Bench to bedside in fungal diagnostics

Transcription

1 Bench to bedside in fungal diagnostics Sharon Chen Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR Westmead Hospital, University of Sydney, NSW, Australia PATHOLOGY UPDATE 2017

2 Overview Existing benchmark tests for invasive fungal disease (IFD) Areas ripe / further development Lateral flow tests (LFAs) Cryptococcus, Aspergillus MALDI TOF MS tips Candida PCR Rapid drug resistance detection enose VOC analyses (moulds) Novel antibody-directed imaging Crossing the Red Sea

3 IFD overall mortality Aspergillosis: 20-60% (AUS: 31% at 30 days) Candidiasis: 30-60% (AUS: 29%) Non-Aspergillus moulds 80 % (AUS 44% at 3 months) Delay in receipt of therapy associated with mortality (yeasts and moulds) Delay is in large due to slow and insensitive diagnostics Garey et al. Clin Infect Dis 2006; Chapman et al. JAC subm; Slavin et al. Clin Microbiol Infect 2015; Kennedy K et al., Clin Microbiol Infect 2016; Teng et al. Haematologica 2015; Cordonnier et al. Clin Infect Dis 2009

4 Caution: May need expert interpretation After Dr. Sophia Koo, with permission, Boston, MA

5 Current diagnostics: consensus Infection Culture/ Histo Aspergillosis Yes - invasive Biomarker (Ab) No Candidaisis Routine Investigational (anti-mannan) Biomarker (Ag) Response to Rx GM/BDG/PCR Increasing evidence PCR/mannan/ BDG Cryptococcosis Routine No Ag/PCR Yes (CSF Ag) Histoplasmosis Culture - delay Mucormycosis Yes - invasive Other moulds Yes invasive No Limited Ag Yes (Ag) No Investigational No No Investigational No

6 Diagnostics: meta-analyses Properties GM PCR BDG Standardised YES Yes for IA No for others YES EORTC/MSG YES Yes* Yes Blood/BAL/CSF +/+/+ (also urine?) +/+ +/-/- False positive Β-lactams, plasmalyte, bifidobacterium Cross reactivity Fusarium,Paecilomyces, Penicillium,Trichosporon Blasto, Histoplasma Sensitivity, specificity 2 consecutive+ * Being updated 78%, 81% Leefang et al Cochrane database syst rev 2008 Colonisation Penicillium 75%, 87% Mengoli et al, Lancet ID 2009 Food, Abs, dressings Bacteremia Panfungal (Crypto, Mucorales) 49.6%, 98.9% Lamoth et al CID 2012

7 GM and Asp PCR together Empirical therapy Earlier diagnosis Aguado Morrissey Barnes Rogers Springer NA NA Yes Yes Yes Yes Yes Antifungal use NA NA Proven/pr obable disease NA NA Aguado et al, CID 2014; Morrissey et al LID 2013; Rogers et al BJH 2013; Barnes et al J Infect 2013; Springer et al JCM 2013

8 BAL GM/PCR in haematology: Melbourne-Sydney experience Diagnostic bronchoscopies in 116 patients from 4 Australian institutions: 40% neutropenic; 68% mould active agents; 36% β-lactam antibiotics at time of BAL excluding GM results in BAL fluid: proven IPA (n=3), probable IPA (n=15), possible IFD n=50) 6 had non-aspergillus mold infections 42 had no IFD. Heng SC et al Diagn Micro Infect Dis 2014

9 BAL GM/PCR in haematology patients: local experience NPVs 85-90% GM BAL (cutoff 0.8) Sensitivity IA 61% 78% Specificity IA 93% 81% PCR BAL Mould-active antifungal therapy did not significantly influence sensitivity Inclusion of GM and PCR results from BAL upgraded 10 (20%) and 18 (36%) possible IFD cases into probable IPA Heng SC et al Diagn Micro Infect Dis 2014

10 Cryptococcal antigen (CRAG): what was & what is CRAG is a good test; pooled sens. 98%,spec. 99%; but LA and EIA methods need heat/enzyme treatment and expertise Since 2011, LFA (IMMY) Strip to which Mabs against polysaccharide antigen is immobilised to Gold particles (all serotypes) Requires no pre-treatment Serum, CSF, urine Rajasingham, J Acquir Immune Defic Syndr 2012

11 Performance: meta-analysis Huang et al., Plos One 2015

12 LFA and LA titres Semi-quantitative by LFA In general, LFA titre is > LA: ratio 1.53 (95% CI ) Correlation (r) = 0.84; (Left): Scatter Plot and (Right): Bland-Altman Plot Novel method of titre-ing: heat signature of laser-irradiated gold (0.01 W, 532 nm, 30s) Temp. change recorded with infrared camera; calculate titre against calibration curve using serial dilutions; R 2 = 0.97 McMullan et al, PloS One 2012; Boulware et al., EID 2014

13 T o C Thermal contrast shows extended dynamic range cf. visual

14 Aspergillus Lateral flow device: 6 years on... Uses mouse mab JF5 to bind Aspergillus glycoprotein in active growth +ve result=germination Semi-qt: -, +, +++ (naked eye) Heat step 100 o C, 3 min (for serum), no heat step for BAL fluid read:10-15 min Chris Thornton, Exeter University X-react with Penicillium spp. J Vis Exp 2012; Mar 22 (61)

15 Aspergillus LFA: animal testing What is known: first iteration of LFA Sensitive and specific for IPA in guinea pigs (cf. GM, B-glucan) Sensitivity in serum reduced if treated with antifungals (posa, vori, LAMB) Sensitivity using BAL fluid similar Urine? (cf. detection GM) Good reproducibility between laboratories Wiederhold et al, Clin Vaccine Immunol 2009; Wiederhold et al, J Clin Microbiol 2013; Dufrense et al, PloS One 2012

16 Aspergillus LFA: clinical studies Prattes J et al. Serum showed less promising results cf. BAL fluid

17 Aspergillus LFA: serum studies use with other tests N=529, 2 results used performed as well as serum Aspergillus GM, Aspergillus PCR Sensitivity 81.8% (cf. 95.5% PCR, 77.3% GM) Specificity 98% (cf. PCR, 96.6%; GM 91.5%) PPV 67% USED in combination with PCR sensitivity 100%, specificity 100%, PPV 80% First iteration of LFA White L et al. JCM May 2013

18 Aspergillus LFA: current status Use of test with BAL fluid >> serum Most promising in non-neutropenic patients (no data on serum LFA in this group) Use in combination with PCR +/- GM Non-specific binding evident even with the CE marked strips observed in some countries now rectified Till more data, small but expected incr. role in IA diagnostics

19 MALDI TOF MS: (replete with publications) ongoing needs Tom Tower, Christchurch College, Oxford

20 Direct detection of yeasts and moulds in blood cultures Why can t we emulate the bacteriologists? Direct application on concentrated aliquot from +ve BC Extraction processes are not as simple Unlike bacterial detection: blood (Hb, albumin) and blood culture (charcoal, cations, media) components give peaks overlapping with yeast spectra or interfere physically Washes, detergents, gel matrices, differential filtration

21 MALDI-TOF: blood culture studies Reference No. Specimen Processing Main results Marinach- Patrice Spiked SDS, ethanol 6 Candida species Ferroni Spiked 5% saponin Candida Yan Clinical Sepsityper Candida, crypto Schubert Spiked Sepsityper 8/15 Candida correct Spanu 12 >300 (2 yr) Can t pick polymicrobial fungemia Clinical Tween 80 C. albicans (96%); nonalbicans (86%) Chalupova J. Biotech Adv, 2014

22 Newer studies: Idelevich: Sepsityper protocol. 24 +BC Candida isolates; score >/=1.5; 2 spots, shortened duration for ID to 23.5 h Farina: in house protocol with SDS. Could only ID 1of 8 Candida strains correctly De Almeida Jr.: In house protocol. SARAMIS database. 7 fungi in blood (Fusarium, Exophiala, Trichosporon) ID to species level with confidence %. Not transferable to Bruker or Vitek MS databases. Idelevich et al. Plos One Dec 2014; Farina et al. New Microbiologica 2015; De Almeida et al Med Mycol 2016; 54:

23 Antifungal (caspofungin) susceptibility: Candida, Aspergillus CCI tool of MALDI Biotyper: maps relatedness of spectra at diff. drug concentrations Expose Candida to caspo for 3 h Categorical agreement 94.1% for Candida De Carolis et al. JCM 2012 July, Vella et al. JCM Sept 2013

24 We describe a simple version of an approach that discriminates susceptible and resistant isolates of C. albicans after 3-h incubation in presence of "breakpoint" level concentrations of caspofungin (CAS). Spectra at concentrations of 0 (null), 0.03 (intermediate), and 32 (maximal) μg/ml of CAS were used to create individual composite correlation index (CCI) matrices for 65 C. albicans isolates, including 13 fks1 mutants. Isolates are classed as susceptible or resistant if the CCI values of spectra at 0.03 and 32 μg/ml are higher or lower, respectively, than the CCIs of spectra at 0.03 and 0 μg/ml The isolate categorizations determined using MALDI-TOF MS-based were consistent with wild-type and mutant FKS1 genotypes and AFST reference methodology.. Vella, et al. JCM Sept 2013

25 Saracli M et al. Med Mycol 2015; 53:

26 Exposure to drug is for 16h at 35 o C, collect cells: FLU example (a)flu-s C. glabrata MIC 16 mg/l; CCI value at 64 and 32 was higher than CCI at 32 and 0 (ie 80.7 is > 37.4) In matrixes: numbers 1, 2, and 3 denote the highest (64), midrange (32) and null concentrations, respectively SARAMIS technology to compare spectra at mid range conc., with that at the null conc. and highest conc.

27 Current consensus (2017) MALDI TOF MS can classify triazole and echinocandin susceptibility Agreement with CLSI varies with drug-bug combination Reproducibility varies between % and also varies with drug-bug combination Need further refinement of technology Need software

28 Rapid resistance detection: I can make this happen Maa m

29 Echinocandin resistance: single aa substitutions Unlike azoles, a single genetic mechanism accounts for clinical resistance to echinocandins FKS1mutations Hot spots (FKS2 mutations also in C. glabrata) Garcia-Effron AAC 2008

30 Spectrum of FKS amino acid changes conferring resistance Frequency of mutations 2:1 FKS2/FKS1 Overall, N = >20 mutations - costs, work flow (multiplex, microarrays [CDC]) Perlin, MCM11, 2015; Garcia-Effron et al, AAC 2009

31 Candida spp. (15 C. glabratas) NGS - Six genes ERG11,ERG3, TAC1, CgPDR1, FKS1, FKS2 40 Candida isolates icnldyign 15 C. glabrrta One C. glabrata R to echinocandins but S to azoles, harboured an FKS2 S663P mutation + novel presumed gain of function CgPDR1 Other strain had new FKS2 mutation S663A, and a new putative GOF CgPDR1 Garnaud et al, JAC 2015

32 LOCAL STUDY Prior AF AMB ANI MIF CAS 5-FC Pos Vrc ITR FLC Pt. ID site Rx (d) 1 CMRL-1 Blood None < CMRL-2 Blood CAS (21) < CMRL-3 Blood None < CMRL-4 Blood CAS (30) >8 < CMRL-5 Pelvic None tissue < CMRL-6 Urine 5-FC, FLC (12) >64 >8 8 >16 256

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34 Azole resistance: A. fumigatus Ahmad et al., JCM, epub April 2014; Van der Linden et al., EID; 2011; 17 Increasingly recognised esp. environmental CYP51A mutations esp. M220 (all azoles), G54 (ITC and POS) amino acid substitutions Mutations in promoter region of CYP51A Predominant mechanism TR 34 /L98H Mutation in L98H coupled with duplication of fragment in promoter (34 bp tandem repeat, TR) up-regulates expression of CYP51A > 8 fold - multi-azole resistance Linked to treatment failure

35 Detection of Resistance PHENOTYPIC METHODS CLSI M38-A2 CLSI M51-A disc diffusion EUCAST Edef 9.1 Agar dilution E-test Agar screening test GENOTYPIC METHODS PCR-based Sequence based

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37 AsperGenius R Commercial kit, PATHONOSTICS Detects relevant Aspergillus spp. Used directly on clinical samples including BAL fluid Detects TR 34 /L98H, TR 46 /Y121F/T289A but not all mutations Costly

38 MOULD METABOLITES Metabolites 2015, 5, ; doi: /metabo Review OPEN ACCESS metabolites ISSN Advances in Electronic-Nose Technologies for the Detection of Volatile Biomarker Metabolites in the Human Breath Alphus D. Wilson Southern Hardwoods Laboratory, Center for Bottomland Hardwoods Research, Southern Research Station, USDA Forest Service, P.O. Box 227, Stoneville, MS 38776, USA; Differential Mobility Spectrometry Tel.: ; Fax: Academic Ionization Editor: Jörg Ingo Baumbach Sample in Source Electric Fields V C + V RF Detector (- Ions) Received: 29 December 2014 / Accepted: 23 February 2015 / Published: 2 March Air Flow + + Air Flow + + CYRANOSE 320 Tunable Ion Filter Detector Abstract: Recent advancements in the use of electronic-nose (e-nose) devices to analyze (+ Ions) human breath profiles for the presence of specific volatile metabolites, known as biomarkers or chemical bio-indicators High Sensitivity of specific - ppb human or less diseases, metabolic disorders and the overall health status of individuals, High Selectivity are providing the potential for new noninvasive tools and techniques useful to point-of-care Detect clinical multiple disease chemicals diagnoses. in a This exciting new area of electronic sample simultaneously disease detection and diagnosis promises to yield much faster and earlier detection of human diseases and disorders, allowing earlier, more effective treatments, resulting in more rapid patient recovery from various afflictions. E-nose devices are particularly suited for the field of disease diagnostics, because they are sensitive to a wide range of volatile organic

39 enose Based on NASA prototype Detects volatile organic compounds (VOCs): breathprint in real time (2-pentyl furans in IA) Proof of concept in Hematology patients 5 min, cheap, breathe into 10L Tedlar bags Pattern recognition software 6 IA, 5 controls 5/5 probable IA, 5/6 controls classified correctly De Heer K, et al. J Clin Microbiol 2013

40 Aspergillus metabolites in breath Koo S, et al. Clin Infect Dis 2014; 59:1733. Sens: 0.94 (0.81, 0.95) Spec: 0.93 (0.79, 0.98) LR+ 13.4, LR-: 0.06

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42 Aspergillus metabolites in breath In vivo VORs 2-PFs, terpines Breath sampler Thermal Desorption tube CG/Mass Spec.

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44 New Imaging techniques for IFD Halos to non-specific consolidation to cavitation: IA vs. other mould infections Current standard of care Caillot et al. J Clin Oncol 2001; Caillot et al. Cancer 2007

45 Can Imaging add specificity: mouse experiment A. fumigatus labelled Mab ([ 64 Cu]DOTAlabelled JF5 Mab) Allowed specific loc. lung infection when combined with PET Able to ddx IA from pneumococcus and other bacterial pathogens STAY TUNED! Antibody-guided immunopet/mr

46 Summary Experience with Aspergillus PCR, GM, serum BDG incr. Aspergillus PCR now EORTC/MSG criterion Cryptococcus LFA YES, implement, screen Aspergillus LFA BAL fluid, use with other biomarkers, more data needed MALDI-TOF MS and molecular AFST - coming Aspergillus VOCs coming, being commercialised, watch this space! Pathogen-specific imaging coming..

47 Spare slides

48 TR 34 /L98H and TR 46 /Y121F/T289A

49 Candida PCR Numerous publications totaling >5000 patients (blood testing) Lack of standardization, clinical validation or demonstrated clinical benefits Varied nucleic acid detection platforms, blood fraction, extraction methods, targets, post-pcr analysis Highly heterogenous study designs, case definitions, types of disease, controls, inclusion of colonization, timing of samples

50 Central challenge in studying Candida diagnostics How do you assess the performance of new diagnostic when the current gold standard is inadequate? How do you interpret blood-culture negative, new diagnostic positive test results? Important question is also if the test can be used to stop AF therapy vis a vis used for diagnosis per se

51 PCR studies: prior to advent of T2 Biosystems Recent meta-analysis Avni 2011 Suspected IC Pooled sensitivity/specificity: 95%/92% Probable IC Sensitivity 85% vs 38% for blood culture Higher sensitivity QIAmp extraction Panfungal rrna or P450 gene targets Candida-specific assay LOD: <10 CFU/mL = similar to blood cultures Trend toward lower specificity with colonization

52 T2Candida Based on changes in excitement of water molecules PCR/T2MR Whole blood assay in self-contained system (1 CFU/ml) FDA approved as adjunct to cultures for candidemia Mylonakis Clin Infect Dis control patients with negative blood cultures for Candida, and 250 spiked blood samples Sensitivity/Specificity: 91%/98% Limited data on clinical samples

53 Clinical studies In one blinded study, T2Candida correctly detected 8 Candida-positive samples from 3 patients in a set of 24 samples Samples from another 1801 patients were tested as part of the T2 pivotal trial. Overall sens. 91.1%, overall spec. 99.4%. T2Candida correctly identified 6 of 7 candidemia patients T2 Candida panel also identified IC in blood culture negative patients with deep candidiasis Also useful in children Pfaller et al., Fut Microbiol 2015; Mylonakis et al., Clin Infect Dis 2015

54 Main results T2Candida directed approach was less costly and more effective than BC-directed approach and fluconazole empiric therapy but Less costly, less effective than echinocandin empiric treatment Cost effectiveness highly dependent on prevalence of Candida BSI Reduced use of unnecessary antifungal therapy by 98% relative to empiric therapy Walker et al., J Clin Microbiol 2016; 54: 718

55 CrAg Lateral Flow Assay CRAG LFA Procedure QUALITATIVE BASIC PROCEDURE For the Detection of Cryptococcal Antigen REF CR2003 INTENDED USE The CrAg Lateral Flow Assay is an immunochromatographic test system for the qualitative or semi-quantitative detection of the capsular polysaccharide antigens of Cryptococcus species complex (Cryptococcus neoformans and Cryptococcus Qualitative Procedure azide. Sodium azide should never be flushed down the gattii) Read in serum, plasma and cerebral at spinal fluid 10 (CSF). minutes (TAT about 20 mins) 1. Add 1 drop of LF Specimen Diluent (REF GLF025) to an The CrAg Lateral Flow Assay is a prescription-use laboratory assay which can aid in the diagnosis of cryptococcosis. SUMMARY and EXPLANATION of the Test REAGENTS Cryptococcosis Semi-quantitative is caused by both species of the Cryptococcus procedure: 4. Wait 10 minutes. species complex (Cryptococcus neoformans and Cryptococcus gattii) (4). Individuals with impaired cell-mediated immunity are at greatest risk of infection (6). Cryptococcosis is one of the most common opportunistic infections in AIDS patients (5). Detection of cryptococcal antigen (CrAg) in serum and 1. LF Specimen Diluent (2.5 ml, REF GLF025): Glycinebuffered saline containing blocking agents and a preservative 2. LF Titration Diluent (6.0 ml, REF EI0010): Glycinebuffered saline containing a preservative followed by 1:2 serial dilutions to 1:2560. initial dilution of 1:5, by 1:2 serial dilutions 3. CrAg LF Test Strips (50 strips in desiccant vial, REF 2. Place 10 micro-centrifuge or test tubes in an appropriate LFCR50) 4. CrAg Positive Control (1 ml, REF CB1020): Glycineto 1:2560 CSF has been extensively utilized with very high sensitivity and specificity (1-3). BIOLOGICAL PRINCIPLES The CrAg Lateral Flow Assay is a dipstick sandwich immunochromatographic assay. Specimens and specimen diluent are added into an appropriate reservoir, such as a test tube, and the lateral flow device is placed into the reservoir. The test uses specimen wicking to capture goldconjugated, anti-crag monoclonal antibodies and goldconjugated control antibodies deposited on the test membrane. If CrAg is present in the specimen, then it binds 2. When handling patient specimens, adequate measures should be taken to prevent exposure to etiologic agents potentially present in the specimens. 3. Always wear gloves when handling reagents in this kit as some reagents are preserved with 0.095% (w/w) sodium drain as this chemical may react with lead or copper plumbing to form potentially explosive metal azides. Excess reagents should be discarded in an appropriate waste receptacle. buffered saline spiked with cryptococcal antigen (strain 184A clinical isolate from Tulane University (Infection & Immunity, June 1983, p )) 5. Package insert MATERIALS NOT PROVIDED PROCEDURE Qualitative testing shown REFER TO REAGENTS SECTION FOR A LIST OF MATERIALS PROVIDED. 1. Pipettor (40-µL and 80-µL) 2. Timer 3. Disposable micro-centrifuge tubes, test tubes, or a appropriate reservoir (disposable micro-centrifuge tube, test tubes, or micro-titer plate, etc.). 2. Add 40 µl of specimen to the container and mix. 3. Submerge the white end of a Cryptococcal Antigen Lateral Flow Test Strip (REF LFCR50) into the specimen. 5. Read and record the results (See READING THE TEST). Semi-Quantitative Titration Procedure 1. Prepare dilutions starting with an initial dilution of 1:5, rack and label them 1-10 (1:5 through 1:2560). Additional dilutions may be necessary if the specimen is positive at 1: Add 4 drops of LF Specimen Diluent (REF GLF025) to tube #1. 4. Add 2 drops of LF Titration Diluent (REF EI0010) to each of the tubes labeled Immuno-Mycologics, Inc., OK, USA 5. Add 40 µl of specimen to tube #1 and mix well. 6. Transfer 80 µl of specimen from tube #1 to tube #2 and mix well. Continue this dilution procedure through tube #10. Discard 80 µl from tube 10 for a final tube volume of 80 µl. India Ink: 20 mins but needs microscope

56 Other tips for BDG testing in BAL fluid Cut off values may differ in different populations NB: blood products (FFP, Packed rbc, human albumin) One study showed that BAL fluid levels could predict mortality at 90 days (>/= 200 pg/ml) Resiches et al., J Infect 2016; Bhaskaran A et al., Med Mycol Aug 2016

57 Recent meta-analysis: 838 patients, 6 studies: pooled sensitivity, specificity, PLR, NLR, DOR were 0.52; 0.58; 1.34; 0.82; 1.71, respectively Conclusions: Accuracy BAL fluid BDG is marginal Shi X et al., Respir Med 2016

58 Does screening matter? Smith R et al. Plos One Vietnam, prevalence 2-4% hence screening considered cost-effective Rajasingham et al. J Acquir Immune Defic Syndr 2012; McKenney et al. Clin Infect Dis 2015

59 ORCAS Slide David Trial, David Boulware: Meya ORCAS (PI) Trial, David Meya (PI) CDC U01GH CDC U01GH Effect of CrAg Screening Kaplan-Meier survival estimates: All Cause Survival in CD4<100

60 Screening to reduce mortality WHO: since 2011, screen if CD4 100/uL IDSA: screen if CD4 is 50/uL USA: CRAG positivity 2.9% - screening is cost effective if prevalence 2%, multiple studies: saves lives by early Rx; At 8% inc: NNS to detect CrAg+ = 11 NNS to detect prevent 1 death from CM = 16 Cf. Colon cancer = 1000 Rajasingham et al. J Acquir Immune Defic Syndr 2012; Aberg CID 2013; McKenney et al. Clin Infect Dis 2015

61 Molecular-based assays

62 Patient LOCAL STUDY MIC (mg/l) Prior AF treatment AMB ANI MIF CAS 5-FC Pos Vrc ITR FLC No. Isolate ID site (days) 1 CMRL-1 a Blood None < < CMRL-2 a Blood CAS (21) b < CMRL-3 a Blood None < CMRL-4 a Blood CAS (30) b >8 < CMRL-5 a Pelvic tissue None < CMRL-6 a Urine 5-FC, FLC >64 >8 8 > (12) b 4 CMRL-7 Blood None < CMRL-8 Fluid None < CMRL-9 Blood None < CMRL-10 Blood None < < CMRL-11 Blood None < < CMRL-12 Blood None <0.06 > ATCC Blood None <