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for Microbiology Improving the stability of microbiology Improving the stability of microbiology EQA specimens: Fungal spore suspensions 12 th October 2010 EQALM Microbiology Working Group

Overview Overview Part 1 - Mycology � Fungi / specimens distributed � New specimen format development New specimen format development � Results from pilot distribution � Experience to date � Experience to date Part 2 - Bacteriology gy � EQA specimen design � Stability results – lyophilised bacteriology specimens � Open discussion 12 th October 2010 EQALM Microbiology Working Group

UK NEQAS Mycology Schemes UK NEQAS Mycology Schemes � Mycology Identification Identification of filamentous fungi and yeasts, Identification of filamentous fungi and yeasts, including dermatophytes and fungi associated with infection in the immuno-compromised 4 specimens distributed 3 times per year i di ib d 3 i � Antifungal susceptibility Identification and assessment of antifungal Id tifi ti d t f tif l susceptibility testing including: Amphotericin B, Fluconazole, Flucytosine, Itraconazole, , y , , Voriconazole and Caspofungin 2 specimens distributed 3 times per year 12 th October 2010 EQALM Microbiology Working Group

Filamentous fungi distributed g 12 th October 2010 EQALM Microbiology Working Group

Participant performance (% correct) – p p ( ) mycology EQA non-dermatophyte fungi

Participant performance (% correct) p p ( ) mycology EQA dermatophyte fungi

Participant performance (% correct) p p ( ) mycology EQA antifungal susceptibility Amphotericin Organism Fluconazole Flucytosine Itraconazole Voriconazole B C. parapsilosis C. parapsilosis 91 91 93 93 94 94 74 74 87 87 C. tropicalis 90 93 96 30 91 C. krusei C krusei 90 90 61 61 23 23 44 44 100 100 R. mucilaginosa 95 100 97 91 22 C neoformans C. neoformans 100 100 86 86 44 44 89 89 100 100 C. albicans 93 98 98 98 96 Overall (%) 93 89 75 71 83 concordance by agent

Participant performance – mycology p p y gy EQA antifungal susceptibility

Developing new specimens Developing new specimens M gypseum M. gypseum A. versicolor 12 th October 2010 EQALM Microbiology Working Group

Review of specimen options Review of specimen options � Lyophilisation � Under mineral oil � Drying on silica gel � Soil storage Soil sto age � Spore suspensions in water � Spore suspensions in water 12 th October 2010 EQALM Microbiology Working Group

Evaluation of the viability of pathogenic filamentous fungi after prolonged storage in sterile water & review of recent published studies p g g p on storage methods Borman et al . Mycopathologia (16) June 2006 Evaluation of the survival and potential morphological alterations of � 45 species of pathogenic filamentous fungi that had been stored in sterile water following Castellani's method in the National Collection of Pathogenic Fungi (NCPF), UK f P th i F i (NCPF) UK Storage duration varied from 2 months to over 21 years � Ninety percent of stored organisms were shown to be viable Ni t t f t d i h t b i bl � Viability was largely independent of the duration of storage, but did � apparently vary to some degree in an organism-specific manner This was especially marked for several isolates of dermatophytes, � where storage resulted in loss of recognisable colonial features, and overproduction of sterile mycelium with aberrant or no conidia These findings suggest that while Castellani's method remains an � easy and inexpensive method for long-term preservation of most fungi, water storage should be supplemented by a second storage method to increase the chances of retaining both viability and th d t i th h f t i i b th i bilit d morphological stability over long periods

Pilot - Dermatophytes selected p y Trichophyton rubrum Trichophyton tonsurans 12 th October 2010 EQALM Microbiology Working Group

Pilot Pilot - Dermatophytes selected Dermatophytes selected Trichopyton interdigitale Epidermophyton floccosum 12 th October 2010 EQALM Microbiology Working Group

Preparation of spore suspensions Preparation of spore suspensions Fl Flood a single colony on the d i l l th � SABM plate with 5mL distilled water Scrape the surface of the colony Scrape the surface of the colony � � to release the spores Take up the water from the plate, � now containing the released spores and transfer to a 5mL spores and transfer to a 5mL bijou Vortex the spore suspension for � one minute to break any o e ute to b ea a y mycelium fragments Inoculate 100µL of the � suspension onto five SABM plates Spread the inoculum plates. Spread the inoculum around the surface of the agar plate using a plastic spreader Incubate the SABM plates for 14 p � days. 12 th October 2010 EQALM Microbiology Working Group

Bulk preparation Bulk preparation After incubation confirm the purity of the � organism g Perform a spore count using a counting � chamber The numbers of spores/ mycelial chamber The numbers of spores/ mycelial fragments found should concur by species with the guidelines provided by the with the guidelines provided by the Mycology Reference Laboratory on spore forming filamentous fungi Prepare a bulk specimen � 12 th October 2010 EQALM Microbiology Working Group

Spore production characteristics of p p some common fungi 12 th October 2010 EQALM Microbiology Working Group

12 th October 2010 EQALM Microbiology Working Group

Pilot specimens Pilot specimens - results results Trichophyton rubrum, T. interdigitale T. tonsurans, Microsporum canis, M audounii to su a s, c ospo u ca s, audou � Every vial in the pilot batch (n= 400) produced viable fungal colonies p g demonstrating typical morphology Epidermophyton floccosum � First batch 50% failed to grow; second � First batch 50% failed to grow; second batch revealed 100% viability with typical morphology

Acknowledgements Acknowledgements Staff and participants of Special thanks to: UK NEQAS for Q Students Students Microbiology Anita Marwaha Kalana Patabendige Kalana Patabendige Nita Patel Malti nakrani Dr Elizabeth Johnson & staff A Ana Deheer Graham D h G h Mycology Mousoumi Reference Laboratory Daschowdhury Daschowdhury Bristol Martha Valencia 12 th October 2010 EQALM Microbiology Working Group

Bacteriology specimens – gy p homogenity and stability EQALM Microbiology Working Group Discussion Discussion

What are the design criteria for EQA g schemes? � Clinically relevant � Homogeneous specimens Homogeneous specimens � No matrix effect � Stable specimens � Adequately characterised � Adequately characterised � Measurement and assessment of performance is possible performance is possible 12 th October 2010 EQALM Microbiology Working Group

0 1 2 3 4 5 6 6 Staphyloc coccus aureus 12 th October 2010 Es scherichia coli Es scherichia coli Klebsiella a pneumoniae Staphyloc coccus aureus Pseudomona as aeruginosa Sta aphylococcus haemolyticus Enteroco occus faecalis Pseudomona as aeruginosa Es scherichia coli Staphyloc coccus aureus Staphyloc coccus aureus Es scherichia coli Es scherichia coli Klebsiella a pneumoniae Microbiology Working Group Staphyloc coccus aureus Staphyloc coccus aureus Serratia a marcescens EQALM Es scherichia coli Pseudomona as aeruginosa Es scherichia coli Acinetobac cter baumanii Sh higella boydii ( (serotype 10) Sten notrophomona as maltophilia Serratia a marcescens Beta-haemol lytic streptoco occus group B Moraxel lla catarrhalis Neisseria gonorrhoeae Bordet tella pertussis Negative Shigella flexne S eri & Salmone ella enteritidis Staphyloc coccus aureus Vibrio o cholerae O1 S Streptococcus s pneumoniae Aspe ergillus flavus Staphy lococcus aure us & Group A… … Lactobacil llus paracasei Plate 5 Plate 4 Plate 3 Plate 2 Plate 1

6 5 4 3 plate 1 2 plate 2 plate 3 1 plate 4 0 plate 5 12 th October 2010 EQALM Microbiology Working Group