2017
DEC 1-3 Non–culture-based identification SPEAKER CONFERENCE,OF Dr Ariya Chindamporn Associate Professor MMTN Department of MicrobiologyAT COPYRIGHT Faculty of Medicine, Chulalongkorn University Bangkok, Thailand
PRESENTED
Presented at MMTN Vietnam Conference 1–3 December 2017, Ho Chi Minh City, Vietnam 2017
DEC 1-3 Hot topics in Asian medical mycology
SPEAKER NON CULTURE BASEDCONFERENCE,OF IDENTIFICATION
MMTN AT COPYRIGHT Ariya Chindamporn Department of Microbiology, Faculty of Medicine, Chulalongkorn University PRESENTED Bangkok, Thailand
MMTN Vietnam Conference December 1-3, 2017 Outline o Antigen-Antibody detection 2017 DEC • ELISA 1-3 • Lateral Flow assay
• Mannan/Galactomannan SPEAKER • (1,3)-β-D-glucan CONFERENCE,OF o DNA based: PCR, Real time PCR MMTN o Histopathology: H&EAT staining, GMS and COPYRIGHT
PRESENTED Fungal Identification & Diagnosis
• Standard method Fungal isolation and cultivation 2017
o Macro- / Microscopic examination DEC o Biochemical test (Yeast) 1-3 Round cells C a p s u l e + ve U r e a s e + ve SPEAKER M e l a n i n + ve CONFERENCE,OF API: Cryptococcus
MMTN KOH prep. www.pinterest.com AT COPYRIGHT
urease Melanin pigment India ink
PRESENTED
100% Cryptococcus neoformans
Aspergillus f u m i g a t u s Cryptococcus n e o f o r m a n s Laboratory Diagnosis APPROACH PRO CON IDENTIFICATION LEVEL 1. CLASSICAL 1.1 Direct examination Rapid (1 hr), Experience Morphology: Yeast, - KOH prep., India ink staining low cost 2017Mold: non septate, - Calcofluor white staining septate: - - Gram stain, MAFB stain DEC (hyaline/dematiaceous) 1-3 1.2 Histopathology Tissue reaction Specialty Tissue reactions - Wright stain Automate & Morphology - H & E, PAS, Gram, GMS, etc 1.3 Culture Low cost, SPEAKERTime Genus, species?? susceptibilityCONFERENCE,OF consuming 2. SEROLOGY One day False positive, Group 2.1 Antigen (cell wall) MMTN False neg, - Mannan, GM, BG, AT COPYRIGHT 2.2 Antibody : Mannan Ag & Ab together Immunocompetent 3. NON - CLASSICAL - High sens., Contamination Genus, level, typing 3.1 DNA based spec. , Accurate PRESENTED - PCR, FISH, qPCR, - Both detection sequencing, etc. & identification 3.2 Protein based Rapid (30 min) Data based, Genus, species, high cost developing to detection, Suscep. Serology Cell wall composition VS diagnosis tool
2017
DEC 1-3
SPEAKER CONFERENCE,OF
MMTN AT COPYRIGHT
PRESENTED
Erwig et al. Nature Reviews Microbiology. 2016. V14: 163-176 Antigen Detection / Antibody detection
• Cryptococcosis 2017 o GXM (Lateral Flow Strip) DEC 1-3 • Histoplasmosis o Histoplasma antigen (EIA) • Invasive Candidiasis SPEAKER CONFERENCE,OF o Mannan (ELISA) / Anti-Mannan • Aspergillosis MMTN AT o Galactomannan (ELISA)COPYRIGHT • Fungal infection o (1,3)-β-PRESENTEDD-glucan (Kinetic ELISA) Cryptococcosis: Lateral Flow immunoassay
2017
DEC 1-3
SPEAKER Clin Infect Dis. 2011 CONFERENCE,OF- Capsular antigen : GXM 4 serotypes - Diagnosis from CSF, Serum, - Urine vs Blood culture: MMTN - 92% sensitivity AT - Urine: LTA vs EIA COPYRIGHT - 72% sensitivity - HIV & Non HIV patients - Rapid (15 min.), user friendly, PRESENTED - Point of care assay - Qualitative, Semi-quantitative assay Histoplasmosis: Histoplasma antigen EIA
- serum (92.3% sens.) & urine (100% sens)
2017
DEC Histoplasmosis Sensitivity (%) 1-3 Disseminated 90-95 Acute pulmonary 80 Chronic pulmonary 20 SPEAKER CONFERENCE,OF
MMTN AT COPYRIGHT
PRESENTED
Connolly et al. Clin Vaccine Immunology 2007 Candidiasis: Mannan
2017
DECCrit Care. 2010;14(6):R222 1-3 • Investigated in 14 studies that comprised 453 patients and 767 controls SPEAKER o specificity(86%) CONFERENCE,OF o sensitivity (83%) - C. albicans (80-100%)MMTN AT - C. tropicalis & C. glabrataCOPYRIGHT(60-75%) - C. parapsilosis &C. krusei (40–50%)
PRESENTED Aspergillosis: Galactomannan
• Major component of fungal cell wall especially Aspergillus spp. and also found in o Family of Trichocomaceae ie. Penicillium spp., Paecilomyces2017 spp.
o Fusarium spp. DEC o Histoplasma capsulatum 1-3 o Pneumocystis jirovecii Tortorano et al. J Clin Microbiol 2012, Huang et al. AIDS 2007, Wheat et al. Clin Vaccine Immunol 2007 SPEAKER • Platelia Aspergillus EIA CONFERENCE,OF (Bio-Rad, France) o Sandwich ELISA MMTN AT o Anti-GM monoclonal COPYRIGHT antibody : mAb EB-A2 (Marisa et al. Sem in Res and Critical care Med 2015)
o FDA approvedPRESENTED : as a diagnostic aids for www.slideplayer.com invasive aspergillosis (IA) (FDA. May 2003) GM: Galactomannan
• Clinical samples interpretation
Recommended Collected samples Sens. Spec. Reference cut-off 2017 Index 0.5 • 2 aliquots: same positive sample + 1 sample collected at a different time point DEC Marisa 2015 Serum 60-80% 80-95% Lamoth 2016 Index 1.0 • 1 single sample 1-3
Guo Y L et al. 2010 BAL Index 0.5-1.0 • 2 aliquots of a single BAL fluid sample 85-90% 90-95% Zou M et al. 2012
SPEAKER CSF* Index 0.5-2.0 • 1 single sample 85-90% 95-100% Chong GM et al. 2016 CONFERENCE,OF
• CSF is validated only for cerebral aspergillosis diagnosis by EORTC-MSG and ECIL so using under MMTN expertise consultation is recommended (De Pauw B et al. 2008, Marchetti O et al. 2012) • CSF has not been approved by FDAAT yet. COPYRIGHT • Two main strategies of use for serum sample: o Serial collection of samples (2-3 times/week) in high risk patients PRESENTED o Intensive testing in symptomatic patients
Marisa et al. Sem in Res and Critical care Med 2015, Lamoth et al. J of fungi 2016 GM detection in other specimens
Urine (Duettmann W et al. Med Mycol 2014) • Study in 75 proven IA cases underlying hematological malignancies 2017 • GM index cut-off : 0.5 DEC o 47.6% Sensitivity 1-3 o 86% Specificity o 24.4 Positive predictive value (PPV)
o 94.5 Negative predictive value (NPV)SPEAKER Moreover, GM positive also reportedCONFERENCE, in OF • Cyst fluids in patients with polycystic kidney disease (Miller-Hjelle MA et al. Emerg Infect DisMMTN 1997) • Subphrenic abscessAT in a pediatric patient with proven IA underlying COPYRIGHT chronic granulomatous (Verweij PE et al. J ClinMicrobiol 2000) • Purulent material, study in patients with fungal rhinosinusitis (Klont RR et al. Clin Infect Dis 2004) PRESENTED However, the study of GM in other specimen is very limited, using for routine diagnosis is not recommended. !! Caution !! GM Negative was found in some species of Aspergillus !!
Features of selected non-A. fumigatus infections (modified from Lass-Florl et al. J Antimicrob Chemother 2017) Species Diseases Specific characteristics Emericella nidulans IA in CGD • More virulent than A. fumigatus 2017 • Higher mortality • Propensity to spread from the lung to adjacent structures and to disseminate • Intrinsic resistance to amphotericinDEC B Emericella quadrilineata IA in CGD and IA • Resistant to caspofungin? 1-3 Aspergillus calidoustus IA • Propensity to disseminate • Intrinsic resistance to azoles • Intrinsic resistance to caspofungin? Aspergillus terreus IA • Propensity to disseminate (63%) • Intrinsic resistance toSPEAKER amphotericin B Aspergillus tubingensis IA, airway colonization • Acquired CONFERENCE,resistanceOF to azoles and ear infections • Lower propensity to disseminate (10%-30%)
Aspergillus lentulus IA • Resistant to azoles and echinocandins • ResistantMMTN to amphotericin B Aspergillus alliaceus IA AT• GM negative •COPYRIGHTHigh MICs of amphotericin B and caspofungin Aspergillus carneus IA • GM low positive Aspergillus novofumigatus IA • Resistant to azoles Aspergillus alabamensis Mainly airway • Resistant to amphotericin B colonization PRESENTED Aspergillus ustus IA • Resistant to amphotericin B, azoles and echinocandins Aspergillus felis IA • High MICs against voriconazole and caspofungin
IA, invasive aspergillosis; CGD, chronic granulomatous disease; GM, galactomannan. !! Caution !! False positive galactomannan test after ice pop ingestion !!
• A 42-year-old woman • Received an HLA-matched hematopoietic stem-cell transplant from an 2017 unrelated donor for a myeloproliferative syndrome. DEC • Serum aspergillus antigen (GM) assessed twice weekly1-3 from the day of transplantation. • GM index increased to 2.22 and 3.01 on days 32 and 34, respectively. SPEAKER • Severe gastrointestinal GVHD. CONFERENCE,OF • Afebrile with no pulmonary or sinus symptoms. • CT scans of the lung, brain, sinusMMTN and abdomen = normal AT • Cultures of sputum samples COPYRIGHT= negative • PCR blood for aspergillus = negative
PRESENTED
Guigue et al. The New England Journal of Medicine. 2013 2017
DEC 1-3
SPEAKER CONFERENCE,OF - 3/37 of ice-pop MMTN - Positive Penicilium spp. AT - showed positive GM COPYRIGHT- In ice-pop - Sod. gluconate (Thickener & stabilizer for water based frozen PRESENTED dessert in Europe & US) BD: 1,3-β-D-glucan • Major component of fungal cell wall but less in o Mucorales ie. Mucor spp., Rhizopus spp. o Cryptococcus spp. and some other Basidiomycota2017ie. Malassezia spp. DEC Commercial Manufacturer Horseshoe Detection Manufacturer Available assay crab substrate system 1-3 cut-off value Associated of Cape Cod US (FDA Fungitell assay Limulus Inc., East Falmouth, MA, Colorimetric 60-80 pg/ml approved in 2003) (Glucatell) USA polyphemus and Europe
Fungitec-G test Tachypleus Seikagaku Corporation. ColorimetricSPEAKER 20 pg/ml Japan MK (G-MK) Tokyo, Japan) tridentatus Wako Pure Chemicals CONFERENCE,OF Beta-glucan Tachypleus Industries Ltd., Osaka, Turbidimetric 11 pg/ml Japan test Wako Japan tridentatus MMTN BGSTAR beta Maruha Nichiro Foods Tachypleus Colorimetric glucan test AT 11 pg/ml Japan Inc.Tokyo, Japan tridentatus Maruha COPYRIGHT (Marisa et al. Sem in Res and Critical care Med 2015) • Fungitell assay (Cape Cod Inc., USA) o FDA approvedPRESENTED as an aid to diagnose deep-seated mycoses and fungimia. o European medical center : presumptive diagnosis of invasive fungal disease (Marisa et al. Sem in Res and Critical care Med 2015) o The EORTC-MSG panel : included a positive BD test as a microbiological criterion of IFI (Lamoth et al. J of fungi 2016) Marisa et al. Sem in Res and Critical care Med 2015
2017
Limulus polyphemus DEC 1-3
SPEAKER CONFERENCE,OF
MMTN AT COPYRIGHT
PRESENTED
Biological cascade-based assay Measuring activation of Factor G through horseshoe crab substrates BD: 1,3-β-D-glucan
• Clinical specimen (Lamoth et al. J of fungi 2016)
Sensitivity Specificity Note Serum 60-80% 80-95% 2017FDA approved
BAL 85-89% 86-95% DEC CSF 85-90% 95-100%1-3
• Unfortunately, BD is not pathogen specific and cannot differentiate fungal species. • Major limitations : its low specificity and frequent occurrence of false-positive SPEAKER reaction CONFERENCE,OF o Non-fungal infection
− Pseudomonas spp. MMTN − Streptococcus spp. o Non-infectious disease conditionsAT COPYRIGHT − Hemodialysis with cellulose membranes − Albumin transfusion − Intravenous immunoglobulin − Gauze packing of serosal surfaces − Use of cellulose filters for PRESENTED − Intravenous amoxicillin-clavulanate intravenous administration
(Marty et al. Med Mycol 2009, Mennink-Kersten MA et al. N Eng J Med 2006, Mennink-Kersten MA et al. Clin Infect Dis 2008) 2017 Diagnostic detection method utilizing miniaturized magnetic resonance technology, measures how water molecules react in the presence ofDEC magnetic fields. 1-3
SPEAKER CONFERENCE,OF
MMTN AT COPYRIGHT
PRESENTED
Moreover, T2MR also can detect the antigen directly from hemoculture. Antibody Detection
• Usually uses in endemic mycoses: Histoplasmosis, Sporotrichosis2017 ,
Coccidioidomycosis etc. in immunocompetent hostsDEC 1-3 o High Specificity
o Sensitivity depends on the phase of disease
• But, not useful for opportunistic fungal infectionsSPEAKER in immunocompromised hosts CONFERENCE,OF
MMTN AT COPYRIGHT
PRESENTED Molecular Technology for 2017 Fungal Identification & DiagnosisDEC 1-3
SPEAKER CONFERENCE,OF
MMTN AT COPYRIGHT
PRESENTED
Eukaryote 2017.03.21 Case 1
. 47 years-old, male . 43 years-old, female 2017 . Dx: Subcutaneous mycosis . Headache, 1 month DEC 1-3 . Specimen: Tissue . Dx: Brain abscess . Request for: . Specimen: Pus SPEAKER - KOH prep. >> Dark septate hyphae . Request for: CONFERENCE,OF - Culture (24d) >> Non-sporulated - KOH prep. >> yeast + hyphaeMMTN AT dark septate hyphae - Culture >> no fungal growthCOPYRIGHT
PRESENTED Molecular Technology (genetic-based study / PCR: polymerase chain reaction)
2017
DEC 1-3
SPEAKER CONFERENCE,OF
MMTN AT COPYRIGHT
PRESENTED
www.users.ugent.be Molecular Technology
Purpose: genotyping
2017
DEC Identification & diagnosis Evolution & Phylogenetic 1-3 Epidemiology
DNA sequence; direct DNA sequence; indirect SPEAKER SNPs CONFERENCE,OF Nuclear ribosomal DNA (rDNA) MMTN Whole genome of fungi varies AT COPYRIGHTfrom 8.97 Mb to 177.57 Mb
Other gene ie. PRESENTED - Chitin synthase II - Cytochrome oxidase II - Tubulin-beta
www.cellsandpathogenstudy.html Nuclear ribosomal DNA
Compose of 2017 1. 18S rDNA DEC 1-3 2. Large subunit of rDNA (LSU) or 26s rDNA 3. Internal transcribed spacer region (ITS: ITS 1 and ITS2) SPEAKER 4. Intergenic spacer regionCONFERENCE, (IGS)OF
MMTN AT COPYRIGHT
PRESENTED 2017
DEC 1-3
18S ITS 28S
SPEAKER CONFERENCE,OF
18S MMTN AT 28S COPYRIGHT
ITS PRESENTED
Localization of universal primer currently used for fungal identification
J.B. Stielow et al. One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes Persoonia. 2015 Dec; 35: 242–263. - + test DNA extraction from PUS
2017
DEC PCR 1-3 amplification and Sequencing process SPEAKER CONFERENCE,OF
MMTN AT Primer LRR/LR3 COPYRIGHT ggaggaaaag aaaccaacag ggattgcctt agtagcggcg agtgaagcgg caaaagctca aatttgaaat ctggcacttt cagtgtccga gttgtaattt gaagaaggta tctttgggtc tggctcttgt ctatgtttct tggaacagaa cgtcacagag ggtgagaatc ccgtgcgatg agatgtccca gacctatgta aagttccttc gaagagtcga gttgtttggg aatgcagctc taagtgggtgPRESENTEDgtaaattcca tctaaagcta aatattggcg agagaccgat agcgaacaag tacagtgatg gaaagatgaa aagaactttg aaaagagagt gaaaaagtac gtgaaattgt tgaaagggaa gggcttgaga tcagacttgg tattttgtat gttactctct cgggggtggc ctctacagtt taccgggcca gcatcagttt gagcggtagg ataagtgcaa agaaatgtgg cactgcttcg gtagtgtgtt atagtctttg tcgatactgc cagcttagac tgaggactgc ggcttcggcc taggatgttg gcataatgat cttaagtcgc ccgtcttga DNA extraction from PUS
2017
DEC PCR 1-3 amplification and Sequencing process SPEAKER https://blast.ncbi.nlm.nih.gov CONFERENCE,OF
MMTN AT COPYRIGHT Blast to Submitted sequence database DataBank PRESENTED 2017
DEC 1-3
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MMTN AT COPYRIGHT
PRESENTED 2 2017
DEC 1-3
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PRESENTED 2017
DEC Thank you1-3
SPEAKER CONFERENCE,OF
MMTN AT COPYRIGHT
PRESENTED
Guarner et al. Clin Microbiol Rev. 2011