Diagnostic Mycology UPHMO PDF

DIAGNOSTIC: MYCOLOGY Patrick Jumar S. Buenaflor, RMT, MSMT(c) Transport and storage of specimen treat all specimen potentially hazardous rapid transport delayed coolbox - refrigerate 4’C - not frozen - tissue, add sterile NSS refrigeration is not an excuse to delay processing...

DIAGNOSTIC: MYCOLOGY Patrick Jumar S. Buenaflor, RMT, MSMT(c) Transport and storage of specimen treat all specimen potentially hazardous rapid transport delayed coolbox - refrigerate 4’C - not frozen - tissue, add sterile NSS refrigeration is not an excuse to delay processing Transport and storage of specimen aseptic condition right side right specimen subtutaneous biopsy cutaneous phlegm sterile containers double protection ziplock Ziplock leak proof screwcap covered with parafilm riplock riplock ex urinecontainer Wolbox yelo 240C  General Recommendations Use agar slants Disinfect all bench tops daily b4 after Do not keep potted plants usually fungilove soil Kaya baka pumunta Do not allow dust to accumulate canbe a vehicle of fungi Examine fungal culture in a hood fungi bacteria in spread Use biosafety hood Autoclave all cultures before discarding  KOH method common methodin the Philippines Fungal Identification Direct Examination don'tneed to culture ex sa balat scrapemyslide addCott the examineunderuterrope Culture for 2weeks 1month – Macroscopic Examination colony – Microscopic Examination Biochemical Test Direct Examination Microscopic examination of fungi in clinical specimen Wet mount with or w/o KOH dye potassiumhydroxide insphalpen KOH w/ or w/o Parker Super Quink blue-black ink underlight microscope  KOH (with or w/o ink) preserve fungal structure Protein, fats and polysaccharides are GINSTE halana makita element solubilized Of KOH parafungal Tissue clears E Fungal cell wall – alkali resistant due to glucans and chitinsno Gentle heating and warming hastens reaction Kott DH D on my m KOH w/ visible hyphae KOH wet mount hair Calcofluor white paraputingpaper nay Optical brightener dye used as whitening agent in textile and paper industry Emmmm Binds cellulose and chitin Yeti in Fluoresce when exposed to UV and short wavelength visible light Uses fluorescent microscope Calcofluor white Alcian Blue Detects C. neoformans in CSF I cneotormans Acid Fast Detection of Mycobacteria and Nocardia I Nocardia Giemsa blood Examination of bone marrow and blood smear I Yffophil 1 yeastcellot Candida Histoplasma capsulatum Gram Stain detection of bacteria and fungi O India Ink INDIRECT MILNOSIOPY Detection of C. neoformans in CSF Used darkfield microscopy Backgroundgung inistain ii To Methylene Blue cutaneous imposes r Detection of fungi in skin scrapings Additive in KOH alternative Methenamine Silver Silverstring will make the fungi black detection of fungi in histologic sections biopsies SEARCH Neuromin girovituit IotatunginHillpatient Papanicolaou forfemale examination of secretions presence of malignant cells Periodic Acid Schiff alsointissue blood sample detection of fungi histochemical stain for glycogen Wright’s Stain Examination of bone marrow or PBS or Asperginus Problems with Direct Examination Negative results never rules out a fungal infection wakuhaan wanton mating my specimen False + findings occur as: - fat droplets as yeast - collagen fibers as Nocardia filaments Equivocal results must be reviewed by more than one reader Cultures should be performed on clinical specimen  Fungal Culture Sufficient amount Pina paede alitin pride weeks month tumubo 1-2 ml to inoculate 3-4 media concentration of fluids by centrifugation or filtration Temperature: Incubate – RT 25⁰C yeart mold Roomtemp to identify it Incubator – BT37⁰C To encourage dimorphic fungi to produce yeast form haventria hasa baba naman bring fungi pay usually Time of Culturing Fungi Dermatophytes: fungal skin infection Canan buni alipunga – at least 2 weeks Opportunists: like asperginuscadda amaranth with same – less than a week Systemic: – 4-6 weeks; if H. capsulatum is suspected up to 12 weeks Prevent dehydration: controlevaporation – thick volume of agar, inside plastic bag to Observe every 2-3 days for growth – wait up to full term of incubation Fungal Culture Media autrient agar Primary recovery media parag Differential test media Primary recovery media BHIA B of prevent contamination I BHI with antibiotics bacteria Fungemia blood samples BHI blood culture for contaminants orbitthegrowthof Inhibitory mold agar Mycosel or mycobiotic agar F SABHI a Sabarant Potato flake agar Yeast extract PO4 agar Primary recovery media samples mi Brain Heart Infusion Agar Primary recovery of saprobic and pathogenic fungi BHI with antibiotics fungiinskin f Recovery of fungi exclusive of dermatophytes BHI biphasic in b.c. bottle Recovery of fungi from blood 0 Ñfhfffhfftaaaa BHI for Blood Culture SABHI BHIA Dermatophyte test medium Screening medium for dermatophytes Inhibitory Mold Agar Recovery of fungi exclusive of dermatophytes Potato Flake Agar Saprobic and pathogenic fungi  Potato Flake Agar Dermatophyte test medium onwards Inhibitory Mold Agar Mycosel or Mycobiotic Agar Primary recovery of dermatophytes SABHI Saprobic and pathogenic fungi Yeast Extract PO4 Agar Recovery of fungi exclusive of dermatophytes  Mycobiotic Agar Agarside coloring Reverse Yungilationto Yung Rulay titingnan SABHI recovery fungifor pathogenic fungi pathogenic Yeast extract agar Cotomy knay Differential Test Media Ascopore agar Corn meal agar Cottonseed conversion agar Czapek’s agar Niger seed agar Potato dextrose agar Rice medium Trichophyton Agar Urea agar Yeast fermentation broth/Yeast nitrogen base Differential Test Media certain yeast species specififora Ascopore Agar Detection of Ascopores in ascoporogenous yeast such as Saccharomyces sp. Corn Meal Agar with Tween 80 and Tryptan blue Identification of C. albicans by chlamydospore production. Identification of C. albicans microscopic morphology Differential Test Media Cottonseed Conversion Agar Conversion of dimorphic fungus B. dermatitidis from mold to yeast form Czapek’s Agar Recovery and differential identification of Aspergillus sp. Czapek’s Agar Bread Differential Test Media Niger Seed Agar Identification of C. neoformans Nitrate Reduction Medium Detection of nitrate reduction in confirmation of Cryptococcus sp. Potato Dextrose Agar Demonstration of pigment production by T. rubrum; preparation of microslide culture red pigmentation PDA Differential Test Media Rice Medium Identification of M. audounii Trichophyton agars 1-7 Identification of members of Trichophyton genus Urea Agar Detection of Cryptococcus spp.; differentiate T. mentagrophytes from T. rubrum; detection of Trichosporon spp. Rice medium parang am Trichophyton Agar Fungi Differential Test Media Yeast Fermentation Broth Identification of yeasts by determining fermentation Yeast Nitrogen Base agar Identification of yeasts by determining carbohydrates assimilation Identification of culture Morphology macroscopic reverse pigment colony microscopic examination kuna colony stain ftp.ihology Physiology (urea utilization, thiamine requirement etc.) howthe yeast heartwiththechemicals in the agar Multiple Test System (API AUX System) Biotech – nuclear probes Co É 0000 fungal sample Nh adddito incubate 2am I  Fungal Culture can be examined microscopically using lactophenol cotton blue stain hindidimanamitsa direct so culture culturestaring lang haypatrobo lactophenol add examine under minnope Specimens Abscess Blood CSF & other sterile body fluids Hair Tdh Provide Nail clippings/shavings Tissue Sputum Specimens stool urine (SPA, clean catch or catheterized) vaginal secretions Skin scrapings T.it hhaoroakho clean lesion with Of scalpel or sterile alassstide periphery (70% ROH) If inflamed or w/ fissures clean with sterile water Use sterile scalpel or microscope slide edge  Scrape active edge center heals first Collect moist exudate for Candida Place between 2 slides, envelope, petri dish Store at room temp dapat makita white powder Nail clippings/shavings clean w/ 70% ROH scrape 4-5 times, discarded From proximal to distal Debris under nail plate should also be collected Small clippings are preferable, for easier KOH digestion Nail clippings/shavings 10-30% KOH; overnight digestion between 2 slides, envelope, petri dish Store at room temp Clean nail w/ alcohol pad Clip toe nail as far as back consider patient’s comfort papGat Use 1 mm curette to collect subungual debris Good specimen in envelope Sputum 1st morning after brushing & mouthwash Wide mouth container 12-72h collection, unacceptable 4’C storage/transport Hair Examine scalp w/ Wood’s lamp for fluorescing hair Sterile forceps, tape Collect fluorescent hair or broken hair Scrape scalp scales from affected area Place between 2 slides, in an envelope or petri dish store RT Tissue collected by physician must consist of normal tissue & both the center and edge lesion H. capsulatum: center ngJugat B. dermatitidis: edge Between 2 moist sterile gauze pads, sterile tubes or vial w/ sterile water Not formalin In willdestroy the fungi Urine sterile containers sent immediately 24h urine unacceptable From catheter bag unacceptable Abscess Aspirated aseptically by physician sterile tubes Blood Aseptic collection Minimum of 5 ml added to broth to get 1:10 to 1:20 1 mL blood blood culturebottle 70mL CSF and other body fluids aseptic collection lumbar puncture process immediately avoid refrigeration SOBRANG NA Vaginal secretions 20% of healthy female, has yeast as normal flora Vaginal candidiasis better diagnosed or established by clinical characteristics and direct examination spot infection na pano it may hypnoe may Stool 40% of healthy and 75% of compromised patients have yeasts in GIT Antifungal Susceptibility Testing M27- A3 for yeast testing M38 - A2 for mold testing M44 - A2 for disk diffusion testing for yeasts M51-A for disk diffusion testing for molds