produces several biochemical, physical and genetic changes triggered by a transcription factor called Ryp1 (Deepe 2012) The H capsulatum cell wall polysaccharides are chitin and glucans; they are very important virulent factors, especially α-glucan, which is the most abundant polysaccharide of the yeast form; ß–glucan is predominant in mycelial form (Negroni 2000; Kauffman 2011) Epidemiology Histoplasmosis is an endemic mycosis with a wide geographical distribution It has been reported in more than 60 countries in the temperate and tropical zones throughout the world It is most common in the American Continent from Canada to Argentina The most important endemic areas are located along the great river valleys in the eastern and central parts of the United States as well as in La Plata River and Serra Mar in South America In the highly endemic zones 80% of adults react positive to histoplasmina skin test (Borelli 1970; Negroni 2004; Negroni 2010a) It should be noted that the incidence of histoplasmin reactivity at any time underestimates the true prevalence of the infection since the skin test may give negative results to years after the infection if the person has not been exposed to new infections (Rippon 1988; George and Penn 1993) Autochthonous cases of histoplasmosis have also been reported in Africa, Australia, India and the Far East (Kauffman 2011) © 2016 by Taylor & Francis Group, LLC 212 Medical Mycology: Current Trends and Future Prospects Histoplasma capsulatum has been isolated from the soil in several endemic regions This microorganism grows well in rich soils with high nitrogen concentration and acid pH, particularly if they are contaminated with bird or bat excreta The majority of the endemic areas are near great rivers or like shores where the mean annual temperature varies between 15 and 20ºC and the annual rainfall oscillates between 800–1,200 mm Some “epidemic spots” have been recorded inside or outside the endemic zones (Larsh 1970; Rubinstein and Negroni 1981; Deepe 2012) These are characterized by heavy soil contamination with H capsulatum and may produce small outbreaks These outbreaks have appeared after the cleaning of poultry or pigeon houses, after entering caves or mines where bats have nested or after any action which leads to the disturbance of the soil where black birds (stornins) or pigeons have roosted (George and Penn 1993; Wheat and Kauffman 2003; Negroni 2010b) H capsulatum may infect bats, which are able to spread the fungus to new locations, both inside or outside the endemic zones (Wheat and Kauffman 2003) The asymptomatic or mild self-limited infections are very common in urban or rural areas of the endemic zones In these areas 20 to 80% of the adult population has positive histoplasmin skin tests The prevalence of this sensitization increases from childhood to 30 years of age (Negroni 1965; Rubinstein and Negroni 1981; Kauffman 2011) Histoplasmosis is not usually transmitted from man to man or from animal to man, but a few cases of histoplasmosis have been recorded in liver transplant recipients who received the liver from an infected person (Silveira and Husain 2007; Deepe 2012) Natural infection with H capsulatum has been found in several animal species, most frequently in dogs and rodents (Rippon 1988) Chronic cavitary pulmonary histoplasmosis is more often observed in adult males above 50 years of age with chronic obstructive pulmonary disease This clinical form is more frequent in Caucasians (Goodwin et al 1981; Rubinstein and Negroni 1981; Negroni 2000) The progressive disseminated forms are related to predisposing factors; they are observed in children under years of age and in adults above 50 years of age, especially in Caucasian males Persons considered at risk of suffering disseminated histoplasmosis are those who have various alterations of cell-mediated immunity such as chronic alcoholism, diabetes mellitus, long-term therapy with corticosteroids, leukemia, lymphomas, treatment with TNF-α inhibitors and those infected with HIV, with less than 150 CD4 + cells/μL (Goodwin et al 1980; Rubinstein and Negroni 1981; Alsip and Dismukes 1986; Negroni et al 1987; Negroni et al 2010a) Those who handle the mycelial form of H capsulatum at the laboratory are at a risk of acquiring a massive primary infection This job should be done in laminar flow chambers (BSL 3), under very strict biosafety conditions (Kauffman 2009) Pathogenesis The infective elements of H capsulatum are the microconidia of the mycelial form which live in the environment, especially in soils with organic decay These spores are inhaled by humans and other animal species, as they float in the air (Negroni 1965; Negroni 1989a; Kwon-Chung and Bennett 1992) © 2016 by Taylor & Francis Group, LLC Classic Histoplasmosis 213 The inhaled spores penetrate the airways until they reach the pulmonary alveoli, where they are phagocytosed, but not lysed, by the alveolar macrophages The phagocytosis occurs after the binding of these spores to the CD18 and CD11 families, adhesion promoting glicoproteins of neutrophils and macrophages (Deepe 2012) Inside the alveolar macrophages the conidia transform into yeast-like elements which multiply by budding In this phase of the infection the yeast form of H capsulatum is able to survive in the phagolysosomes of macrophages by using several mechanisms, including its ability to resist being killed by oxygen radicals and to modulate the intraphagosome pH Iron and calcium acquisition by yeasts is also an important survival tool that allows the growth of this microorganism inside the macrophages (Negroni 2000; Kauffman 2009; Kauffman 2011; Deepe 2012) The regulatory genes controlling production of 60 KDa heat-shock proteins by the yeast cell wall play a fundamental role in the transformation of the mycelial form into the yeast form The enzymes produced during this process are involved in the sulfhydrilic link amino acids, like cisteine and biotin These, in addition to the temperature of 37ºC, are the two most important factors in the dimorphic process (Negroni et al 2010a; Kauffman 2011; Deepe 2012) The yeast form of H capsulatum is initially found inside alveolar macrophages and polymorphonuclear neutrophils, as we have already mentioned The initial stage of the inflammatory response involves polymorphonuclear neutrophils, these cells are able to decrease the growth of H capsulatum by producing a respiratory burst and by releasing azurophil granules, but they are unable to control the progress of the infection The fungistatic activity of the azurophil granules of the neutrophils is not related to nitric oxide or to the action of toxic radicals of oxygen In this early part of the infection macrophages permit a rapid reproduction of the H capsulatum yeast form inside the phagolysosomes of macrophages, as we have previously said When the number of yeasts inside these cells is very high, they are liberated from them and rapidly captured by other macrophages to which they adhere by ß2 integrins (Negroni 2000; Negroni 2010; Deepe 2012) These yeast cells also adhere to dendritic cells by binding fibronectin The dendritic cells are in charge of the antigens presentation to the TCD4 + lymphocytes, giving way to the specific cell-mediated immunity (Negroni 2000; Deepe 2012) Lung infection during this initial phase progresses by contiguity, continuing through the lymphatic system to mediastinal lymph nodes and finally, to the blood stream This hematogenous dissemination is asymptomatic in the majority of the infections and H capsulatum yeasts colonize the reticuloendothelial system (Kwon-Chung and Bennett 1992; George and Penn 1993) The activation of specific cell-mediated immunity in immunocompetent hosts is evident within two to three weeks after infection The immunological response involves the production of Th1 type of cytokines, which effectively dominate the infectious process In this stage of the infection, macrophages become activated, especially by the action of the INF-γ , IL3, IL12 and TNF-α produced by T CD4 + cells Activated macrophages are able to kill yeast cells through nitric oxide activity The maturation of cell-mediated immunity becomes evident by the production of compact epithelioid granulomas in affected tissues, by the delayed hypersensitivity skin test with histoplasmin turning positive, and by the blastogenic response of the lymphocytes © 2016 by Taylor & Francis Group, LLC 214 Medical Mycology: Current Trends and Future Prospects against specific antigens becoming evident Other cytokines such as IL1 and GM-CSF also aid to contain the infection (Negroni 2000; Kauffman 2009; Deepe 2012) The role of T CD4 + cells in the defensive mechanisms against histoplasmosis is seen in athymic mice and AIDS patients, both of whom have serious forms of the disease The role of T CD8 + cells does not appear to be significant in host survival, but, apparently, these cells are necessary for optimal defense The importance of NK cells, which kill extra cellular yeast, is not absolutely clear nor is the mechanism of cytokines in the transformation of macrophages into activated macrophages (Wheat and Kauffman 2003; Negroni et al 2010a; Deepe 2012) Nevertheless, γ-INF plays a protective role in experimental animal models of histoplasmosis When cell-mediated immunity mechanisms are normal the infection progresses to a latent stage which probably persists for a lifetime In this latent stage epithelioid granulomas with a caseous center, which present viable yeast-like elements inside, can be detected in different organs (Salfelder et al 1990) These granulomas are surrounded by a fibrous capsule which calcifies with time The reactivation of this latent infection may occur if the host becomes immunocompromised (Negroni 2000) According to this pathogenesis model, several clinical forms of histoplasmosis have been accepted They are presented in Table (Goodwin et al 1980; Goodwin et al 1981; Alsip and Dismukes 1986; Negroni 2000) Table Histoplasmosis Clinical Forms Histoplasmosis in immunocompetent host 1.1 Asymptomatic or mild self-limited respiratory primary infection 1.2 Symptomatic pulmonary primary infection 1.3 Primary infection complications and sequelae 1.4 Re-infection 1.5 Progressive chronic pulmonary disease Histoplasmosis in immunocompromised host 2.1 Acute disseminated histoplasmosis 2.2 Subacute disseminated histoplasmosis 2.3 Chronic disseminated histoplasmosis Immunologically-mediated disease Clinical Manifesta ons AsymptomaƟc or mild respiratory infecƟon More than 95% of primary infections belong to this group Sometimes, they present mild respiratory alterations like those of influenza, which are self-limited The course of the primary infection depends upon the number of inhaled macroconidia as well as on age and previous clinical and immune status of the host Occasionally, they may produce pneumonitis and enlargement of the hiliar lymph nodes (Negroni 1965; Goodwin et al 1981; Negroni 2000) The incubation period varies from to 21 days; it is shorter in re-infections and in massive infections © 2016 by Taylor & Francis Group, LLC Classic Histoplasmosis 215 These mild cases have retrospectively been identified during the epidemiological research by the positive histoplasmin skin tests and calcified lesions in the lungs, lymph nodes or spleen These calcifications appear in approximately a quarter of those infected, one or two years after the infection Only 20% of such cases present positive serologic reaction to histoplasmin As we have already said, healing is spontaneous (Kauffman 2006; Deepe 2012) Acute pulmonary infecƟon These are symptomatic primary infections; their severity is related to the quantity of inhaled macroconidia Their clinical characteristics are similar to the pneumonia produced by Legionella, Mycoplasma, Chlamydia or viral infections The symptoms most often observed are fever, asthenia, myalgia, night sweats, dry cough, dyspnea and pleuritic or non-pleuritic chest pain (Rubinstein and Negroni 1981; Negroni 1989a) Chest radiographs frequently show bilateral patches of pulmonary infiltration and hiliar or mediastinal adenopathies (Deepe 2012) Severe acute pulmonary infections are observed in people who are exposed to heavy inoculum of H capsulatum In these cases, dyspnea, cough and fever are more severe, hepatosplenomegaly is detected and the chest radiology exam shows a micronodular intersticiopathy with a diffuse, bilateral, reticulonodular pattern and hiliar adenomegalies (Figs and 4) Most of the serious cases are reported during histoplasmosis outbreaks and some of these patients develop an adult respiratory distress, which requires mechanical respiratory assistance This extremely severe infection may rarely be fatal (Kauffman 2009; Negroni 2010b) Approximately 6%–10% of the infected persons may present clinical manifestations associated with hypersensitivity, such as erythema nodosum or multiform, polyarthritis, pleural and pericardial effusion Joint involvement is usually symmetric and the fluids recovered from arthritis, pleural and pericardial effusions are xantochromic or serofibrinous containing lymphocytes and polymorphonuclear Figure X-ray examination of a severe primary pulmonary infection, showing micronodularinterstitiopathy © 2016 by Taylor & Francis Group, LLC ... 27 8, 28 0, 28 6 Cochliobolus 147–149, 1 52 Co-infection 26 4 4 32 Medical Mycology: Current Trends and Future Prospects Confocal microscopy 38, 39, 51, 67 Conidia 60– 62, 25 7 25 9, 26 1, 26 2 Conidia-to-yeast... 71, 26 2, 26 5, 26 6 Anthropophilic dermatophytes 3, 5–7, 9, 11, 14, 15, 23 Anti-Biofilm therapeutics 305 Antibody therapy 26 6 Antifungal drugs 21 25 , 25 6, 26 2, 26 6 Antifungal resistance 22 , 25 Antifungal... 3 72, 3 82 384 P Panophthalmitis 59, 72 Paracoccidioides brasiliensis 25 5 Paracoccidioides lutzii 25 5, 26 6 Paracoccidioides spp 25 4, 25 5, 25 7 25 9, 26 4 Paracoccidioidomycosis 25 4, 25 6, 25 9 26 1, 26 3 26 5,