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Candida Hyaline Molds Pigment 4/6/2016 ACCME/Disclosures The USCAP requires that anyone in a position to influence or control the content of CME disclose any relevant financial relationship WITH COMMERCIAL INTERESTS which they or their spouse/partner have, or have had, within the past 12 months, which relates to the content of this educational activity and creates a conflict of interest. Drs. Guarner and Bryan declare that they have no conflict(s) of interest to disclose. FUNGI Case 49 years old HIV positive woman with relapsed Morphology and culture multiple myeloma presents with respiratory and Jeannette Guarner, MD renal failure. Emory University Vital signs show hypotension and on physical exam a palpable neck lymph node is noted. Molecular diagnostics The lung X‐ray showed multiple nodules. A fine needle aspirate of the neck lymph node is Andrew Bryan, MD, PhD performed. University of Washington 1 4/6/2016 Molds = hyphae Five days later the case grows Candida Hyaline molds Pigment Septation Dematiaceous Pauciseptated Septated Madurella spp, Mucorales genera: Aspergillus spp., Fonsecaea spp, Rhizopus, Fusarium spp., Cladophialophora spp, Rhizomucor, Scedosporium spp., Exserohilum spp, Mucor, Trichoderma spp., Curvularia spp, Cunninghamella, Paecilomyces spp. and Bipolaris spp, and Lichtheimia (Absidia). others including others. Candida spp. 2 4/6/2016 Candida, Microorganism identification several species Matrix‐Assisted Laser Desorbtion Ionisation ‐ Time Candida albicans, C. Of Flight Mass Spectrometer glabrata, C. parapsilosis, C. tropicalis, and C. krusei. (MALDI‐TOF‐MS) Histopathologically: Candida glabrata All others MALDI‐TOF and Candida sp. # cases Concordance Sensitivity Candida albicans 195 187 95.9 P. aeruginosa Candida glabrata 26 22 84.6 Salmonella serotype B Candida guilliermondii 10 6 60.0 K. pneumoniae Candida krusei 86 75.0 E. coli Streptococcus Candida parapsilosis 69 65 94.2 S. aureus Candida tropicalis 32 28 87.5 total 346 316 91.3 SY Hsieh, et al. Highly efficient classification and identification of human pathogenic bacteria by MALDI‐TOF MS. Molecular & Cellular Proteomics T Spanu, et al. Mass spectrometry assay of blood culture broths for rapid 2008;7:448–456. identification of Candida species causing bloodstream infections: an observational study in two large microbiology laboratories. J Clin Microbiol 2012;50:176-179 3 4/6/2016 Candidiasis Epidemiology: Candida .Superficial: candida in Who: mouth (thrush), vaginal ◦ Immunosuppressed/ neutropenic individuals. infections, ◦ Others: Patients with central catheters, on broad onychomycosis. spectrum antibiotics, on parenteral nutrition, .Invasive hemodialysis, or after surgery. Easy to diagnose in the “usual” locations: GI tact. Final identification Candida glabrata JA Cortes et al. Infecciones micóticas del torrente sanguíneo en hospitales de tercer nivel en Colombia. Rev Iberoam Mycol 2011; 28: 74 4 4/6/2016 What happens if Antigenemia tests for Candida cultures aren’t and many other fungi submitted? 1,3 β‐D‐glucan in serum: ◦ Cell wall component ◦ Limited or no detection of Zygomycetes, Cryptococcus, Blastomyces ◦ Sensitivity 50–100%, specificity 44– 92% in one specimen if cut off values are high (80pg/ml) for candidemia. ◦ High rates of false positives in critically ill patients, patients with bacterial infections, medical devices with glucans PMIDs 19375641, 26910223, 23949603 Why we speciate: Magnetic resonance Candida sp. have predictable for speciation of Candida directly from blood susceptibility patterns Flucon‐ Itracon Voriconazole & Flucyt‐ Ampho‐ Echino‐ Speciate five Candida sp. azole ‐azole posaconazole osine tericin B candins Direct on blood without C. albicans & SS S S S S need for extraction C. tropicalis C. paropsilosis SS S S SS to R Reduce time to diagnosis compared to culture C. glabrata S‐DD to S‐DD to S‐DD to R SS to IS R R FDA‐cleared C. krusei RS‐DD to S Ito R S to IS Unclear cost‐effectiveness R C. lustaniae SS S SS to R S S‐DD = Susceptible, dose‐dependent S = Susceptible PMIDs 26371384, 24135411 R = Resistant 5 4/6/2016 Mass spectrometry (MS) for speciation of fungi In situ hybridization for speciation of fungi from both fresh specimens and FFPE from both fresh specimens and FFPE • MALDI-TOF on blood (PMID 25636941) FISH to fungal rRNA DNA sequences has been used for identification of • PCR-electrospray ionization MS on FFPE Candida and Histoplasma from (PMID 23985922 (Pritt)) blood culture bottles, Cryptococcus from CSF, etc - m/z ratios of small PCR amplicons from several loci -> fingerprint Sensitivity 50 ‐ 95%, specificity > 90% - Good agreement with culture; detected environmental Probe intensity more limited in contaminants FFPE, but have been designed for PMID 22584138 range of yeast and molds • MALDI tissue imaging on FFPE Sequence similarity may limit (PMID 26831708, 24287632) speciation of closely related FFPE pulmonary mixed Candida and - On the horizon? organisms Aspergillus infection (PMID 26781382) PMID 23574773 (Guarner), 21791040, 26781382 PMID 25621874 PCR followed by sequencing for speciation of Representative work‐flow of fungi from direct specimens and FFPE broad‐range fungal PCR • Analogous to prokaryotic (bacterial) 16S rRNA sequencing • 28S rRNA DNA sequences are highly conserved, but cannot always distinguish species • Internal Transcribed Spacer (ITS) sequences are nonfunctional linkers, thus are highly polymorphic, facilitating speciation • Amplification and sequencing of ITS1 and/or ITS2, with database match for species identification Modified from S. Salipante 6 4/6/2016 Specimen type and DNA Extraction impact Additional handling of blocks increases risk of contamination diagnostic yield of PCR (PCR true positive, biologic false positive) Fluids: centrifuged (more is typically better) Pathogen burden Tissue: 30 –50 mg 200µL elution 5µL/rxn Consider pre‐test probability: Histopath + ≠ FFPE ~17% positive rate for broad range PCR non‐FFPE ~9% positive rate Histopath ‐ Bone: Unknown Malassezia sp. Bead‐based lysis: efficient, but releases calcium (inhibitory) 6.2% of non‐FFPE cases P < 0.05 EDTA decal: 12.9% of FFPE cases Strong acids: Shave off the top to reduce contaminants Of all positive broad‐range fungal PCRs medibiztv.com Balancing pre‐test probability and Initial PCR amplification of conserved risk of contamination of blocks sequences 28S, ITS1, ITS2 Duplicate patient samples Tissue permitting: Inhibition control Consider adjacent sections ITS1 –Freeze tissue (for PCR) – FFPE (histopath) BAL (Polymicrobial): Comsol.com Multiple bands Can’t proceed with Sanger sequencing, but still can perform species‐specific assays 7 4/6/2016 Databases queried with patient sequences PCR product is sequenced by Sanger to determine species of organism method & chromatographs reviewed Organism %ID Length Journal evalue Also search internal UW database of clinical isolates If match > 99.5% &all other species < 98.5%, report name most commonly used in clin micro otherwise generate tree If simple database search is ambiguous, selected sequences If simple database search is ambiguous, are used to generate tree tree is generated to determine where sample clads C. dubliniensis C. tropicalis C. albicans C. parapsilosis C. glabrata Modified from PMC4629792 8 4/6/2016 Case is reviewed and signed out Edible fungi • Multiple independent reviewers, including director‐level sign‐out • If alignment 97 – 99.7% with multiple species, use tree and genus +/‐ species level name most commonly used in clinical microbiology • If no clear match, report higher level taxonomy • Candida albicans DNA detected with ITS primer set Case 47 year old woman with aplastic anemia treated with eculizumab, awaiting a bone marrow transplant. He presents with fever and an X‐ray showed a pneumonia. Erythematous skin lesions are also noted. Laboratory: 300 WBC/ mL, Hb 8 g/dL and 13,000 platelets/mL 9 4/6/2016 Molds = hyphae Diagnosis: Hyaline molds Pigment Angioinvasive fungal elements. Septation Dematiaceous Pauciseptated Septated Madurella spp, Mucorales genera: Aspergillus spp., Fonsecaea spp, Rhizopus, Fusarium spp., Cladophialophora spp, Rhizomucor, Scedosporium spp., Exserohilum spp, Mucor, Trichoderma spp., Curvularia spp, Cunninghamella, Paecilomyces spp. and Bipolaris spp, and Lichtheimia (Absidia). others including others. Candida spp. Pathologic misclassifications Misclassifications occur when: Retrospective studies that correlate Frequent misclassifications when: few, folded, culture results with histopathology and fragmented or necrotic fungal elements. cytology show: ◦ accuracy ranges from 20 to 80%. There is a false sense of ability to name the fungal genus based on morphology alone (hyaline septated The lowest correlation: invasive septate hyphae rather than Aspergillus spp.). molds. Lack of knowledge of morphologic mimics of yeasts Special stains do not improve and hyphal forms. pathologists’ diagnostic capabilities. Patel AJ, et al. Am J Surg Pathol 2010;34:256-261. Sangoi AR, et al. Am J Clin Pathol 2009;131:364-375. 10 4/6/2016 Case continues Identification 5days after the skin biopsy was obtained the blood culture bottles showed: What happens Direct tests for Aspergillus if it all goes antigen in patient fluids into formalin? Galactomannan in serum or other fluid: ◦ ELISA for Aspergillus galactomannan ◦ Sensitivity 40‐100%, specificity 56‐100% ◦ More specific than 1,3 β‐D‐glucan, but still not entirely specific to Aspergillus ‐ May also detect Penicillium, Alternaria, Paecilomyces, Geotrichum and Histoplasma ‐ False‐positives in 50% of patients taking
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