第六届真菌会6号上午b1-cidm china fungus

1、Dr Fanrong Kong .auFanrong.K.au Research interests: Clinical & Molecular Microbiology. 117 SCI publications - e visit my Researchgate website. 2014-2016Visiting Professor of Department of Clinical Laboratory, Peking Union Medical College Hospital.2004-nowPermanent Senior Hospital Sc

2、ientist at CIDM, ICPMR-Pathology West, Westmead Hospital, University of Sydney.2000-2004PhD of Medicine, University of Sydney.1991-1994Master of Medicine, Beijing Medical University.1985-1991Bachelor of Medicine, Beijing Medical University.1982-1985Tianjin Nankai Middle School.74 generation grandson

3、 of Confucius family.The Mycology Laboratory, Lab Diagnosis & Research on Fungal Infections in AustraliaFanrong KongSenior ScientistCIDM, ICPMR, Westmead Hospital, NSW 2145, Australia .auFanrong.K.au One HealthTravel easier - infection transmission & outbreak. munication easier - wo

4、rk together for the many challenges in diagnosis, treatment, prevention of transmissible infectious diseases.China is the most promising developing country & should contribute more to promote the whole human beings health.You All and CIDM, Westmead Hospital, Sydney University, Australias colleagues

5、as parts of the One Health team players.Small Mycology Reference LabCIDM had 140 staffs.2-4 staffs in Mycology Reference Lab.15 m2 private conventional space for Mycology Reference Lab.Sharing spaces & platforms with many other labs:DNA extraction, PCR set-up, post PCR rooms, RT-PCR & MALDI-TOF, etc

6、.Mycology Research LabsCIDM Director Sharon Chen - Mycology Reference Lab related research.CIDM Director Sharon Chen & Professor Wieland Meyer - Australian Invasive Fungus studies: Candidemia, other invasive fungal infections, Scedosporium, Aspergillus, Cryptococcus , etc.Professor Wieland Meyers gr

7、oups 2 staffs & students & visitors - taxonomy & typing.Professor Tania Sorrell & Dr Julianne Djordjevics groups 2 staffs & students - phospholipase & antifungal drugs.Challenges of Mycology Reference Lab Australian Public Health System - government funding.70% clinical diagnosis based on Pathology

8、information. 5% hospital cost from Pathology service.Global Financial Crisis - budget cut popular.Diagnosis labs - need save more money & cut staff no. & provide more efficient services.Research projects with limited funding.Challenges in Medical MycologySignificant opportunistic fungal infections,

9、in promised hosts.Environmental fungi - emerged/life-threatening. Emerging pathogenic fungi could be inherently resistant to antifungal drugs.Empirical treatment risk of antifungal resistance.Clinical and economic consequences.Morphological knowledge Taxonomy changing/Species definition - controvers

10、ial. Fungal DiversityHow many fungi species exist?Estimate (Hawksworth 1991): 1.5 million species (0.69.9 millions). Hawksworth DL (1991) The fungal dimension of biodiversity: magnitude, significance, and conservation. Mycological Research 95: 641655. - a landmark paper. Hawksworth DL (2001). The ma

11、gnitude of fungal diversity: the 1.5 million species estimate revisited. Mycological Research 105: 14221432.Professor David Hawksworth was the last Director of the International Mycological Institute (Kew and Egham, 1983-97), and is former Council Member of English Nature (1996-99). He has also serv

12、ed as President or Chair of the International Union of Biological Sciences (IUBS), IUBS/IUMS International Committee on Bionomenclature, International Mycological Association, British Mycological Society, and British Lichen Society.Taxonomy ChallengesOnly 100,000 species described worldwide.100-200

13、species relatively regularly described in human/animal mycoses.Updated - relatively often - fast molecular progress; - sometimes clinicians unaware. Too diverse species - cut-off? Over classified genus/species - informative/signature SNPs?Why we need fungal pathogens names?Confirm & facilitate accur

14、ate diagnosis. Initiate early intervention with aggressive antifungal treatment. Reduce empiric use of antifungal agents.Reduce antifungal agents resistance.Identify risk & prevent infections.Select correct techniques for strain typing to investigate outbreaks or the source of ial infections.Why we

15、need molecular diagnostic tools?Conventional identification could be laborious, slow & incorrect, but molecular diagnostic tools couldachieve rapid & accurate detection & identification from cultures & in particular clinical specimens; sequence based ID is the new “gold standard” for species ID. How

16、ever, there are potential problems with sequence based ID!DNA sequencing - gold standards alternatives:PCR (conventional, allele-specific, MAS-PCR, etc.)?PCR-SSCP (single-strand conformation polymorphism) ?PCR-RFLPRT-PCR (real-time PCR & reverse transcription PCR for RNA) PCR-SCGEPCR-HRMA (high-reso

17、lution melting analysis)RCABiochipRLBGenomic sequencingMolecular ToolsSequencingConventional phenotype based Clinical Mycology is still VERY important & sometimes are still “gold standard” - like antifungal drug susceptibility tests.BUTMany experienced mycologists are retiring.Genes, genomics sequen

18、ces/sequencing are more accessible/cheaper than ever before.Much more objective & easier to compare - but need to be standardized as barcoding.ITS, D1D2, IGS, etc., ITS-barcoding.Quality ITS controlled database are being developed by ISHAM JCM paper submitted.Public Databases: GenBank=EMBL=DDJBLack

19、of a universally accepted good/gold genetic locus.A huge amount of ITS sequences are available in the public database but WITH PROBLEMS.Many ITS sequences containing mistakes as a result of: - experimental errors. - misidentification of the species/taxonomy changes. - swap of cultures. Limited taxon

20、omic coverage. - the total number of currently available ITS sequences represents - less than 1% of the estimated 1.5 million fungal species.Sequence ID is meaningful ONLY if:Well-curated, robust & reliable databases - available. Easy use & public free access & updatable.Populated with sequence data

21、 from: - type or reference strains (where possible); - a wide range of clinical strains; - a wide variety of target species.Strains were rigorously validated in terms of their nomenclature. (MALDI-TOF - the similar consideration for databases build up).Barcoding - ITSISHAM - The International Societ

22、y for Human and Animal Mycology.ISHAM working group (dozens of international medical mycology reference laboratories) was formed to establish a quality controlled ITS database for “DNA barcoding of human and animal pathogenic fungi”.It contained 2800 ITS sequences representing 421 species, provides

23、the medical community with a freely accessible tool to rapidly and reliably identify most agents of mycoses, accessible at or .But some important pathogenic fungal species, such as the dermatophytes & emerging yeast pathogens entries/ID were less satisfied, for which additional molecular methods/gen

24、etic markers are required to reliably identify these isolates from clinical and veterinary specimens.Cut-off 98-99% is incorrect?Fungal species ITS 1&2 regions intra-species variation species-dependent.Clinical samples sequences variation is much higher than those type strains sequences.Population b

25、ased intra-species sequence variation ranges from 08.35%.The currently used cut-off 98-99% is incorrect.? A new cut-off point of 91% (?), depending on the species tested.Other Well-curated ITS DatabasesCBS Fungal Biodiversity Centre database for medical fungi ( )Fusarium spp. (FUSARIUM-ID v. 1.0; )

26、Phaeoacremonium spp. ( ) Trichoderma spp. ( )Mycorrhizal fungi (UNITE, )Standardization - ITS 100200 copies - same/different alleles.PCR primers, condition, sequencing primers, etc.Database: ISHAM, CBS, NCBI, in house? Comparison length - with same start/stop position.Cut off value - 98.5%? or speci

27、es-specific?Algorithms: - most species isolates are very straightforward; - limited species need other targets to further ID.QAP regularly.AutomationTrend in Clinical Microbiology.Increased activity, reduced financial & human resources.Save labour cost & more objective.Comprehensive systems have bee

28、n developed by several big companies.Plate setup/reading/reporting/etc.Validation & Standardization needed.Human beings are still the final “gold standard” in many areas/foreseeable future.Post-genomic ParadigmGenomic sequencing is much cheaper than before, but analysis is still time & money consumi

29、ng, store & sharing the huge amount data - a challenge.More genome sequences are available in GenBank or other databases.As gold standard - to confirm or against some previous assays/results - MLST, microsatellites, PFGE, RAPD, etc.To develop novel & practical diagnostic & epidemiological typing res

30、earch tools. To investigate antifungal drug resistant mechanisms.To explore novel potential antifungal drug targets.MALDI-TOF - A Revolution!MALDI-TOF MS enabled rapid, reliable identification of clinically-important yeasts. The addition of spectra to databases and reduction in identification scores

31、 required for species-level identification may improve its utility. Matrix-assisted laser desorption ionization-time of flight mass spectrometry identification of yeasts is contingent on robust reference spectra. Pinto A, Halliday C, Zahra M, van Hal S, Olma T, Maszewska K, Iredell JR, Meyer W, Chen

32、 SC. PLoS One. 2011;6(10):e25712. doi: 10.1371/journal.pone.0025712. Epub 2011 Oct 13.MALDI-TOF MS is a rapid identification tool for the correct recognition of the two currently recognized human pathogenic Cryptococcus species and offers a simple method for the separation of the eight major molecul

33、ar types and the detection of hybrid strains within this species complex in the clinical laboratory. The obtained mass spectra provide further evidence that the major molecular types warrant variety or even species status.MALDI-TOF MS enables the rapid identification of the major molecular types wit

34、hin the Cryptococcus neoformans/C. gattii species complex. Firacative C, Trilles L, Meyer W. PLoS One. 2012;7(5):e37566. doi: 10.1371/journal.pone.0037566. Epub 2012 May 29.Filamentous fungi - Aspergillus, Penicillium, Fusarium, Scedosporium & dermatophytes - promising but with challenges, need in-h

35、ouse database.MALDI-TOF Infancy?Further evaluation based on real Clinical Mycology practice are needed.Vitek or Bruker or China home made?Multicentre results comparison published - but need more.Cut-off value need to be standardized? 1.7? or 2.0?In-house databases are needed for Filamentous fungi -

36、Aspergillus, Penicillium, Fusarium, Scedosporium & dermatophytes. - but how to standardization is a challenge.MALDI-TOF Standardization - In Detail Needed!Machine? Software (database)s version.Protein extraction protocol (for Bruker).Database - commercial, NIH, in house?Cut-off value - 1.7? or 2.0?

37、or in-house?“QAP” regularly to compare results based on the “same set/identical copy samples.”ID is for objective/accurate diagnosis & communication. Standardization - a prerequisite to avoid wrong diagnosis & Misunderstanding.Validation & Standardization - Very Important!It needs a lot of efforts/t

38、ime/money!But it is important for getting correct results/promote communication/collaboration.Pathology/Mycology is only part of the clinical medical practice.Common/biological senses are still needed. Be careful when you refer published data - many could be without Validation & Standardization - ev

39、en Misleading.PUMCH need/can do more for the purposes - through the networks like CHIF-NET, etc.Nothing Is Perfect!Conventional bench work are still most commonly used, usually cheaper, quicker but subjective.Sequencing is very objective but slow, experience. Other molecular assays - PCR, RT-PCR, PC

40、R-CGE, mPCR, PCR/RLB, PCR/biochip, etc. are still important working assays - need more bench places, good practice, etc.MALDI-TOF is promising but equipment is expensive and in-house databases are still needed for some genus/species.“Cocktail” to make the best of use the available assays - more prac

41、tical approach.Identification & TypingCustomers requirements in mind.Your purposes?Not over-do it for clinical?Over-do it for research/epidemiological?Using most straightforward ways or most accurate way or most comprehensive ways?Genomic sequencing, when is necessary?Antifungal Drug ResistanceConve

42、ntional MIC is still the gold standard.Automation only in part of bench work.in vitro & in vivo difference!Mechanisms to assist break points or Epidemiologic Cutoff Values (ECVs)?Genomics mining for further direction.Networks Build-upCHIF-NET TeamIn vitro susceptibilities of yeast species to flucona

43、zole and voriconazole as determined by the 2010 National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) study.Wang H, Xiao M, Chen SC, Kong F, Sun ZY, Liao K, Lu J, Shao HF, Yan Y, Fan H, Hu ZD, Chu YZ, Hu TS, Ni YX, Zou GL, Xu YC.J Clin Microbiol. 2012 Dec;50(12):3952-9. doi: 10.1128/JC

44、M.01130-12. Epub 2012 Oct 3.Yeast identification algorithm based on use of the Vitek MS system selectively supplemented with ribosomal DNA sequencing: proposal of a reference assay for invasive fungal surveillance programs in China.Zhang L, Xiao M, Wang H, Gao R, Fan X, Brown M, Gray TJ, Kong F, Xu

45、YC.J Clin Microbiol. 2014 Feb;52(2):572-7. doi: 10.1128/JCM.02543-13. Epub 2013 Dec 11.China-SCAN TeamJ Antimicrob Chemother. 2013 Jul;68(7):1660-8. doi: 10.1093/jac/dkt083. Epub 2013 Mar 29.Invasive candidiasis in intensive care units in China: a multicentre prospective observational study.Guo F1, Yang Y, Kang Y, Zang B, Cui W, Qin B, Qin Y, Fang Q, Qin T, Jiang D, Li W, Gu Q, Zhao H, Liu D, Guan X, Li J, Ma X, Yu K, Chan D, Yan J, Tang Y, Liu W, Li R, Qiu H; China-SCAN Team.J Antimicrob Chemother. 2014 Jan;69(1):162-7. doi: 10.1093/jac/dkt330. Epub 201