1. ‘A COMPARISON OF THREE RAPID DIAGNOSTIC TECHNIQUES FOR THE IDENTIFICATION OF YEASTS DIRECT FROM BLOOD CULTURE' Rebecca Gorton Centre for Clinical Microbiology meeting 11 January 2012

2. Candidemia  High morbidity and mortality rates 32-50% mortality rates.  Significant proportion of Candidemia is caused by ‘non- albicans’ species. may be resistant to Fluconazole and other azole antifungal agents.  Substantial financial burden. Echinocandin use equates to a 26 fold increase in costs compared with Fluconazole

3. Current diagnostic technique  Current identification techniques require 24-72 hours for speciation. Candida albicans ~24 hours - CHROMagar™ Candida. ‘Non-albicans’ 72 hours – Auxacolor™/CMA agar.

4. Rapid identification techniques  Turn around time < 2 hours.  Cost  Technical requirement Gram’s stainPNA-FISHMALDI-TOF

5. Samples  50 spiked blood cultures Isolates collected from Candidemia cases at RFHNHS  Each investigator was blinded to the yeast identification in all cultures.  Included 2 polyfungal cultures in the cohort. Table 1. Yeast blood culture isolates identifications using conventional methods Conventional identification* Number (n = 50) C. albicans17 C. tropicalis4 C. glabrata9 C. krusei4 C. parapsilosis9 C. guillermondii2 C. lusitaniae1 C. pelliculosa1 C. neoformans2 B. capitatus1

6. Gram’s stain C. tropicalisC. albicansC. glabrataC. krusei Use identification keys whilst looking at slides to identify the yeast species. Included 7 yeasts species within the ‘database’

7. PNA-FISH Peptide nucleic acid fluorescence in-situ hybridisation PNA probes Peptide backbone – neutrally charged Hybridise to rRNA molecules

8. MALDI-TOF Matrix assisted laser desorption ionisation time of flight mass spectrometry  Sepsityper kit Red cell lysis Detergent wash  Formic acid extraction on yeast pellet.  Over 100 species in the database.

9. Results

10. Table 2. Frequencies of classification, misclassification and non-recognition for Gram’s stain, MALDI-TOF and PNA-FISH Classification results Correctly identified Isolates misclassified UnknownSuccess rate (%) (a)Performance total Gram’s stain3613172 (40 $ ) PNA-FISH432586 MALDI-TOF28 (38)*220 (10*)76 (b)Performance only on species in respective method database Gram’s stain3610176.6 (42 $ ) PNA-FISH4300100 MALDI-TOF28 (38)*220 (10*)76 (c) Performance total (including ‘unknown’ as the correct identification for isolates absent from the database) Gram’s stain3613174 (41 $ ) PNA-FISH482096 MALDI-TOF28 (39)*220 (10*)76 $ Inexperienced health care scientist

11. Table 3. Isolates missed or misidentified by Gram’s stain, PNA-FISH and MALDI-TOF Conventional identification nGram’s stain nPNA-FISHnMALDI-TOF n C. albicans17C. tropicalis2-Missed4 C. parapsilosis1- Missed1- C. tropicalis4C. albicans1--- C. glabrata9C. parapsilosis1-Missed3 C. krusei4C. parapsilosis3- 9C. albicans1-Missed2 C. guillermondii2C. glabrata1--- C. lusitaniae1C. glabrata1-Missed1 C. pelliculosa1C. glabrata1-Missed1 C. neoformans2-C. glabrata/krusei1Missed1 B. capitatus1-C. glabrata/krusei1-- Both reported as negative on repeat cc

12. Table 4. Cost, turn around time and technical requirement for Gram’s stain, PNA-FISH and MALDI-TOF Gram’s stainPNA-FISHMALDI-TOF Cost per test£0.10*£70.00*£1.30* Turnaround time 15 minutes90 minutes Technical requirement Experienced $ Inexperienced & Inexperienced

13. Discussion 1. Specificity for yeast biomarkers -RNA PNA-FISH probes are highly specific and blood becomes background ‘noise’ against fluorescence.  MALDI-TOF MS detects high abundance human proteins which mask the yeast proteins. Software cannot exclude these proteins from analysis and therefore negatively scores them leading to poor results.

14. Discussion 2. Ease of interpretation PNA-FISH system is ‘easy’ to interpret as a 3 colour Traffic light system requiring little expertise.  Gram’s stain  Mis-identification was frequent  A high level of experience was required to achieve a success rate close to that required

15. However.... PNA-FISH..... 1. Only includes 5 species within its database 14% of isolates were unknown in this study 2. Is expensive at £70 per test. 3. Unclear clinical impact Choosing fluconazole over an echinocandin is not only based on the yeast identification but also the patient.

16. Conclusions  MALDI-TOF has the greatest potential Improved proteins extraction Advances in software  Gram’s stain analysis Mis-identification not clinically significant for any C. albicans isolates so could be used as a C. albicans/Non albicans indicator where an experienced HCS is available  PNA-FISH Requires a multi-centre trial to assess clinical impact of the assay.

17. Acknowledgments  HCS involved in the trial  Purnima Ramnarain, Niel Stone and Kevin Barker  Professor Kibbler and Dr T. McHugh.  Microbiology department.  Bruker and AdvanDX for technical support.