• Proper diagnosis of the infections aids in the treatment procedure. So, the combination of clinical observations along with laboratory investigations is crucial for the diagnosis of the infection.
• Different diagnostics approach applied to fungal infections are:
  1. Clinical approach for diagnosis of fungal disease
  2. Conventional Methods for diagnosis of fungal disease
  3. Molecular Methods for diagnosis of fungal disease
  4. Recently developed new techniques for diagnosis of fungal disease
  5. Other Miscellaneous Methods

Clinical approaches for diagnosis of fungal disease:

• The characteristics of lesions produced by superficial and subcutaneous mycoses suggest their fungal etiology though it may resemble other diseases too.
• There is no particular sign and symptom in the case of systemic mycoses. The infection is similar to bacterial, viral, or parasitic disease.
• Modern imaging techniques aids in the early diagnosis of fungal disease.
• Based on it and clinical significance, the suspected disease can be further identified by laboratory investigations.

Conventional (microbiological) approaches for diagnosis of fungal disease: 

Based on Sites and Types of Specimen:

• For the proper diagnosis of fungal disease, the specimen type and the site of collection should be accurate.
• a) Superficial Mycoses:
  • The site should be cleaned using 70% alcohol. Before taking the specimen it should be allowed to evaporate.
  • The material should be collected at the folded square of paper because it:
  • Permits drying of the specimen
  • Reduces bacterial contamination
  • Stores for a long time without losing the viability of fungi
  • Dermatophytic lesions should be collected using the scalpel blade which is held at 90°C to the skin surface. From the edges of the active lesion, materials should be scrapped outward and collected.
  • In case of lesions on glabrous skin ( smooth or hairless), cellophane tape can be used.
  • Scalp specimen should be collected with blunt scalpel including hair-stubs, contents of plugged follicles, and scales.
  • Instead of cutting, hair should be plucked.
  • Ringworm infection of the scalp is detected by Wood’s lamp examination. The infection hair produces fluorescence.
  • To obtain a specimen for fungal culture, a hairbrush sampling technique can be used. In this technique, the scalp is brushed thoroughly which is then used for inoculation on culture media.
  • Patients should be off antifungal agents, one week before the collection of specimens in the case of onychomycosis ( fungal infection of nails ).
  • The nail should be disinfected with 70 % alcohol and clipped from the free edge.
  • Culture can also be done from the borings taken from the base of the nail.
  • Scrapings are preferred over the swab for the infection of the mucous membranes.
• b) Subcutaneous Mycoses:
  • For the microscopy and culture, scrapings taken from the superficial parts of the subcutaneous lesions may be satisfactory.
  • Contamination is common with bacteria and saprophytic fungi. The aspirated sample of pus and/or biopsy material is valuable.
  • If sporotrichosis is suspected then a biopsy may be avoided. It is because of the chances of spread of infection and the lesions will also be healed slowly.
• c) Systemic Mycoses:
  • Specimen: Biopsy, pus, feces, urine, sputum, spinal fluid, blood, scrapings, or swabs from the edge of lesions.
  • The urine taken from the catheter bag and twenty-four hours’ sputum is unsatisfactory. It is because the commensal yeast can multiply rapidly.

Collection and transport of different fungal specimens

• a) Respiratory specimens:
  • Specimens: sputum, tracheal secretions, bronchoalveolar lavage (BAL), lung biopsy
  • An early morning sputum sample is collected.
  • In case of non-productive cough, sputum may be induced
  • Bronchoscopy is also used for the examination of lesions and collection of specimens in respiratory mycoses.
  • For concentrating the specimens:
    • Add 0.5 gm of N-acetyl L-cysteine (NALC) in sodium citrate buffer which is prepared freshly.
    • Then vortex for 10-30 seconds
    • Then M/15 phosphate buffer with pH 7.0 is added. Its volume should be double the volume that is already present in the tube.
    • Then centrifuge at 1000 rpm for 15 minutes.
    • The supernatant is discarded and the sediment is used for smear preparation and in media inoculations.
  • Opportunistic pathogens like Candida can be present in the sputum as the oropharyngeal contaminant. The presence of Candida in the respiratory specimens is not clinically significant unless it is found in tissue. Bronchial brushing and lung biopsy may also be used.
• b) Cerebrospinal Fluid:
  • For the culture of CSF, it should be centrifuged and the sediment is inoculated in the agar.
  • Inhibitory agents should not be used in the media because, in normal conditions, CSF is always sterile.
  • CSF should not be processed immediately. It should be kept at room temperature or incubated at 30°C
• c) Blood Culture:
  • Biphasic Brain-Heart Infusion Agar Broth can be used.
  • Though most of the fungi can be detected within the first four days of incubation, an occasional isolate of Histoplasma capsulatum may require 10 to 14 days.
  • Blood culture media should be incubated at both the 25°C and 37°C temperature.
  • Subculture should be done at two days and seven days respectively.
  • After the seven days of subculture preliminary report and after 28 days of subculture final report is sent.
• d) Tissue, Bone Marrow, and Body Fluids:
  • From the pyogenic and the necrotic areas of the wounds, tissue specimens should be taken.
  • Povidone-iodine should not be used as the chances of isolation of fungi will be bleak. It may be applied after the collection
  • The tissue specimen should be minced before culture. Then it should be inoculated in appropriate culture media and incubated at 37°C for 4 weeks.
  • Bone marrow can be directly inoculated on media and incubated.
  • Before the culture, centrifugation should be done for the body fluid which is collected from a sterile site.
• e) Semen Culture:
  • Done in Histoplasmosis and Cryptococcosis when the disease is somewhat hidden and patient without any improvement has taken repeated courses of antitubercular treatment.
• f) Skin:
  • For dermatophyte infection, skin scrapping should be used.
• g) Nail:
  • The nail should be clipped from the discolored, dystrophic, or brittle parts.
  • Since, in the distal part of the nail, the fungus is non-viable, it fails to grow in culture.
  • Before the inoculation in the suitable culture media, nails should be cut into pieces.
  • It can be only visualized by microscopy.
• h) Hair:
  • Instead of cutting, hair needs to be plucked with forceps.
  • It is then kept in a sterile petri dish or paper envelope.
  • Since low temperature may be detrimental to the dermatophytes, it should not be refrigerated.
  • Dermatophytes are cultured in Sabouraud dextrose agar with chloramphenicol and cycloheximide.
  • Before reporting them as sterile, cultures should be incubated at 25°C, 30°C, and 37°C for a minimum period of four weeks.
• i) Urine culture:
  • Twenty-four hour’s urine is not useful for the fungal culture.
  • If a delay is anticipated, it should be refrigerated at 4°C. It can be kept for up to 12 hours.
  • Before the culture, the urine sample needs to be centrifuged and the sediment is inoculated.
  • Culture media should contain antibacterial antibiotics to prevent bacterial contamination and isolate the fungi in pure form.
• j) Vaginal Secretions:
  • Clinical features along with the direct smear of secretions help in the diagnosis of vaginal candidiasis.
  • About 20 % of healthy females have yeasts as the normal flora, so culture can be misleading too.
  • Cultures can help monitor the therapy and for the management of chronic recurring diseases.
• k) Stool culture:
  • Biopsy of tissue is done than the culture of stool specimens for the diagnosis of fungal infections in the gastrointestinal tract.
  • Since yeast colonizes as the commensals in 40% of healthy individuals and 75% of compromised patients, positive cultures may be misleading.
• l) Eye:
  • Corneal scrapings are taken in the case of keratomycosis.
  • Kimura’s spatula is used aseptically to take the sample from the base and margin of the ulcer.
  • 4% xylocaine is used as the local anesthetic.
  • In keratomycosis, aspiration of hypopyon is done using the sterile needle.
  • In the case of fungal endophthalmitis, the posterior chamber may also be aspirated.

Sample preparation for laboratory diagnosis of Fugal disease

a) Direct Microscopic examination:

• The direct demonstration of fungi in the clinical specimen is taken as the “gold mine”.
• Fungi can be observed directly in the clinical specimens by:
  • Wet Mounts
  • Histopathology
  • Frozen-Section Biopsy
  • Fluorescent-Antibody staining

i)  For Wet Mounts preparation:

• Slide and tube KOH Mounts are prepared.
• Sodium hydroxide may also be used as an alternative.
• After the partial digestion with 10-20% KOH, specimens can be examined in wet mounts.
• On the slide, specimens like hair, nail, skin are mounted in KOH under the coverslip.
• Materials are digested under 5-20 minutes depending on the thickness.
• Under the low flame, it can be lightly warmed but should not be overheated.
• DMSO can be supplement in KOH to increase clearing of the fungi in the skin scrapings.
• Calcofluor and Blankophor are also used to prepare wet mounts. It offers excellent visualization of the fungi.
• The fungal cell wall under ultraviolet illumination, fluoresce brightly under the fluorescence microscope.
• For the detection of Cryptococcus wet mounts of India ink and Nigrosin staining are used.
• For evaluation of the viability of fungi, neutral red staining can be used.
• For the demonstration of fungi like Malassezia , Candida spp. and dermatophytes, Vinyl Adhesive Tape (VAT) preparation is also used.

ii) For Histopathology:

• Demonstration of the fungi in the tissue sections aids in diagnosis.
• If histopathology shows neither the fungal elements nor the tissue reaction, the fungal isolate can be the contaminant.
• Histopathological examination of the biopsy and autopsy specimens is the best method for the diagnosis of mycotic infections.
• In the histopathology laboratory, H&E stain is a routine procedure.
• For demonstrating fungi in tissue-specific fungal stains, such as Periodic Acid-Schiff (PAS), Grocott-Gomori’s methenamine silver stain, and Gridley stains are widely used.
• For a demonstration of capsular material of Cryptococcus and endospores and sporangia of Rhinosporidium seeberi, Mayer’s mucicarmine can be used.
• To demonstrate acid mucin, Alcian blue staining may be done.
• Disadvantage of use of special stain:
• Mask the natural color of fungal elements making it difficult to decide if it is hyaline or naturally pigmented.

iii) Frozen-Section Biopsy:

• This modality is adopted for making an intra-operative diagnosis of suspected malignancy.
• In mucormycosis and fungal rhinosinusitis, good results have been obtained.
• It is a useful tool to guide the extent of surgical debridement and/or onset of antifungal therapy.
• Frozen sections describe the morphology and infectious process.
• The evaluation of the frozen section has an important function in surgical pathology for diagnosis in tissue while the patient is undergoing an operative procedure.

iv) Fluorescent-Antibody Staining:

• It is used for the detection of fungal antigen in clinical material such as pus, blood, CSF, tissue impression smears, and in paraffin sections of formalin-fixed tissue.
• For the sputum specimen, it is less satisfactory.
• Advantage: detects fungus when few organisms are present.

b) Fungal Culture:

• Commonly employed medium is Emmons’ modification of Sabouraud Dextrose Agar.
• To minimize bacterial contamination, gentamicin and chloramphenicol can be supplemented in the media.
• To inhibit the saprotrophic fungi, cycloheximide can be supplemented in the media.
• Some fungi like Cryptococcus, Talaromyces marneffei, Aspergillus, or Scytalidium are sensitive to cycloheximide. So, cycloheximide should not be used in this case.
• For the isolation of the particular pathogens, a special medial can be used.
• For isolation of neoformans, Birdseed agar, Niger seed agar, Sunflower seed agar can be used.
• neoformans develop brown-colored colonies.
• Candida , Aspergillus spp. , Rhizopus spp. grows within 24-72 hrs of incubation.
• For the dimorphic fungi, incubation should be done at:
• 37°C for the isolation of yeast.
• Room temperature for the isolation of molds.

Techniques for detection of fungi from culture:

• • Scotch Tape preparation
  • Wet Mount preparation
  • Lactophenol cotton blue staining
  • Germ Tube production test

i. Scotch Tape preparation:

• Over the growth of filamentous fungi, the sticking surface of scotch tape is touched.
• Then it is placed on a glass slide and observed under the microscope.
• Characteristics shape and arrangement of spores, type of hyphae and conidia, etc can be observed.
• Advantages:
  • It can be prepared quickly and easily.
  • Slide can be preserved for a longer time.
  • Fungi can be seen with their own pigmentation.
• Disadvantages:
  • Sample won’t be adequate if it is not pressed firmly.

ii. Wet Mount preparation:

• It is done for the observation of spores that were not seen by scotch tape.
• Example: Microconidia of Histoplasma capsulatum can be observed by the wet mount method.
• Procedure:
  • Bent wire of 90° is used.
  • Small portion is cut at the intermediate point of the center and periphery of the isolated colony.
  • It should contain a small amount of the supporting agar.
  • Then a drop of KOH is added.
  • It is covered by a coverslip and observed under the microscope.
• Disadvantages:
  • If pressure is applied to the coverslip, the characteristic arrangement of spores will be disrupted. In such a condition, definitive identification of fungi can’t be done.

iii. Lactophenol cotton blue staining:

• Fungal cytoplasm can be stained against which the walls of hyphae can be readily seen.

iv. Germ Tube production test:

• It is used for the definitive identification of fungi within 3hrs.
• Example: Yeast, Candida albicans
• Germ tube is the elongated tube-like structure originating from yeast cells.
• Nucleus is absent.
• It has half the width and 3-4 times greater the length of the yeast cell.
• Procedure:
  • Suspend the inoculums from the isolated colony of yeast cells in 0.5 ml sheep or rabbit serum.
  • Incubate the tube at 37°C for 3-4 hrs.
  • Then take the suspension and observe under the microscope.

c) Serological test:

• Agglutination tests:
  • Whole-cell agglutination (WCA)
  • Latex particle agglutination (LPA)
• Passive haemagglutination (PHA)
• Immunodiffusion (ID)
• Counterimmunoelectrophoresis (CIE)
• Complement fixation (CF)
• Indirect fluorescent antibody (IFA)
• Enzyme-linked immunosorbent assay (ELISA)
• Radioimmunoassay:
  • Solid-phase
  • Competitive RIA
• Complement fixation test (CFT):
  • Coccidioidomycosis, Histoplasmosis, Blastomycosis
  • Presence of the polysaccharide capsular antigens of  Cryptococcus neoformans in the CSF can be detected by the latex agglutination test.

Skin Test for fungal disease:

• Individuals infected with the Histoplasma or Coccidioides develop a Delayed-Type-Hypersensitivity (DTH) reaction with in1-14 days and may persist for many years
• Appropriate fungal antigen is inoculated intradermally.
• Within 24-72 hours induration and erythema occur.
• Skin test is used for:
• Establishing the etiological diagnosis
• Conducting the epidemiological survey
• Immunological classification of the subjects like atopic and non-atopic groups
• Find out the immunological status of the patients as in the immunodeficiency diseases

Various skin tests and fungal antigens used:

Molecular approaches for diagnosis of fungal disease:

1. Hybridization methods
2. Amplification methods
   • Broad range PCR
   • Nested PCR
   • Multiplex PCR
   • Nucleic acid Sequence-based Amplificatio
   • Fluorescence Resonance Energy Transfer
   • TaqMan
   • Molecular Beacons

3. Sequencing-based methods
   • Sanger’s Sequencing
   • Pyrosequencin
   • Next-Generation Sequencing
   • Ultra-Deep Sequencing
   • DNA Bar Coding

Recently Developed new Techniques for diagnosis of fungal disease:

• AccuProbe
• PNA FISH
• MALDI-TOF Mass Spectrometry
• Quantamatrix Multiplexed Assay Platform

Miscellaneous Methods:

• Point of Care Testing
• T2Candida Panel
• Biosensors for Medical Mycology
• Epidemiological Markers of Fungi
• Quality Control in Medical Mycology

Epidemiological Markers Used in Fungal Infections:

• Phage typing
• Secreted lethal factor typing
• Serotyping
• Morpho typing
• Mating typing
• Resistotyping
• Biotyping
• Protein electrophoresis (Immunoblot)
• Isoenzyme typing
• Restriction fragment length polymorphism (RFLP)
• Karyotyping
• Nucleic acid probes

The post Different approaches for Fungal Disease Diagnosis: Clinical, conventional and molecular approaches appeared first on Online Biology Notes.

• Mucormycosis is an opportunistic fungal infection which is rare but serious infection.
• It is also called as black fungus infection which has been reported recently in the recovered patients of COVID-19.
• Mucormycosis is sometimes called as zygomycosis.
• It is caused by mucormycetes which is group of fungi.
• Several fungi belonging to the phylum Glomeromycota cause it. They are the saprophytic fungi and are present ubiquitously in the soil and environment.
• The first authentic human case of mucormycosis was reported in 1855. It was described by Kurchenmeister in a patient of neoplastic lung on the basis of histopathology.
• Pulmonary mucormycosis was described by Furbringer in 1876 for the first time.
• Mycology:
  • Mucormycetes is a group of lower fungi.
  • Their hyphae are generally non-septate, sparsely septate or pauci-septate.
  • Reproduction occurs asexually by sporangiospores and/or by means of conidial development.
  • Reproduction occurs sexually by the formation of zygospore.

Common causative agents of Mucormycosis:

• Rhizopus arrhizus ( Old name Rhizopus oryzae )
• Rhizopus microspores var. rhizopodiformis
• Mucor racemosus
• Rhizomucor pusillus
• Lichtheimia corymbifera ( Mycocladus corymbiferus or Absidia corymbifera )
• Apophysomyces elegans
• Cunninghamella bertholletiae
• Saksenaea vasiformis
• Cokeromyces recurvatus
• Syncephalastrum recemosum
• Absidia corymbifera

Modes of Transmission of Mucormycosis:

•  Mucormycosis is caused by:
  • Inhalation of spores present in air.
  • Ingestion from contaminated food
  • Inoculation into skin amd soft tissues by trauma Trauma inoculates in skin and soft tissue.
  • Person to person and between animals and persons transmission doesn’t occur.

Virulence factor of Rhizopus arrhizus

• Rhizopus arrhizus is the commonest encountered mucormycetes which has several virulence factors such as:
  • Angioinvasive nature
  • Growth at the body temperature or above
  • Destructive enzymes production
  • Dormant spores
  • Active ketone reductase system
  • Hydroxamate siderophores

Pathogenesis of Mucormycosis:

• Infection can occur by either inhalation, percutaneous inoculation or ingestion of fungal spore.
• Phagocytes, polymorphonuclear neutrophils and macrophages play a critical role in patients of mucormycosis.
• When the spores are inhaled into lungs then alveolar macrophages ingest it.
• After ingestion, macrophages inhibit the germination of spores but the activity to kill them is limited. So, they can escape out from the antifungal activity of the macrophages and germinate into mycelia form .
• Then the expected to work against the fungi are polymorphonuclear neutrophils and peripheral monocytes.
•  The leukocytopenic patients are susceptible to this disease because they lack the sufficient cell like these which perform the antifungal activity.
• There is also the risk of mucormycosis to the patients of ketoacidosis ( metabolic acidosis due to accumulation of ketone bodies in the blood).
• It may be due to the release of iron bound to the protein.
• The low serum pH due to the ketoacidosis decreases the phagocytic effect of macrophages, chemotactic and oxidative burst of neutrophils.

Risk factor for Mucormycosis

1. Diabetes patients with diabetes kitoacidosis:
   • The patients of diabetes are at risk of mucormycosis when they have got ketoacidosis.
   • It is because diabetic patient have the high glucose level in their blood and Rhizopus  can thrive in it too.
   • Dye to the presence of active ketone reductae system Rhizopus can survive in high glucose and acidotic conditions.
   • Due to the impaired glutathione pathway, these patients also have decreased phagocytic activity.
   • Though the exact phenomenon is unknown, it might be due to the metabolic abnormalities in combination with diabetic patients.
   • Similarly, the invivo growth of fungus is not supported solely by hyperglycemia o acidosis.
   • Acidosis without hyperglycemia has been reported with the invasive mucormycosis.
   • Rhizopus gets inhibited at the normal serum but the growth is stimulated in the patients of diabetic ketoacidosis.
   • It has been found that the patients on dialysis and iron overload, who are being treated with deferoxamine, an iron chelator are susceptible to mucormycosis.
   • It may be due to Mucorales which to obtain more iron use the siderophore as the chelator.
2. Other risk factors includes:
   • Neutropenia ( abnormal decrease of the neutrphil in circulating blood )
   • High-dose systemic steroids
   • Protein-calorie malnutrition
   • Solid organ and bone marrow transplantation
   • Immunodeficiency
   • Leukemia (blood cancer caused by increase in White blood cell )
   • Intravenous drug use

Histopathology of Mucormycosis:

• The primary histopathological features of mucormycosis are:
  • Angioinvasion: ( spread of tumor into a blood vessel, also called as vascular invasion)
  • Thrombosis: (formation of the fibrinous clot in any part of the circulatory system)
  • Ischemia: (deficiency of blood supply to an organ or tissue)
  • Tissue necrosis: (death of tissue in the living body)

Clinical features of Mucormycosis:

• It is the fatal disease which progresses rapidly due to its involvement in blood vessels and being angioinvasive in nature.It is of six types:
  • Rhinocerebral Mucormycosis
  • Pulmonary Mucormycosis
  • Cutaneous Mucormycosis
  • Gastrointestinal Mucormycosis
  • Isolated Renal Mucormycosis
  • Disseminated Mucormycosis

i) Rhinocerebral Mucormycosis:

• It is the most common and fulminating type of mucormycosis.
• If it is left untreated it may lead to fatal consequences within a week.
• It occurs when the inhaled spores germinates in the nasal passage then making spreading from the nasal mucosa to the turbinate bones, paranasal sinuses, orbit and palate.
• It then spreads upto the brain where its invades blood vessels massively causing the major infarct (area of necrosis due to deficient blood supply )
• The imaging techniques like CT or MRI shows the destruction of bones.
• Mostly it is caused by Rhizopus arrhizus and others etiological agents are also reported.
• Symptoms includes:
  • Facial pain
  • Head-ache
  • Lethargy
  • Loss of vision
  • Brownish, bloodstained nasal discharge
  • Black eschar on palate ( due to hemorrhage and tissue necrosis )
  • Fixed and dilated pupil
  • Global proptosis and ptosis
  • Dysfunction of cranial nerves ( especially 5^th and 7^th nerves )
  • Extensive and rapid destruction to the surrounding tissues.
  • Sometimes the infection may spread to other parts like lungs, gastrointestinal tract, skin.
  • Symptoms of orbital (related to orbit which is the socket in the skull where eyeball is present) mucormycosis:
  • Chemosis (oedema of the conjunctiva producing the pronounced ring around the cornea)
  • Periorbital cellulitis
  • Ophthalmoplegia ( partial or total paralysis of the muscles that moves the eye)
  • Proptosis ( forward displacement of any organ )
  • Ptosis ( drooping or falling or lowering out of eyelids or other organs )
  • Abrupt visual loss
  • Orbital pain
  • Facial hypoesthesia ( decreased sensitivity particularly of touch )
  • Infection may spread from orbit to brain which may lead to frontal lobe necrosis and abscesses formatin.
  • Invasion of the fungi in blood vessels causes necrotizing (causing the death of tissues ) vasculitis ( inflammation of blood vessels ) which results in the thrombosis (formation of fibronous clot in any part of circulatory system ) of vessel lumen.

ii) Pulmonary Mucormycosis :

• It occurs through the inhalation of the sporangiospores.
• The invasion of the blood vessel may lead to the destruction of lung parenchyma.
• From the onset of the illness to the terminal stage of illness it takes about 1 to 4 weeks.
• Symptoms includes:
  • Chest pain
  • Dyspnea
  • Hemoptysis
  • Infiltration can be seen in the chest x-ray which shows the progressive infectiom from an anatomical site to the multiple adjoining areas on the same lung.
• The pulmonary mucormycosis and its extension is best determined by the HRCT (High Resolution Computed Tomography) scan.
• When the patients have a reverse halo sign on CT of the chest, this entity is suspected.
• Rarely the members of the order Mucorales are involved in forming the fungus ball like that of aspergilloma.
• It has been reported that the Rhizopus caused the hypersensitivity pneumonitis in Scandinavian Sawmill workers (so-called Wood trimmer’s disease) and in farm workers.

iii) Cutaneous Mucormycosis:

• It occurs in the patients of severe burns which spread to the underying tissue.
• The severe underlying necrosis develops.
• In the diabetic patient cutaneous lesions may occur at the site of injection.
• It may occur when the contaminated surgical dressings or the splints are applied in the skin.
• Clinical manifestation may vary from the pustules or vesicles to wounds with wider areas of necrotic zones.
• Lesions in the early stages resembles ecthyma gangrenosum. In this condition, cotton like growth can be seen over the surface of the tissue. This clinical sign is called as “hairy pus”.
• Cutaneous mucormycosis can be primary infection or it can be secondary to the disseminated form.
• For the primary cutaneous mucormycosis, Saksenaea and Apophysomyces should be suspected as the causative agent. It occurs when there is contamination of the wound and traumatic injured areas with dust or soil. After few days blistering and necrotic lesions occurs.
• Apophysomyces tends to invade the vascular lumen causing thrombosis leading to ischemia and finally tissue necrosis.

iv) Gastrointestinal  Mucormycosis:

• Rarely occurring mucormycosis which is about 7 % of all the mucormyccosis.
• It most often involves in stomach.
• It occurs in patients with extreme malnutrition.
• Ingestion of foods like fermented milk, porridge, and alcohol made from corn and herbal products  contaminated with fungal spores causes gastrointestinal mucormycosis.
• Lesions occurs  in stomach which are followed by colon, ileum and esophagus.
• Invasive mucormycetes either colonize or invade gastric mucosa.
• Non-specific symptoms include:
  •  Abdominal pains
  • Diarrhea
  • Hematemesis
  • Melena
• Patients on dialysis who are undergoing treatment with desferrioxamine are also reported to develop mucormycosis.
• Causative agents of gastrointestinal mucormycosis:
  • Lichtheimia corymbifera
  • Basidiobolus ranarum

v) Isolated Renal Mucormycosis:

• It is one of the emerging clinical entity.
• It is an unusual cause of renal infarction, if not detected in time may be fatal.
• It infects the kidney.
• Symptoms includes:
  • Flank pain
  • Fever
  • Pyuria
  • Infarct of renal tissue leading hematuria
  • Initially patents are assumed to be infected with bacterial pyelonephritis and does not respond to the antibiotics.
  • Acute pyelonephritis occur in immunocompromised patients.

vi) Disseminated Mucormycosis:

• Portal of entry is skin which may be from trauma. It then invades blood vessels and disseminates.
• Dissemination may occur to different parts of body which affects lungs, kidney, gastrointestinal tract, heart and brain.
• Lungs is the most commonly involved site.
• Symptoms includes:
  • Pneumonia
  • Stroke
  • Subarachnoid hemorrhage
  • Brain abscess
  • Cellulitis
  • Gangrene of skin
  • Gastrointestinal bleeding
  • Peritonitis
  • Acute myocardial infarction
  • Hepatitis
• Disseminated Mucormycosis is caused by:
  • Apophysomyces
  • Saksenaea
  • Rhizopus
• Cerebral infection is distinct from the rhinocerebral mucormycosis because it leads to the focal neurological signs.
• Disseminated infections by Mucor species:
• Endophthalmitis
• Prosthetic mitral valve mucormycosis

Laboratory diagnosis of mucormycosis:

• Diagnosis is slightly difficult because of rapid and fulminant course of disease.
• Doubtful significance of isolates because they are usually encountered as laboratory contaminants.

Specimens:

• Nasal discharge
• Biopsy
• Sputum
• Bronchoalveolar lavage
• Necrotic lesions
• CSF

Direct Examinations: by Microscopy

• KOH Wet mount preparation of the nasal discharge or biopsy material shows  broad, non-septate, thick walled ribbon-like hyphae with wide-angle or right-angle branching at irregular intervals which are the characteristics of  hyphal elements of mucormycetes. It is  different  from other fungi. For example: Hyphae of Aspergillus, Fusarium and/or Scedosporium spp.have  slender hyphae with regular dichotomous branching and frequent septation.
• Microscopic morphology of Saksenaea vasiformis shows a typical trumpet-shaped sporangiophore.
• Calcofluor white staining
• Well stained in H & E but poorly stained with PAS
• Other staining: Gridley, Gram’s and GMS
• Immunochemical staining methods
• Frozen section evaluation while patient is undergoing operative procedure

Fungal Culture:

• Specimens should be directly inoculated in the culture media avoiding grinding. It is because the hyphal elements are prone to physical damage.
• Mucormycetes grows at conventional media like Sabouraud Dextrose Agar (SDA) with antibiotics at 25°C and 37°C.
• BHI broth can be used for biopsy material.
• Portion of tissue can be kept in water which is added with  malt yeast broth. Then molecular techniques can be done directly from the sample.
• Fibrous and cotton-candy growth in media. Also called as “lid-lifters”.
• Apophysomyces spp. , Saksenaea spp. and Mortierella wolfii under routine cultural conditions on SDA, CMA or PDA may produce only sterile hypahe without any spores.
• For the induction of sporulation various induction techniques are designed:
• Czapek Dox agar and slide culture in humid atmosphere
• Padhye and Ajello technique: use of saline agar (1%) supplemented with grass and hay
• Soil extract medium

Serological test:

• No routine serological test

Treatment of Mucormycosis:

• Rapid correction of underlying predisposing factor of the host like diabetic ketoacidosis
• Surgical debridement of necrotizing tissue
• Antifungal therapy
• Consideration of adjunctive therapy such as hyperbaric oxygen
• Drugs: Intravenous amphotericin B, Posaconazole and Isavuconazole

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• Procedure:
  • Sterile slide—> add the molten agar and allow to solidify —> cut the material making two half —-> place cover slip ——> seal the coverslip with wax or petroleum jelly making small area free at the side if cut —-> buried in a soil gently in a tray à allowed to incubate for few days ——-> remove gently ——> remove coverslip and observe under microscope.
• 1. Bait method:
  • Many molds have quite specific nutrient requirements and are specialized to use materials that other fungi use with difficulty or not at all.
  • We can isolate fungi by presenting a particular substance to the environment for colonization then later recovering it for isolation of the fungi that occupied it.
  • Different types of baits might be pieces of wood, insects, carrot chunks, plastics, hair etc.
  • The bait can be submerged in a specific habitat in nature or in a moist chamber.
  • E.g. To isolate dermatophytes, we can place a hair on moist soil in a most chamber and examine it periodically for sporulating molds.
  • Many cellulolytic fungi can be isolated from cellulose containing material.
  • Then the mycelium is transferred into medium like PDA, SDA, Czapels agar for cultivation.
• Soil dilution plating:
  • If hyphae cannot be dissected from field material for identification, they must be induced to grow cut as visible colonies onto an artificial culture medium.
  • The dilution plate method, in which a dilution series is prepared from a soil suspension and the selected dilution incorporated in an agar medium (PDA-SDA).
  • After few days of incubation, colonies will appear in varying densities and the number of spores present in the original sample can be calculated.

II. Method of isolation of fungi from Organic components:

• Fungi can also be isolated from rotten fruits, from roots etc.
• Surface sterilized —-> crushed in distilled water —–> inoculate in suitable agar medium.

III. Method of Fungi isolation from clinical specimens: Isolation of pathogenic Fungi

• Fungal cultures are microbiology laboratory tests to detect or role out the presence of fungi in specimens taken from patients or animals.
• The laboratory employs optimal conditions to grow and identify any fungus present in the specimen.
• The specimen is cultured on various agar media and then incubated and examined.
• Specimen could be the skin scrapping, nail scrapping, infected hair etc.
• Plate exposure method:
  • Useful method for airborne fungi.
  • Certain molds are likely to get their spores into the air and these spores may be serve as an infective agent of plant diseases and same allergies.
  • These can be isolated by —> plate containing PDA is exposed to air for few minutes —-> covered à incubate at 25^oC or room temperature for few days —-> observe.
• Imprint method:
  • Fungi present on leaf surface or root of the plant can be isolated by pressing leaf or root on a suitable culture media and then incubate at 28^oC or at room temperature.
• Other methods:
  • Direct transfer, moist chamber, direct soil plate method etc.

Identification of fungi:

• Criteria for identification of Fungi

Methods of Identification of Fungi

1. Wet mount (tease mount) method for fungal hyphae identification:

• Procedure of wet mount preparation:
  • Take a grease free slide and plate with fungus culture.
  • With the help of sterile scalpel or 90^o bent wire, remove fungal colonies from plate (which might contain a small amount of supporting agar.
  • Place the portion of culture into a slide to which has been added to a drop of lactophenol cotton blue or aniline blue.
  • Place the coverslip into position and apply the gentle pressure to dispose the general growth and agar.
  • Examine microscopically.
• Drawbacks:
  • Characteristic arrangement of spores might be disrupted when pressure is applied to the coverslip.

2. Adhesive (scotch) tape preparation for fungal spore identification:

• Procedure of scotch tape preparation:
  • Touch the adhesive side of a cellophane tape to the surface of the colony.
  • Adhere the tape to the surface of a microscopic slide to which has been added a drop of lactophenol cotton blue or aniline blue.
  • For the characteristic shape and arrangement of the spores, microscopical examination is required.
  • This method allows to observer in a way it sporulates in culture and easy method to identify organism.
  • In instances where spores are not observed, a wet mount should be made as a backup step.

3. Microslide culture technique for fungi identification :

• Procedure of microslide culture technique:
  • Take sterile petri dish—-> remove its lid —–> place sterile filter paper over it and add distilled water to moisten the filter paper —–> put glass slide over glass rod ——> add 5mm² PDA agar on glass slide from agar plate ——> inoculate spores of fungi on that agar and cover with coverslip —–> incubate at room temperature for 7 days ——> remove coverslip and mounted in next slide and observe under microscope.

4. Coverslip culture technique for fungi identification :

• This technique is simple, less time-consuming technique which produces high quality permanent mounts and is suitable for clinical isolate identification, student teaching, examination of fungi at different stages of their development without disturbing the arrangement of spores and hyphal structure.
• It is advantageous over slide culture technique that if the first preparation fails to demonstrate adequate sporulation, there are still left to be examined of weekly intervals.
• The ability of aerial mycelia to adhere to a glass surface has been utilized as a basis of this technique.
• Procedure of coverslip culture method:
  • Insert 4-5 coverslips on petri-plate containing POA at an angle of 45^o —–> inoculate organism at the point of media and coverslip ——>   incubate the plate at room temperature for 7 days ——> after incubation remove the coverslip gently and mounted with lactophenol cotton blue and observe under microscope.

Isolation and Identification of Fungi from soil and clinical specimens

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• Mycotoxins are toxic chemical produced by some pathogenic strain of fungi.
• Many strains of fungi contaminate the food and produce potent mycotoxin in food.
• Disease resulting from ingestion of mycotoxin in food is called mycotoxicosis. E.g. mushroom poisoning.
• One important characteristic of most mycotoxin is that they are heat stable.
• Therefore, if mycotoxin is produced in food, it is not damaged easily during normal cooking of food.
• Most of the mycotoxin cause mutation and are associated with various types of cancer.
• Mycotoxins are secondary metabolites of fungi.
• Metabolites produced by microorganisms are divided into two types i.e. primary and secondary metabolites.
• Metabolites produced by certain microorganisms that serve as a growth factor for other microorganisms are called as primary metabolites. E.g. vitamins, amino-acids, carbohydrate etc.
• Primary metabolites are produced during log phase.
• Metabolites produced by certain microorganisms which are not needed for growth if other organisms are called secondary metabolites. E.g. toxin, antibiotic etc.
• Secondary metabolites are produced during stationary phage of growth.

Types of mycotoxin:

1. Aflatoxin:

• It is produced by Aspergillus flavus, A. parasiticus and some Penicillium spp.
• It is located from variety of mold contaminated food like peanuts, rice etc.
• Two major types of toxin include B₁ and G₁ that show blue and green fluorescence when exposed to UV lights.
• Other types of aflatoxin include B₂, G_2, M₁, M₂, and P₁ which are derivatives of B₁ and G_1.
• Among them B₁ is most toxic.
• After ingestion, aflatoxin binds to chromosomal DNA and causes point mutation and frame-shift mutation.
• By inducing mutation, they cause cancer of liver.
• Aflatoxin is toxic to wide variety of animals like cat, chicken, cattle, human beings etc.

2. Patulin:

• It is produced by wide variety of mold including Penicillium expansum, P. patulum, P. melini, P. equinum, Aspergillus clavatus, A. terreus etc.
• It is isolated from many molds contaminated food like bread, sausages, fruits etc.
• It is white crystalline solid.
• It is sensitive to SO₂ and alkali but resistant to acid.
• Patulin binds to NH₂ and -SH functional group of biomolecules and causes chromosomal aberration.
• It affects wide variety of animals and plants as well as bacterial cells like E. coli and Staphylococcus aureus.
• Therefore, originally it was classified as antibiotics.
• Tissue damage caused by patulin include oedema of brain, hemorrhage of lungs, damage of blood capillary, spleen and kidney.

3. Penicillic acid:

• It is produced by Penicillium roqueforti, P. cyclopium, P. morteneii, Aspergillus flavus, A. ochraceus.
• It is isolated from many molds contaminated food like tobacco.
• Penicillic acid binds to –SH and –NH₂ group of biomolecules forming covalent bond.
• It is carcinogenic and affects wide variety of animals including rat.

4. Citrinin:

• It is produced by Penicillium citrinum, P. viridicatum and other species.
• It is isolated from many mold contaminated foods like- polished rice, bread, meat and meat products
• It is carcinogenic to wide variety of animals.

5. Ochratoxin:

• There are atleast seven types of structurally related ochratoxin, of which type A is the most common and most toxic.
• It is produced by many Aspergillus and Penicillium species like- A. ochraceus, A. alliaceous, A. mellis, P. viridicatum, P. cycloplum.
• Like other mycotoxin, it is heat stable and is not damaged during cooking of food.
• When ochratoxin is ingested it induces mitosis and cause cancer of kidney.

6. Sterigmatocystin:

• It is produced by Aspergillus versicolor, A. nidulus, A. regulosus and other.
• It causes cancer of liver by inhibiting DNA synthesis.

Types of Mycotoxin

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• Tinea versicolor is a common, mild, but often recurrent infection of the stratum corneum because of the lipophilic yeasts of the genus Malassezia.
• It is also termed as Pityriasis versicolor.
• Less commonly, these organisms cause serious systemic infection in low-birth-weight infants and other immune-compromised and debilitated individuals.
• Geographical distribution:
  • The disease is distributed worldwide, but is much more prevalent in tropical and subtropical regions.

What causes Tinea versicolor?

• Etiology:
  • Till now three Malassezia species were identified:
    – two lipid-dependent species, M. furfur and M . sympodialis, and one non-obligate lipophile, M. pachydermatis.
  • The genus has now been enlarged into seven species following genomic and ribosomal sequence comparisons of a large number of human and animal isolates. It comprises of the three former taxa, M. furfur, M. pachydermatis and M. sympodialis, and four new taxa, M. globosa, M. obtusa, M. restricta and M. slooffae.
  • Six of the seven Malassezia species are lipid- dependent with the exception of M. pachydermatis.
  • Molecular methods have been found to be a rapid and reliable method for the differentiation of Malassezia species.

Epidemiology of tinea versicolor:

• Malassezia species form part of the normal microbial flora of the skin of humans and other warm-blooded animals and most infections are endogenous in origin.
• The prevalence of skin colonization with these organisms depends on age, anatomical site, and, to a lesser degree, race.
• The condition of skin colonization rises from around 25% in children to almost 100% in adolescents and adults.
• In post-pubertal individuals the density of colonization is greater in anatomical sites that contain pilosebaceous glands.
•  Malassezia species have been isolated from 100% of samples from the backs of adults, but from only 75% taken from the face and scalp.
• It is assumed that colonization with Malassezia species primarily occurs at the time of puberty when the sebaceous glands become active and the concentration of lipids on the skin increases.
• The accurate conditions which results in the development of pityriasis versicolor and other forms of superficial Malassezia infection have not been defined, but host and environmental factors both seem to be essential.
• The lesions of pityriasis versicolor and seborrhoeic dermatitis have a preference for sites well supplied with sebaceous glands, such as the chest, back and upper arms.
•  It has been seen that patients with seborrheic dermatitis have higher concentrations of lipids on their skin than do other individuals.
• In-case if the non-cohabiting members of the same family have developed pityriasis versicolor it suggests a genetic pre-disposition.
• The relationship between Malassezia species and the immune system is essential.
• It is suggested by increased incidence of Malassezia folliculitis and seborrhoeic dermatitis in persons with the acquired immune-deficiency syndrome (AIDS) and those receiving corticosteroid or other immunosuppressive treatment.
• Pityriasis versicolor is worldwide in distribution, but is most prevalent in hot, humid tropical and subtropical climates, where 30-40% of the adult population may be affected.
• In temperate climates, the disease affects 14%of the adult population, but is most common during the hot summer months.
• Malassezia folliculitis is also more prevalent in tropical countries and, in temperate regions.
• It is more common during the summer months.
• Transmission of Malassezia species is occurs, either through direct contact or via contaminated clothing or bedding.
• In practice, however, infection is endogenous in most cases and transmission between persons is uncommon.

Clinical manifestations of Tinea versicolor:

• Pityriasis versicolor is a harmless condition.
• Lesions:
  • The lesions are characterized by patches of fine brown scaling, especially on the trunk, neck and upper portions of the arms.
  • The lesions may become confluent and progress to cover large areas of the trunk and limbs.
  • In the tropics the lesions are more commonly localized on the face.
  • In light-skinned subjects, the affected skin may appear darker than normal.
  • The lesions are light pink in colour but grow darker, turning a pale brown shade.
  • In dark-skinned or tanned individuals, the affected skin loses colour and becomes depigmented.
  • The same patient may have lesions of different shades, the colours depending on the thickness of the scales, the severity of the infection and the inflammatory reaction of the dermis.
  •  The amount of exposure to sunlight also affects the shade of lesions.
  • The disease is aggravated by sunlight and sweating.
  • The clinical manifestations of tinea versicolor in immunocompromised persons are alike to those observed in normal individuals.
  • However, the lesions are often more erythematous and seem to be raised.
  • In most cases the lesions show a pale yellow fluorescence under Wood’s light, allowing the extent of the disease to be examined.

Differential diagnosis of tinea versicolor:

• Hyperpigmented lesions must be differentiated from a number of conditions, including erythrasma, naevi, seborrhoeic dermatitis, pityriasis rosea and tinea corporis.
• Hypopigmented lesions can be confused with pityriasis alba and vitiligo.

Lab diagnosis of Tinea versicolor

• Specimens:
  • The scraping from the affected skin acts as material for direct microscopic examination.
• Microscopy:
  • Pityriasis versicolor lesions consists of a mixture of budding yeast cells, typical of the organism seen in normal skin sites.
  • They appear as numerous short, broad unbranched hyphae.
  • These hyphae, which are assumed to be the same organism in its pathogenic phase, are not observed at unaffected skin sites or in culture.
  • Direct microscopic examination of scrapings from lesions is enough to allow the diagnosis of pityriasis versicolor if clusters of round or oval budding cells and short hyphae are seen.
• Culture:
  • Because Malassezia species are part of the normal cutaneous flora, their isolation in culture does not contribute to diagnosis.
  • Besides, with the exception of M . pachydermatis, these organisms cannot be isolated on routine mycological media unless lipid is added.

Treatment of tinea versicolor:

• If left untreated pityriasis versicolor will remain for long periods.
• Most patients respond to topical treatment, but more than 50% relapse within 12 months.
• Oral treatment is recommended in patients with extensive or recalcitrant lesions.
• There are various topical agents which can be used to treat pityriasis versicolor.
• Selenium sulphide (2%) shampoo should be applied at night and washed off the following morning.
• The treatment should be repeated 1 and 6weeks later.
• Ketoconazole shampoo should be applied once daily for 5 days.
• It should be left in contact with the lesions for 3-5min before being rinsed off.
• Other topical imidazoles, such as bifonazole, clotrimazole, econazole, miconazole and sulconazole, should be applied morning and evening for 4-6weeks.
• Topical terbinafine should be applied to the lesions each morning and evening for 2 weeks.
• Pityriasis versicolor is often a difficult disease to clear and topical preparations may need to be reused at intervals to ensure that the infection is eradicated.
• Oral antifungal treatment should be employed for patients with extensive lesions or recalcitrant infection that is unresponsive to topical treatment.
• Both itraconazole (200mg/day for 1 week) and ketoconazole (200mg/day for 1 week) are effective treatments.
• Oral griseofulvin and terbinafine are inactive in patients with pityriasis versicolor.

Tinea (Pityriasis) versicolor: etiology, clinical manifestations diagnosis and treatment

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• The term tinea unguium refers to dermatophyte infections of the fingernails or toenails.
• Onychomycosis is a less specific term used to describe fungal disease of the nails.
• In addition to dermatophytes, it can be caused by a number of other moulds and by Candida species.
• This condition is common in men and older adults and is seen more in people with weakened immune system such as individuals with diabetes, psoriasis, peripheral vascular disease.
• Geographical distribution:
  • Distributed worldwide.

Epidemiology of Tinea unguium:

• Etiology:
  • Onychomycosis is the most usual nail disease in adults, responsible for up to 50% of all nail diseases.
  • The most commonly asssociated dermatophyte is the anthropophilic species, Trichophyton rubrum, followed by T. mentagrophytes var. interdigitale.
  • Onychomycosis is most prevalent in older adults but, due to the limited number of large-scale studies, the actual incidence of the condition is difficult to assess.
• Various risk factors for onychomycosis have been identified. They include:
  – male gender
  – increasing age peripheral vascular disease
  – hyperhidrosis
  – tinea pedis and dystrophic nails
• The difference between the incidence of onychomycosis in men and women might be a reflection of the degree to which individuals are concerned about the appearance of their nails.
• Likewise the higher incidence of onychomycosis in older individuals could be due to the greater likelihood of younger patients seeking treatment at an earlier stage.

Clinical manifestations of Tinea unguium:

• There are four identified clinical patterns of dermatophyte onychomycosis:
  • distal and lateral subungual onychomycosis
  • superficial white onychomycosis
  • proximal subungual onychomycosis
  • total dystrophic onychomycosis
• It is very rare to see a patient with finger- nail infection without toenail involvement.

1. Distal and lateral subungual disease:

• It is the most common presentation.
• The affected nail becomes thickened and discoloured, with a varying degree of onycholysis (separation of the nail plate from the nail bed).
• Toenails are more commonly affected than fingernails.
• The infection of toenails is usually secondary to tinea pedis, whereas fingernail infection generally follows tinea manuum, tinea capitis or tinea corporis.
• Tinea unguium may infect a single nail, more than one nail, both fingernails and toenails, or in exceptional circumstances, all of them.
• The first and fifth toenails are more frequently affected, probably because footwear causes more damage to these nails.
• Fingernail infections are usually unilateral.

2. Superficial white onychomycosis:

• The infection initiates at the superficial layer of the nail plate and migrates to the deeper layers.
• Crumbling white lesions is seen on the nail surface, especially the toenails.
• These slowly spread until the entire nail plate is engaged.
• This condition arises normally due to Trichophyton mentagrophytes var. interdigitale infection.

3. Proximal subungual onychomycosis:

• Most cases of proximal subungual onychomycosis involve the toenails.
• This infection originates in the proximal nail fold, with subsequent penetration into the newly forming nail plate.
• The distal region of the nail stays normal until late in the course ofthe disease. T. rubrum is often the cause.
• Although proximal subungual onychomycosis is the least common presentation of dermatophyte nail infection in the general population, it is common in persons with the acquired immunodeficiency syndrome (AIDS).
• It has sometimes been considered a useful marker of human immunodeficiency virus (HIV)infection.
• In AIDS patients, the infection often spreads rapidly from the proximal margin and upper surface of the nail to produce gross white discoloration of the plate without obvious thickening.

4. Total dystrophic onychomycosis:

• These different clinical forms of nail disease may eventually lead to total dystrophic onychomycosis, in which the whole of the nail bed and nail plate is involved.
• The pattern of infection is variable.
• In occasional cases, pockets of tightly packed hyphae develop in the subungual space leading to a dense white lesion visible beneath the nail.
• The type of infection can be resistant to antifungal treatment without prior removal of the lesion.
• This appearance is most often seen in the great toenail.
• In total dystrophic onychomycosis, the infected nail generally begins to lift up from the nail bed due to an accumulation of debris (hyperkeratosis) under the nail.
• The nail becomes thickened and yellow or brown in colour.
• The nail plate may crumble, beginning at the free end.
• The infection may be confined to only one nail, but more commonly several nails on one or both feet are affected.
• Most patients have concurrent interdigital or moccasin tinea pedis, and some may also have tinea cruris.
• Most patients tell about nail discomfort, especially when cutting, and many may experience pain during activities such as running and jogging.

Differential diagnosis of Tinea unguium:

• The clinical signs of tinea unguium are often difficult to distinguish from those of a number of other infectious causes of nail damage, such as Candida, mould or bacterial infection.
• Unlike dermatophytosis, candidosis of the nailsusually initiates in the proximal nail plate, and nail fold infection (paronychia) is also present.
• Bacterial infection, particularly when due to Pseudomonas aeruginosa, tends to result in green or black discoloration of nails.
• Sometimes bacterial infection can coexist with fungal infection and may require treatment in its own right.
• Many other non-infectious conditions can produce nail changes that mimic onychomycosis, but the nail surface does not usually become soft and friable as in a fungal infection.
• Non-fungal causes of nail dystrophies include onychogryphosis, psoriasis, chronic eczema and lichen planus.

Lab diagnosis of Tinea unguium

• Microscopy:
  • The clinical diagnosis of fungal infection is confirmed by direct microscopic examination.
  • It is sometimes possible to distinguish Cundidu infection, or infection due to moulds such as Scopuluriopsis brevicuulis from tinea unguium.
• Culture:
  • Isolation of the aetiological agent in culture will permit the species of dermatophyte involved to be determined.
  • It is essential to inform the laboratory if nail material is suspected of being infected with non- dermatophyte moulds, so that duplicate plates with and without cycloheximide (actidione) can be inoculated.
  • The results of culture can be positive even if microscopic examination is negative, but it is more common for microscopic examination to be positive while culture is negative.

Treatment of Tinea unguium:

• Tinea unguium is a tough condition to treat.
• In general, onychomycosis should be treated with an oral anti- fungal agent.
• However, localized distal nail disease can sometimes be treated with topical antifungals such as amorolfine, ciclopirox, or tioconazole solutions.
• Topical: amorolfine should be taken at weekly intervals or tioconazole, twice daily for 6 months for fingernails and 9–12 months in case of toenails.
• Oral: itraconazole 2 or 3 pulse treatment 400 mg/day for 1 week in 4, or continuous 200 mg/day for 3 months.
• Oral griseofulvin applied for 4–8 months.
• Oral terbinafine should be taken 250 mg/day for 6–12 weeks for fingernails, 12 weeks or longer for toenails.
• Oral fluconazole 150–450 mg once weekly for 6–9 months in toenail infections, 3 months for fingernails.  
• Ciclopirox solution must be applied once daily for at least 6 months.
• Response rates are low compared with oral agents used in nail infections, and topical agents are usually reserved for infections of limited extent or where they can be combined with nail removal.
• Two new oral antifungal agents, terbinafine and itraconazole, are effective in onychomycosis and have been approved for use in adults with this indication.
• Cure rates with these agents approach 80% in most trials.
• The allylamine terbinafine is now the treatment of choice for patients with dermatophytosis of the finger- nails or toenails.
• Treatment with oral terbinafine will also clear associated cutaneous lesions without additional topical treatment.
• Terbinafine has proved to be effective in HIV-infected individuals and no interactions or significant adverse effects related to the drug have been reported.
• Itraconazole is other effective alternative for dermatophyte nail infection.
• This drug persists in nail for at least 6 months and pulsed treatment (in which 1week of treatment is alternated with 3 weeks without treatment) has given encouraging results.
• Fluconazole has proven to be less effective than terbinafine or itraconazole for onychomycosis.
• With griseofulvin, up to 90% of fingernail infections can be cured in 4-8 months, but its low cure rate of 20-40% in toenail infections means that it is now less appropriate than terbinafine or itraconazole.
• The application of 40% urea ointment to the nail under occlusion for 4-7 days allows the nail to be excised after this treatment.

Prevention:

• Many individuals with onychomycosis are unaware of their fungal infection and some believe that dystrophic nails are simply part of the ageing process.
• Patients should be made aware that the disease is contagious, and can be spread to those around them.
• Individuals with untreated or partially treated plantar or interdigital tinea pedis should be informed of the risk of developing onychomycosis.
• Measures that can help to control nail infection or prevent reinfection include:
• application of antifungal powders to the feet after bathing
• wearing of absorbent cotton socks
• frequent changing of socks
• application of antifungal powders to footwear
• avoidance of occlusive footwear that increases sweating
•  and wearing of protective footwear in hotels, changing rooms, gymnasiums and other public facilities.
• It is also important to keep the nails as short as possible and to avoid sharing nail clippers with other household members.

Tinea unguium: etiology, clinical manifestation, diagnosis and treatment

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• The term tinea manuum refers to dermatophyte infections of one or both hands.
• Tinea refers to ringworm and manuum refers being on hand.
• Geographical distribution:
  • It is distributed worldwide.

Epidemiology of tinea manuum:

• Etiology:
  • The commonest causes of tinea manuum are anthropophilic dermatophytes Trichophyton rubrum and T. mentagrophytes var. interdigitale.
  • Less commonly, the condition is caused by zoophilic dermatophytes, such as Microsporum canis and T. verrucosum, or geophilic dermatophytes, such as M. gypseum.
• Dermatophytosis of the hands can be acquired as a result of contact with another person, an animal or soil, either through direct contact, or via a contaminated object such as a towel or gardening tool.
• Autoinoculation from another site of infection can also take place.
• Manual work, profuse sweating and existing inflammatory conditions, such as contact eczema, are predisposing factors.

Clinical manifestations of tinea manuum:

• Tinea manuum is usually unilateral, the right hand being more commonly affected, and is characterized by a dry scaling eruption of one palm.
• Lesions on the dorsum of the hand or in the interdigital spaces appear similar to those of tinea corporis.
• They have a distinct margin and central clearing may occur.
• Two clinical forms of palmar infection may be distinguished:
  • the dyshidrotic or eczematoid form
  • the hyperkeratotic form
• It is not un- usual for one form to turn into the other.
• The dyshidrotic or eczematoid form of tinea manuum:
  • In this condition, periods of partial remission occurs between successive exacerbations.
  • It is characterized in the acute stage by vesicles which tend to appear in an annular or segmental pattern.
  • These are localized to the edges of the hand, to the lateral and palmar aspects of the fingers, or to the palm itself where the vesicles are rather larger, tense, often single, and contain a clear viscous fluid.
  • Removal ofthe top of the vesicles exposes a pinkish-red weeping surface with fine scaling margins.
  • Pruritus, formication and burning are common symptoms.
• The hyperkeratotic form of tinea manuum:
  • It is a subacute or chronic condition.
  • It begins as a succession ofadjacent vesicles which desquamate.
  • This results in a reddened scaling lesion which is round or irregular in outline and enclosed by a thick white squamous margin from which extensions run straight towards the centre.
  • Once the chronic stage is reached, the disease involves most or all of the palm and fingers.
  • The dry hyperkeratosis, with underlying erythema, readily causes fissuring in the palmar creases.
  • The hand has a mealy appearance because of the furfuraceous scales that remain adherent to the horny layer. This is thickened and black in the creases.

Differential diagnosis of tinea manuum:

• Tinea manuum needs to be differentiated from other forms of dyshidrosis.
• This condition is usually bilateral or even symmetrical.
• In its particular form, clear vesicles are placed on the lateral and volar aspects of the fingers as well as on the palm.
• There is little or no inflammation of the base.
• Dyshidrotic eczema is usually bilateral, but mycological examination is frequently required to differentiate it and other conditions (such as psoriasis, whether pustular or not) from tinea manuum.

Lab diagnosis of Tinea manuum:

• Microscopy:
  • Direct microscopic examination of infected material, such as vesicle tops and contents and skin scales, should expose the branching hyphae characteristic of a dermatophyte infection.
• Culture:
  • Isolation of the aetiological agent in culture will allow the species of fungus engaged to be detected.

Treatment of Tinea manuum:

• Tinea manuum often coexists with tinea pedis.
• Local treatment with a topical imidazole, such as clotrimazole, econazole, miconazole or sulconazole, or an allyllamine, such as naftifine or terbinafine, will often suffice.
• Oral terbinafine (250mg/day for 2-6 weeks), or itraconazole (100mg/day for 4 weeks) should be advised in cases that fail to respond to topical treatment,.

Prevention:

• It is essential to recognize potential sources of infection and, in the case of household pets, take the animal for treatment of suspected dermatophytosis to prevent transmission or recurrence of zoophilic tinea manuum,
•  Infection with anthropophilic species, such as T. rubrum, seldom follows transmission from another infected body site, such as the feet, groin or nails.
• These sites should be examined and treated if dermatophytosis is present in order to prevent recurrence.

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• It is commonly termed as athlete’s foot.
• The term tinea pedis refers to contagious dermatophyte infections of the feet.
• These infections often involve the interdigital spaces, but chronic diffuse peeling can affect the entire sole.
• It is mostly seen in individuals whose feet are sweaty because of tightfitting shoes.
• Geographical distribution:
  • It is distributed worldwide and is most commonly found in hot, tropical, urban regions.

 Epidemiology of Tinea pedis:

• Etiology:
  • The commonest causes of tinea pedis in Europe and North America are the anthropophilic dermatophytes Trichophyton rubrum, T. mentagrophytes var. interdigitale and Epidermophyton floccosum.
  • T. rubrum is the principal cause of chronic tinea pedis.
• Tinea pedis being a contagious condition it is easily transmitted from person to person.
• Transfer within house- holds has been reported, but the main spread occurs in communal bathing places and showers.
• The infection is usually taken by walking barefoot on contaminated floors.
• Tinea pedis is a modern disease, linked with the wearing of occlusive footwear.
• The growth of the fungus requires heat and moisture and the frequency of infections in temperate climates increases during the summer months.
• Tinea pedis is more frequently seen in men than women.
• It often initiates in late childhood or adolescence and is most frequent between the ages of 20 and 50.
• The disease is most prevalent among individuals of places where bathing facilities are shared such as swimming pools and fitness centres, involved in sporting activities, or living in closed communities, such as residential schools and prisons.
• Tinea pedis has been found to affect about 15-20% of adult males.
• The prevalence of infection is much higher among industrial workers such as coal miners, who are exposed on a daily basis to infection.
•  It has been estimated that about one-third of patients with tinea pedis also have a fungal nail infection.

Clinical manifestations of tinea pedis:

• Bilateral involvement of tinea pedis is more common but may be unilateral too.
• Three main clinical forms may be distinguished:
  • 1)  acute or chronic interdigital infection
  • 2) chronic hyperkeratotic (moccasin or dry type) infection of the soles and lateral borders of the feet
  • 3) vesicular (inflammatory) infection of the instep and sole.
  • 4) Occasional patients develop an acute ulcerative infection of the soles.
• It is usual for patients to develop a combined clinical presentation involving two or even three of these forms of tinea pedis.

1. Acute or chronic interdigital infection:

• It is the commonest form of tinea pedis characterized by itching, peeling, maceration and fissuring of the toe webs.
• The skin beneath the whitish build-up of debris may appear red and weeping.
• A foul odour is sometimes present.
• The cleft between the fourth and fifth toes is most often involved, but the infection may spread to adjacent areas of the feet, including the toenails.
• In a patient with supposed chronic tinea pedis, an absence of nail involvement makes the diagnosis of dermatophytosis questionable.

2. Chronic hyperkeratotic infection:

• It is characterized by areas of pink skin covered with fine white scaling.
• Vesicles and pustules are absent.
• Hyperkeratosis is usually limited to the heels, soles and lateral borders of the feet.
• The infection may seem patchy in distribution or involve the entire weight-bearing surface, in which case the disease is termed ‘moccasin’or ‘dry type’ tinea pedis.
• The condition is usually bilateral and may be asymptomatic.

3. Vesicular infection:

• Vesicular or vesiculobullous tinea pedis is characterized by the development of vesicles, usually beginning on the sole, the instep and the interdigital clefts.
• The eruptions vary in size, may be isolated or coalesce into vesicles or bullae, and are initially filled with a clear fluid.
• After rupturing, the lesions dry, leaving a ragged ring-like border.
• The disease may resolve without treatment, but often recurs.

4. Ulcerative infection:

• It is characterized by maceration and ulceration of large areas of the soles.
• White hyper- keratosis and strong odour are common.
• Bacterial superinfection, usually with gram-negative organisms, is frequent and should be taken into account in treating this condition.
• In certain parts of the world, concomitant mould, Candida and/or bacterial infection is relatively common in patients with tinea pedis.
• These conditions usually represent secondary infection following fissuring or maceration of a toe cleft.
• The secondary infection may induce inflammation and further maceration.

Differential diagnosis of tinea pedis:

• The symptoms and clinical signs of tinea pedis can be difficult to distinguish from those of a number of other infectious causes of toe web intertrigo such as bacterial or Candida infection.
• Non-infectious conditions that resemble tinea pedis include contact dermatitis, eczema and psoriasis.
• Candidosis most often presents as mild interdigital erosion and maceration.
• It sometimes occurs in patients with diabetes mellitus and is more common in hot climates.
• It often occurs in conjunction with a dermatophyte infection.
• Other moulds which produce lesions indistinguishable from tinea pedis include Scytalidium dimidiatum (Hendersonulatoruloidea) and S. hyalinum.
• Dermatophytosis of the feet is often associated with itching.
• In contrast, gram-negative bacterial infection tends to produce more painful inflammatory lesions, often with marked erosion of the skin.
• The clinical appearance of erythrasma (Corynebacterium minutissimum infection) is difficult to distinguish from interdigital tinea pedis.
• Laboratory tests should be performed in any patient with foot lesions of undetermined origin.

Lab diagnosis of tinea pedis:

• Microscopy:
  • Direct microscopic examination of infected material should confirm a clinical diagnosis of dermatophyte infection.
  • It is sometimes possible to distinguish a yeast infection from tinea pedis.
• Culture:
  • Isolation of the aetiological agent in culture will permit the species of fungus involved to be determined.
  • Media containing cycloheximide (actidione)should not be used if infection with a Scytalidium species is suspected.
• Wood’s light examination of the lesion should be performed to establish whether the patient has erythrasma.
• The coral red fluorescence characteristic of this condition does not, however, exclude coexistent tinea pedis.

Treatment of tinea pedis:

• Interdigital tinea pedis will often respond to topical treatment with an imidazole compound, such as bifonazole, clotrimazole, econazole, isoconazole, miconazole, oxiconazole, sulconazole, terconazole or tioconazole, or an allylamine, such as naftifine or terbinafine.
• Terbinafine, applied to the toe clefts and other affected sites
  –  morning and evening for upto 2 weeks.
• Imidazoles, used for up to 4 weeks.
• To help prevent the infection from spreading, it is beneficial to apply cream to the soles.
• The recurrence rate is quite high, and chronic infection with minor scaling may persist.
• Exacerbations of previous infection may also occur.
• The common infections are mixed fungal and bacterial infections of the feet.
• Thus, topical antifungal preparations that are effective against dermatophytosis and candidosis, and those possessing some antibacterial action (such as miconazole) are often advised.
• Oral treatment with terbinafine (250mg/day for 2-6 weeks) or itraconazole (100mg/day for 4 weeks) should be given in addition to topical treatment (which should be continued for 8 weeks or longer) if the disease is extensive, involving the sole and dorsum of the foot, or if there is acute inflammation.
• However, relapse is common.
• Chronic tinea pedis is often linked with infection of the nails.
• Insufficient treatment of onychomycosis may result in reinfection of the feet.
• Tinea pedis is a chronic condition which seldom resolves if left untreated.
• Exacerbations, that occurs in the summer, alternate with partial remissions.
• Nevertheless, the prognosis in general remains benign.

Prevention of tinea pedis:

• It is essential to inform the patient of measures that can help to control the infection or prevent reinfection.
• These consist of
  –  antibacterial soaps
  – daily bathing of the feet
  – followed by thorough drying of the toes and inter- digital spaces
  – liberal application of antifungal powders to the feet after bathing
  – wearing of cotton socks to absorb sweat
  – frequent changing of socks
  – application of antifungal powders to footwear
  – avoidance of occlusive footwear that increases sweating
  – wearing of protective footwear in hotels, changing rooms, gymnasiums and other public facilities.
• Educating infected individuals not to expose others to their infection by not walking barefoot on the floors of communal changing rooms and by avoiding public baths and showers can help to reduce the spread of tinea pedis.
• Some helpful preventive measures are frequent hosing of the floors of public baths and the discouraging of antifungal foot dips near communal baths.

Tinea pedis: etiology, clinical manifestation, diagnosis and treatment

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• The term tinea cruris refers to the dermatophyte infections of the groin, perianal and pubic region.
• It is commonly known as jock itch and is a form of ringworm.
• It is characterized often by red and itchy rash in warm areas of body and it is prone to people with diabetes and are obese.
• Geographical distribution:
  • It is distributed worldwide.

Epidemiology:

• Etiological agent:
  • The dermatophytes most often encountered in tinea cruris are the anthropophilic species, Trichophyton rubrum and Epidermophyton fzoccosum less commonly Trichophyton mentagrophytes and Trichophyton verrucosum are involved.
• The development of the infection is favoured by warm moist conditions resulted by maceration and occlusion of the skin in the groin.
• The disease is more prevalent in tropical climates.
• Tinea cruris is mostly seen in men and is seen 3 times more than women.
• The infection often follows spread from another infected site, such as the feet, in the same individual.
• It can also be spread from person to person through direct contact, or through indirect contact via contaminated fomites such as towels, clothing and bed linen.
• Tinea of the groin is a highly contagious condition and outbreaks of infection have sometimes occurred in schools and other closed groups such as sports teams.

Clinical manifestations of tinea cruris:

• Tinea cruris is usually seen as one or more rapidly spreading, erythematous lesions with central clearing on the inside of the thighs.
• The lesions, which tend to coalesce, have a raised erythematous border which encloses a brown area of scaling.
• Patients often complain of intense pruritus.
• Scratching may result in small satellite lesions which sometimes fuse with the primary lesion altering its outline.
• The infection may spread from the inside of the thigh to the scrotum, penis, natal cleft and gluteal folds, as well as to the anterior and posterior aspects of the thighs.
• Localized scrota1 infection is quite common: the clinical signs are often inconspicuous.

Differential diagnosis of Tinea cruris:

• Tinea cruris can be difficult to differentiate from other causes of erythematous groin lesions, such as bacterial and Cundidu intertrigo, erythrasma, psoriasis and sebhorrhoeic dermatitis.
• Women with a crural dermatosis are often infected with Cundidu, whereas men are more likely to have a dermatophyte infection.
• Thus, laboratory tests should be performed in any patient with groin lesions of undetermined origin.

Lab diagnosis

• Microscopy:
  • Direct microscopic examination of infected material should reveal the branching hyphae characteristic of a dermatophyte infection.
• Culture:
  • Isolation of the aetiological agent in culture will allow the species of fungus involved to be determined.

Treatment of Tinea cruris

• Most patients with tinea cruris will respond to local antifungal treatment within 2-4 weeks.
• Topical imidazoles, such as clotrimazole, econazole, miconazole or sulconazole, and allylamines, such as naftifine or terbinafine, should be applied morning and evening for at least 2 weeks.
• Hygiene measures such as thorough drying and using separate towels for the groin area should prevent spread.
• There is a recurrence in 20–25% of patients.
• If this happens, patients should be given further antifungal treatment and advice about non-pharmacological control measures should be repeated.
• To prevent re-infection, treatment should be continued for at least 1 week after the lesions have cleared.
• Oral treatment should be given if a patient has extensive lesions, or fails to respond to topical treatment, or has tinea pedis as well.
• Itraconazole (100mg/day for 2 weeks) and terbinafine(250mg/dayfor2-4 weeks) have proved more effective than griseofulvin (10mg/kg per day for 2-6 weeks).

Prevention:

• To prevent reinfection following treatment, the patient should be advised to dry the groin thoroughly after bathing and to use separate towels to dry the groin and the rest of the body.
• The feet should be examined and treated if tinea pedis is present.
• Occlusive or synthetic garments should be avoided.
• In case if the patient is obese, weight loss might be of benefit by reducing chafing and sweating.

Tinea cruris: etiology, clinical manifestation, diagnosis and treatment

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• The term tinea corporis refers to dermatophyte infections of the trunk, legs and arms, except the groin, hands and feet.
• It is characterized by either inflammatory or noninflammatory lesions on glabrous skin.
• Geographical distribution:
  • It is distributed worldwide however, it is mostly prevalent in tropical and sub-tropical regions.

Epidemiology:

• Etiological agents:
  • Epidermophyton floccosum and many species of Trichophyton and Microsporum are causative organisms of Tinea corporis.
  • Infection with anthropophilic species, such as E. floccosum or T. rubrum, often follows spread from another infected body site, such as the feet.
• Tinea corporis caused by T. tonsurans is seldom observed in children with tinea capitis and their close contacts.
• Tinea corporis can also occur following contact with infected household pets or farm animals.
• Less commonly, it results from contact with wild animals or contaminated soil.
• Microsporum canis is a common cause for human infection, and Trichophyton verrucosum infection is frequent in rural districts.
• Tinea corporis is more common among individuals who are in regular contact with animals or with the soil.
• Human-to-human transmission of infection with geophilic or zoophilic species is uncommon.

 Clinical manifestations of Tinea corporis:

• Tinea corporis may affect any body site, but infections with zoophilic species are more likely to occur on exposed parts such as the face, neck and arms.
• Patients may complain of mild pruritus.
• The clinical manifestations are variable, depending on the species of fungus involved and the extent of progression.
• i) Lesion:
  • In typical cases, round scaling lesions which are dry, erythematous and clearly circumscribed are seen.
  • The fungus is more active at the margin of the lesions and hence this is more erythematous than the middle, which tends to heal earlier.
  • As the first ring of advancing infection continues to spread outwards, it may become surrounded by one or more concentric rings or arcuate patterns.
  • Adjacent lesions may fuse producing gyrate patterns.
  • In some instances, particularly when a zoophilic dermatophyte is involved, the lesion can become indurated and pustular.
  • The lesions of tinea corporis are often more extensive but less obvious in immunosuppressed individuals.

Differential diagnosis of Tinea corporis:

• Tinea corporis can be difficult to distinguish from other causes of erythematous, scaling skin lesions such as discoid eczema, impetigo, psoriasis and discoid lupus erythematosus.
• Thus, laboratory tests should be performed in any patient with skin lesions of undetermined origin.

lab diagnosis of Tinea corporis

• Specimens:
  • Specimens should be collected from the raised border of the lesion by scraping outwards with a blunt scalpel held perpendicular to the skin.
  • If vesicles are present, the entire top should be submitted for examination.
• Microscopy:
  • The branching hyphae is a characteristic of a dermatophyte infection which must be revealed during direct microscopic examination.
• Culture:
  • Isolation of the aetiological agent in culture will allow the species of fungus engaged to be determined.
  • This will give information as to the source of the infection and support in the selection of appropriate treatment.

Treatment of Tinea corporis

• Topical antifungal preparations are the treatment of choice for localized lesions.
•  Numerous imidazole compounds (including bifonazole, clotrimazole, econazole, isoconazole, miconazole, oxiconazole, sulconazole, terconazole and tioconazole) and two allylamine compounds (naftifine and terbinafine) are available in different countries in a number of topical formulations.
• All provide similar high cure rates (70-100%) and side- effects are unusual.
• The drugs should be taken morning and evening upto 2-4 weeks.
• After the clearation of lesions, treatment should be continued for at least 1 week and the medication should be applied at least 3 cm beyond the advancing margin of the lesion.
• Oral treatment is recommended if the lesions are extensive or the patient fails to respond to topical preparations.
• Itraconazole (100mg/day for 2 weeks) and terbinafine (250mg/day for 2-4 weeks) have proved more effective than griseofulvin (10mg/kg per day for 4-6 weeks).

Prevention:

• For the prevention of spreading or recurrence of zoophilic tinea corporis, it is essential to recognize potential sources of infection and, in the case of household pets, take the animal for treatment of suspected dermatophytosis.
• Infection with anthropophilic species, such as T. rubrum, sometimes follows spread from another infected body site, such as the scalp, feet, hands or nails.
• These sites should be examined and treated if dermatophytosis is present in order to prevent reinfection.
• Tinea corporis can also result from close body contact with other infected individuals, who should be identified and treated if possible.

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