improving the stability of microbiologyimproving …€¦ · improving the stability of...

for Microbiology

Improving the stability of microbiologyImproving the stability of microbiology EQA specimens:

Fungal spore suspensions

12th October 2010 EQALM Microbiology Working Group

OverviewOverview

Part 1 - MycologyFungi / specimens distributedNew specimen format developmentNew specimen format developmentResults from pilot distributionExperience to dateExperience to date

Part 2 - BacteriologygyEQA specimen designStability results – lyophilised bacteriology specimensOpen discussion


UK NEQAS Mycology SchemesUK NEQAS Mycology Schemes

Mycology IdentificationIdentification of filamentous fungi and yeasts, Identification of filamentous fungi and yeasts,

including dermatophytes and fungi associated with infection in the immuno-compromised

i di ib d 3 i4 specimens distributed 3 times per year

Antifungal susceptibilityId tifi ti d t f tif l Identification and assessment of antifungal

susceptibility testing including: Amphotericin B, Fluconazole, Flucytosine, Itraconazole, , y , ,Voriconazole and Caspofungin

2 specimens distributed 3 times per year


Filamentous fungi distributedg


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

Organism Amphotericin B Fluconazole Flucytosine Itraconazole Voriconazole

C. parapsilosis 91 93 94 74 87C. parapsilosis 91 93 94 74 87

C. tropicalis 90 93 96 30 91

C krusei 90 61 23 44 100C. krusei 90 61 23 44 100

R. mucilaginosa 95 100 97 91 22

C neoformans 100 86 44 89 100C. neoformans 100 86 44 89 100

C. albicans 93 98 98 98 96

Overall (%) concordance by agent

93 89 75 71 83

Participant performance – mycology p p y gyEQA antifungal susceptibility

Developing new specimensDeveloping new specimens

M gypseumM. gypseum

A. versicolor


Review of specimen optionsReview of specimen options

Lyophilisation

Under mineral oil

Drying on silica gel

Soil sto ageSoil storage

Spore suspensions in waterSpore suspensions in water


Evaluation of the viability of pathogenic filamentous fungi after prolonged storage in sterile water & review of recent published studies p g g pon storage methodsBorman 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 f P th i F i (NCPF) UKof Pathogenic Fungi (NCPF), UK

Storage duration varied from 2 months to over 21 years

Ni t t f t d i h t b i blNinety percent of stored organisms were shown to be viable

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

th d t i th h f t i i b th i bilit d method to increase the chances of retaining both viability and morphological stability over long periods

Pilot - Dermatophytes selectedp y

Trichophyton rubrum

Trichophyton tonsurans


Pilot Dermatophytes selectedPilot - Dermatophytes selected

Trichopyton interdigitale

Epidermophyton floccosum


Preparation of spore suspensionsPreparation of spore suspensions

Fl d i l l thFlood a single colony on the SABM plate with 5mL distilled water

Scrape the surface of the colonyScrape 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 5mLspores and transfer to a 5mL bijou

Vortex the spore suspension for one minute to break any o e ute to b ea a ymycelium fragments

Inoculate 100µL of the suspension onto five SABM plates Spread the inoculumplates. Spread the inoculum around the surface of the agar plate using a plastic spreader

Incubate the SABM plates for 14


pdays.

Bulk preparationBulk preparation

After incubation confirm the purity of the organismg

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


Spore production characteristics of p psome common fungi


Pilot specimens resultsPilot specimens - results

Trichophyton rubrum, T. interdigitale T. tonsurans, Microsporum canis, M audouniito su a s, c ospo u ca s, audouEvery vial in the pilot batch (n=400) produced viable fungal colonies p gdemonstrating typical morphology

Epidermophyton floccosumFirst batch 50% failed to grow; second First batch 50% failed to grow; second batch revealed 100% viability with typical morphology

AcknowledgementsAcknowledgements

Staff and participants ofUK NEQAS for

Special thanks to:StudentsQ

MicrobiologyStudentsAnita MarwahaKalana Patabendige

Nita PatelMalti nakraniA D h G h

Kalana Patabendige

Dr Elizabeth Johnson & staffAna Deheer GrahamMousoumi

Daschowdhury

MycologyReference Laboratory

DaschowdhuryMartha Valencia

Bristol


Bacteriology specimens –gy phomogenity and stability

EQALM Microbiology Working GroupDiscussionDiscussion

What are the design criteria for EQA gschemes?

Clinically relevantHomogeneous specimensHomogeneous specimensNo matrix effectStable specimensAdequately characterisedAdequately characterisedMeasurement and assessment of performance is possibleperformance is possible


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