Ongoing Quality Assurance for - The NELAC...

Ongoing Quality Assurance for Cryptosporidium Analysis

Leah Fohl Villegas Ph.D.

2

▪ 2002: EPA Cryptosporidium Lab Quality Assurance Program

– Cryptosporidium Lab evaluations initiated by the EPA

▪ 2006: Beginning of LT2 Compliance Monitoring for Public Water Systems

▪ 2009: PTs provided by ACLASS certified PT provider

– TNI FoPT table

▪ 2010: Training State Certification Officers for Cryptosporidium

▪ 2015: State Certification Programs for Cryptosporidium

Cryptosporidium Lab Certification History

1623

Method:

1623.1

1623 & 1623.1

1623 & 1622

1623.1, 1623, & 1622

Envirochek HV

Filter:

Filta-MaxCFC

Grants Pass

Truckee

SNWS

San Francisco

BioVir

CH Diagnostics

Accurate

State Hygienic Lab

MilwaukeeWSLH

McCoy & McCoy

SMIEurofins

EAL

ASIMohawk

Philadelphia

PA DEP

Charlotte-MecklenburgESC

GA DNR

Orange County

Tampa

EMSL

MI State UNYC DEP

PRASA

Montgomery

TTL

NYS DOH

Cel-Analytical

Lab/Cor

Santa Clara

San Diego

Metropolitan

Scottsdale

Phoenix

Citizens

Irvine Ranch

LA DWP

BCS

Eurofins-CA

LA DHH

OK DEQ

44 Certified Cryptosporidium Labs in EPA Regions

4

▪ Method 1622

– Cryptosporidium Only

▪ Method 1623

– Cryptosporidium and Giardia

▪ Method 1623.1

– Cryptosporidium & Giardia

– Modification of 1623

Approved Methods for Cryptosporidium Detection

5

Cryptosporidium Detection Methods Overview

10 L 100 mL

Sample

Immunomagnetic

Separation

Centrifuge Tubes

Filter

Slide

6

Cryptosporidium PT Recoveries for Approved Laboratories

Method 1623

1st round monitoring

begins

OPR criteria raised to 22%

Method 1623.1 published


ends


2nd round monitoring

begins

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

Per

cen

t R

eco

very

Month and Year of Test Event

Cryptosporidium Recovery for LT2 Laboratory Proficiency Tests

Avg % Crypto Recovery

St Dev Crypto Recovery

* indicates PT samples contain a standard matrix

Method 1622/1623


begins


Method 1623.1 published


ends


2nd round monitoring

begins

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

Per

cen

t R

eco

very

Month and Year of Proficiency Test Event

Avg % Crypto Recovery St Dev Crypto Recovery

* indicates PT samples contain a standard matrix

7

Method 1623 2005 Version

Federal Register Feb. 25, 2009

Federal Register Sept. 5,2013

CryptosporidiumOPR Minimum

Acceptance Criteria 11% 22% 33%

Number of samples

293 333 753

Number of Labs 6 58 56

Blind vs. Unblind Unblind Blind Blind

Acceptance limit increased to 33%

A World of SolutionsTM 8

▪ Conformity with EPA Drinking Water Laboratory Certification Manual

▪ Certification Officer passed EPA Cryptosporidium CO Training Course

▪ Federal Register Vol. 78, No. 172, September 5, 2013. 54643

▪ Train certification officers

▪ Provide written guidance

▪ Provide technical support for states, regions and laboratories

▪ Review/assist regional programs

▪ Maintain a list of links to state websites naming certified labs

State Oversight for Cryptosporidium Laboratories

State Program Equivalence Technical Support Center


▪ Checklists A, B, & C

▪ Positive Staining Control and OPR slide review by experienced microscopist

▪ Blind spikes proved by 3rd

party vendor

▪ Microscopy Skills Evaluation by 3rd party expert

▪ Federal Register Vol. 74, No. 36, February 25, 2009. 8531

Substantial Conformity


▪ Courses offered in Cincinnati

– 2010

– 2011

– 2013

▪ Courses offered online

– 2013

– 2014

– 2015 (twice)

– 2016

– 2017

– Next offering September 2017

Cryptosporidium Certification Officer Training


What can I learn at the Cryptosporidium CO Course?

▪ Audit a lab’s ability to follow the method consistently

▪ Identify discrepancies between lab practices and written procedures

▪ Recognize proper lab techniques that reduce the loss of organisms


What can I expect from the Cryptosporidium CO Course?

▪ Course is 4 weeks of online lessons

– 8-10 hours study/week

▪ Weekly webinars

▪ Online open-book exam and mock laboratory

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▪ Continuous access to CO Course materials

▪ Videos of Method and modifications

▪ Training tools for microscopy, IMS, and sampling

– Note reviewing this material is not a substitute for completing or auditing the course

Website updates for continued training


▪ Online Analyst Skills Evaluation

– Every Analyst in the country has completed the online assessment

Audit Support: Analyst Skills Evaluation


▪ Evaluation of Microscope Slide Preparation

▪ Subject mater expert evaluates the OPR and positive staining control using a defined rubric, and scores 1 to 4.

Audit Support: Slide Review

FITC 1000X DAPI 1000X DIC 1000X

16

▪ Bubbles can be seen with the naked eye

▪ DAPI and DIC do not work correctly in a bubble

Common concerns with slides: Bubbles

1000X DIC

Bubble

17

Common concerns with slides: Debris

• Positive Staining Controls and

OPRs should have minimal

debris

• No debris should fluoresce

like a Cryptosporidium oocyst

or Giardia cyst

FITC 1000X DAPI 1000X DIC 1000X

20X FITC

Cryptosporidium

Debris shown below

18

Common concerns with slides: Debris

FITC 200X

Bright field 200XDAPI 200X

▪ Reagent Water OPRs should have little to no debris


Organisms Outside of the Well

FITC 1000X

DIC 1000XDAPI 1000X


▪ Call log for reported laboratory concerns or issues

– Recovery drops when we run multiple samples?

– Presence of magnetic particles interfering with recovery.

– Product concerns or issues

▪ Technical Support

– Method questions

– Tips and Tricks

– Research

– Webinars

– Microscope assistance

Laboratory Support


▪ Skill sharing forums with experienced analysts

▪ Hands-on workshops for newer analysts

– Microscopy technique

– IMS tips and tricks

– Productivity

– Recovery

▪ Workshops offered

– 2015

– 2016

Analyst Training Workshops and Forums

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Cryptosporidium and Giardia Recoveries can be matrix dependent

0

5

10

15

20

Pe

rce

nta

ge o

f th

e T

ota

l

Matrix Spike Recovery Range

24

Matrix Effects Can Impact IMS Bead Steps

N.J. Shaw et al. / Journal of Microbiological Methods 75 (2008) 445–448

25

Lot to lot variation has been reported

0.0

20.0

40.0

60.0

80.0

100.0

Suspect Lot (12) New Lot (17) Suspect Lot (11) New Lot (13)

IMS Control Full Method

Cry

pto

spo

rid

ium

Per

cen

t R

eco

very

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Three Beads Evaluated

Dynabeads

*Scale bar is 5 microns for each image

Alternate B Alternate C

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Reagent Water IMS Control Recoveries

0

20

40

60

80

100

Perc

ent

reco

very

Alternative IMS Beads

Cryptosporidium Giardia

*

* Giardia IMS beads are not available with this product

n ≥ 4

Change from IMS Controls to

Full Method

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Method 1623.1 Recovery in Reagent Water

0

20

40

60

80

100Pe

rcen

t re

cove

ry

Cryptosporidium Giardia n ≥ 6

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Method 1623.1 Recovery in Ohio River Water

0

20

40

60

80

100P

erce

nt

reco

very

Cryptosporidium Giardian = 4

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▪ The Dynabeads resulted in higher average Cryptosporidium recoveries compared to both alternative beads in all conditions.

▪ The alternative products either resulted in significantly lower Giardia recoveries in all matrices or did not have a Giardiabead.

▪ Overall, tests of the alternate IMS beads did not demonstrate that they were comparable to results obtained using Dynabeads

Result of Laboratory Support Study

32

▪ TSC provides technical assistance to laboratories and certification officers

– Audit support

• Slide reviews

• Microscopy skills evaluation

▪ Frequent communication with vendors

▪ Certification Officer training offered online

Continuing Certification Officer and Laboratory Support

Thank You!!

• For more information contact:

– [email protected]

Ongoing Quality Assurance for - The NELAC...

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