Introduction

Those infections, which are found in patients with underlying predisposing conditions. Produced by relatively non pathogenic or contaminant fungi in a host whose immunological defense mechanisms are weakened by endogenous or exogenous causes. Normally most of them found to be nonpathogenic to human beings but wherever an opportunity arises, it becomes a significant pathogenic agents.

Opportunistic mycoses

Candidiasis Cryptococcosis Pneumocystosis Penicilliosis marneffei Aspergillosis Zycomycosis

Apergillosis

Aspergilli & Penicillia commonest moulds seen on damp dread / any other organic matter. Of the 300 spp of Aspergilli, A.fumigatus highly pathogenic for birds & occasionally causes invasive disease in human beings. Few other species may also Cause opportunistic human disease. Common human disease otomycosis.

Systemic aspergillosis occurs as the following clinical types

1. Pulmonary aspergillosis * Aspergillus asthma * Bronchopulmonary aspergillosis * Colonising aspergillosis ( Aspergilloma ) 2. Disseminated aspergillosis

1. Aspergillus asthma
Occurs in atopic individuals following sensitisation to inhaled aspergillus spores. 2. Bronchopulmonary aspergillosis Heavy & repeated exposure to the spores of Aspergillus species Allergic alveolitis in which individuals suffers from breathlessness, fever & malaise after few hrs of exposure. The fungus grows within the lumen of bronchioles, which may be occluded by fungus plugs. The fungus demonstrated in sputum.

3. Colonising aspergillosis
Usually develops in pre existing pulmony cavities tuberculosis / cystic disease. The fungus grows into large balls Aspergilloma. Surgical removal becomes necessary as the disease commonly causes massive hemoptysis. Invasive aspergillosis fungus actively invades the lung tissue. 4. Disseminated aspergillosis Involving the brain, kidney & other organs fatal complication debilitated patients on prolonged treatment with antibiotics, steroids & cytotoxic drugs.

Lab diagnosis

Specimens: Sputum, bronchoalveolar lavage fluid (BAC) / Transbronchial biopsy depending on the site of involement. 1. Microscopic examination 2. Culture 1. Microscopic examination 10% KOH Hyaline septate hyphae of Aspergillus species Septate hyphae 3-6m in diameter with dichotomous branching. Other stains Calcoflour white stain, Fluorescent antibody techniques.

Fungal culture

Pathogenic Aspergillus species grow easily & relatively quickly. Sample SDA with antibiotics & without cycloheximide at 250 & 370 C examined daily for a week & twice a week. Potato dextrose agar useful for inducing sporulation. Identification monographs & taxonomic keys. Aspergillus flavus Colony velvety yellow to green / brown. Reverse golden to red brown. Conidiophores variable length, rough, pitted & spiny. The phialides single & double cover entire vesicle, point out all directions.

Aspergillus fumigatus

Colony velvety / powedery smoky green Reverse white to tan. Conidiophore smooth, phialides single ( Uniseriate), usually cover upper half vesicle, parallel to the axis of stalk.

Aspergillus niger
Colony woolly white to yellow dark brown to black. Reverse white to yellow. Conidiophore variable length, phialides biseriate arrenged in 2 rows, covered the entire vesicle fo radiate head.

Aspergillus terreus

Colony velvety cinnamon brown Reverse white to brown. Conidiophore short smooth, phialides in 2 rows compactly columnar.

Aspergillus glaucus
Colony green with areas occasionally brown. Reverse yellowish to maroon Conidiophores variable length, smooth. Phialides single, radiate to very loosely columnar.

Immunodiagnosis

Patients with Aspergilloma demonstrate precipitation IgG ab. ABPA - +ve skin test reaction to aspergillus Ag & elivated levels of IgE & specific IgE & IgG precipitating Abs. 1. Detection of Ab. Immunodiffusion test.

2. Detection of Ag

Demonstration of Abs against aspergilli may be absent in patients with immunosuppression. In patients with invasive infection, Ag detection may be helpful. Latex agglutination, RIA, ELISA, BALISA detection of soluble Ag of Aspergillus spp in serum,urine,other body fluids & even within host phagocytic cells. Western blot analysis Ag detection in CSF. Treatment & prophylaxis Amphotericin B drug of choice for disseminated Aspergillosis. Oral itraconazole.

Penicilliosis marneffei

> 250 known spp of the genus Penicilliumdistributed in nature,soil & decomposing organic debris. Non pathogenic & used in manifacturing antibiotics like P.notatum for penicillin P.griseofulvum for griseofulvin antifungal antibiotic Some species involved in allergic fungal diseases & mycotoxicosis due to their potential mycotoxin producing capacity.

Infection may be caused by Penicillium species involving various tissues & the disease hyalohyphomycosis.

P. marneffei dimorphic fungus of this genus causing Peniciliosis marneffei an emerging systemic fungal disease.
Acquired immunodeficiency syndrome acted as a trigger to the rapid spread of penicilliosis marneffei as a secondary opportunistic infection.

Pathogenesis

The ability of yeast forms of P. marneffei to proliferate inside macrophages may effectively protect organisms from the hosts cellular immune responses. Inhalation of airborne conidia alveoli Bind laminin via sialic acid dependent process (Laminin extracellular matrix glycoprotein present in basement membranes & may exposed after tissue damage) Primary site of infection is reticuloendothelial system.

3 distinct histopathological patterns in disseminated P. marneffei infections

1. Granulomatous 2. suppurative 3. Necrotizing reaction 1. Granulomatous reaction. Occurs in reticuloendothelial system BM, liver, spleen & lymphnodes of immunocompetent patients. Yeast cells invade macrophage, survive & multiply intracellularly. CMI to fungi granuloma formation ( macrophage containing P. marneffei, lymphocytes, plasma cells & occasional giant cells.) Granulomas enlarge & become necrotic in the centre & the yeast cells are released.

2. Suppurative reaction Inflammatory response to yeast forms involving accumulation of N & fibrin. Occurs typically in lungs, skin & subcutaneous tissue of immunocompetent individuals.

3. Necrotizing reaction

Focal necrosis with surrounding macrophage containing yeast forms with little / no granuloma formation. Lung, liver & skin mainly involved.

Clinical features Systemic penicilliosis marneffei chronic illness low grade fever, chills, malaise, marked weight loss, anemia, cough, generalized papular skin lesions with central umbilication, lympadenopathy & hepatosplenomegaly.

Lab diagnosis
Specimens

HIV -Negative - body fluids / tissue biopsy. Skin purulent materials from skin lesions, sputum, bonemarrow, lymphnode,liver & blood. HIV +ve Sputum, skin lesions & BM Direct examination 1. Wrights stain Wright stained impression smear of skin, lymphnode biopsy, BM aspirate. 2. Hematoxylin & eosin stain 3. Periodic acid schiff & 4.Grocott Gomoris methenamine silver stain Yeast like tissue forms of P. marneffei that multiply by transverse fission & have a prominent central septum.

Fungal cultre

sample SDA

Incubate at 250C

Fungal culture

Sample SDA & BA at 250 C greyish white colony wooly downy to granular in texture & yellow orange , yellow green, grey / green in the centre. Periphery of colony white, grey orange / purple orange & has radial folds. Wrinkled, folded & have red stained mycelia & sporulate poorly with greenish conidia Reverse bright rose color due to diffusion of a water soluble brick red pigment into the surrounding agar medium. Hyaline, short hyphae septate & branched. Conidiophores located laterally & terminally. Mycelial phase include chains of elliptical or lemon shaped, smooth walled phialoconidia.

Contd..
P. marneffei at 370 C yeast form on SDA, BHIA, CSA, SBA pinkish white LCB ellipsoidal to rectangular yeast cells that reproduce by a fission process. Treatment & prophylaxis Amphotericin B Itraconazole.

Mucormycosis

Invasive disease caused by phycomycetes Rhizopus, Mucor & Absidia. Incidence of the diseases increase as a result of widespread use of antibiotics, steroids & anti metabolites. Normally avirulent & invade tissues when general resistance is extremely low. Primary infection upper respiratory tract / nose where the spores germinate & mycelia invade the adjacent tissues orbit, sinuses & brain. Lung fungi invade arteries to cause thrombosis & infarction.

Diagnosis

Histological examination of autopsy material by presence of broad, nonseptate mycelia in tissues. SDA without cycloheximide. Mucor branched sporangiophores arising randomly along aerial mycelium Rhizoids absent Rhizopus rhizoids & sporangiophores arise in groups directly above to rhizoids.

Pneumocystosis

Pneumocystosis opportunistic fungal infection of respiratory system leading to intestitial plasma cell pneuminia (Intense plasma cell infiltrate in malnourished infants) Primarily a disease of alveoli Atypical pneuminia immunocompromised individuals. Organism Pneumocystis carinii PCP.

Life cycle of Pneumocystis carinii

1. Asexual phase of cell division. 2. Sexual phase leading to genesis of a thick walled reproductive cyst with 8 intracystic bodies. Organisms now classified as fungus but description of sexual form is still used as protozoan terminology trophozoite, cyst & sporozoite ( intracystic body )

Contd

P. carinii unicellular eukaryote. Intrapulmonary life cycle consisting of trophozoites & cysts. 3 main stages 1. Trophozoite ( vegetative) 2. Cystic 3. Sporozoite Transition phase b/w trophic & cystic stages precyst ( Sporocyst )

1. Trophozoite

Principal substance of foamy structures filling alveoli of the involved lungs. Pleomorphic, tiny bodies 2-5m in size & exist in clusters. Covered with tubular projections which facilitate attachment to epithelial cells & increase absorptive surface. Have a thin flexible, fragile external layer containing glucan & glycoprotein

Precyst ( sporocyst)

Intermediate stage of sexual phase of reproduction leading to cyst formation. Mating event first occurs to produce a zygote that initiates sporogenesis. Zygotic nucleus undergoes meiosis & a subsequent mitosis take place with in the sporocyst.

Cystic form

Frequently observed stage of organisms in clinical samples. Large, disc like structures, 4-6 m in size, oval shaped, thick walled & possessing upto 8 intracystic bodies / sporozoites. Sporozoite oval, amoeboid / peach shaped &1-2m long.

Contd.

Giemsa stain basophilic cytoplasm. Reddish purple nuclei in eosinophilic mass. Sporozoites extruded through cyst wall after the division of mature cyst. They are subsequently converted into trophozoites. Empty cysts navicular structures black by Grocotts methanamine silver stain / Reddish purple by Toluidine blue O Asexual phase Trophic forms multiply by binary fission & not by budding.

Life cycle of P.carinii

Precyst

Sexual phase

Asexual phase

Pathogenesis

Inhalation P. carinii alveoli proliferate in lung Pneumonitis.

alveolar type 1 epithelial cells &

Clinical features

Affect lung Most of the time the infection asymptomatic. Incubation period 4-8 weeks.

Clinical manifestations divided into 2 broad categories:1. Pulmonary manifestations Pneumonia, cough & dyspnoea over a period of weeks / even months. Progressive nonproductive cough followed by dyspnoea, fever, tachypnea, tachycardia & occasionally sputum production / hemoptysis. 2. Extrapulmonary manifestations. Extrapulmonary sites involved are lymphnodes, BM, spleen, liver, stomach, small intestine, pancreas, thyroid & eyes.

Labdiagnosis

Pulmonary
Immunofluorescent stain of BAL PCR Induced sputum.

Extrapulmonary

Demonstration of P. carinii cysts / trophozoites in body fluids. Direct examination Gomoris methenamine silver stain Toluidine blue O Cresyl echt violet Stain wall of the cyst Gram weigert stains Immunofluorescence assay trophozoites & cystic stages. Hematoxylin & eosin stain tissue typical, granular, foamy, honeycombed material Calcofluor white stain BAL.

Fungal culture Treatment

Not useful for routine diagnosis of P. carinii infections. Organisms has not been successfully grown.

Trimethoprim & sulfamethoxazole