Ongoing Quality Assurance for Cryptosporidium Analysis. Leah Fohl Villegas Ph.D.

Transcription

1 Ongoing Quality Assurance for Cryptosporidium Analysis Leah Fohl Villegas Ph.D.

2 Cryptosporidium Lab Certification History 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 2

3 44 Certified Cryptosporidium Labs in EPA Regions Lab/Cor Grants Pass Mohawk ASI MI State U EAL NYC DEP WSLH Milwaukee NYS DOH BioVir Truckee San Francisco Cel-Analytical Santa Clara CH Diagnostics State Hygienic Lab SMI Eurofins Citizens PA DEP EMSL Philadelphia SNWS McCoy & McCoy LA DWP Irvine Ranch Method: 1623 Eurofins-CA Metropolitan San Diego & & , 1623, & 1622 Phoenix Scottsdale OK DEQ Accurate LA DHH ESC GA DNR TTL Montgomery Charlotte-Mecklenburg BCS Orange County Tampa Filter: Envirochek HV Filta-Max CFC PRASA

4 Approved Methods for Cryptosporidium Detection Method 1622 Cryptosporidium Only Method 1623 Cryptosporidium and Giardia Method Cryptosporidium & Giardia Modification of

5 Cryptosporidium Detection Methods Overview Sample Filter Centrifuge Tubes Immunomagnetic Separation 10 L 100 ml Slide 5

6 Percent Recovery Percent Recovery Cryptosporidium PT Recoveries for Approved Laboratories Avg % Crypto Recovery St Dev Crypto Recovery Cryptosporidium Recovery for LT2 Laboratory Proficiency Tests st round 70.0 monitoring begins st round monitoring begins Avg % Crypto Recovery St Dev Crypto Recovery 1st round monitoring 1st round monitoring ends ends 2nd round monitoring begins 2nd round monitoring begins Method 1623 Method /1623 OPR criteria raised to 22% OPR criteria raised to 22% Method published Method published OPR criteria raised to 33% OPR criteria raised to 33% indicates PT samples contain a standard matrix * indicates PT samples contain a standard matrix * Month and Year of Test Event Month and Year of Proficiency Test Event 6

7 Acceptance limit increased to 33% Method Version Federal Register Feb. 25, 2009 Federal Register Sept. 5,2013 Cryptosporidium OPR Minimum Acceptance Criteria Number of samples 11% 22% 33% Number of Labs Blind vs. Unblind Unblind Blind Blind 7

8 State Oversight for Cryptosporidium Laboratories State Program Equivalence Conformity with EPA Drinking Water Laboratory Certification Manual Certification Officer passed EPA Cryptosporidium CO Training Course Technical Support Center 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 Federal Register Vol. 78, No. 172, September 5, A World of Solutions TM 8

9 Substantial Conformity Checklists A, B, & C Positive Staining Control and OPR slide review by experienced microscopist Blind spikes proved by 3 rd party vendor Microscopy Skills Evaluation by 3 rd party expert Federal Register Vol. 74, No. 36, February 25, A World of Solutions TM 9

10 Cryptosporidium Certification Officer Training Courses offered in Cincinnati Courses offered online (twice) Next offering September 2017 A World of Solutions TM 10

11 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 A World of Solutions TM 11

12 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 A World of Solutions TM 12

13 Website updates for continued training 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 13

14 Audit Support: Analyst Skills Evaluation Online Analyst Skills Evaluation Every Analyst in the country has completed the online assessment A World of Solutions TM 14

15 Audit Support: Slide Review Evaluation of Microscope Slide Preparation Subject mater expert evaluates the OPR and positive staining control using a defined rubric, and scores 1 to 4. FITC 1000X DAPI 1000X DIC 1000X A World of Solutions TM 15

16 Common concerns with slides: Bubbles Bubbles can be seen with the naked eye DAPI and DIC do not work correctly in a bubble Bubble 1000X DIC 16

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 20X FITC Debris shown below Cryptosporidium FITC 1000X DAPI 1000X DIC 1000X 17

18 Common concerns with slides: Debris Reagent Water OPRs should have little to no debris FITC 200X DAPI 200X Bright field 200X 18

19 Organisms Outside of the Well FITC 1000X DAPI 1000X DIC 1000X A World of Solutions TM 19

20 Organisms Outside of the Well FITC 1000X DAPI 1000X DIC 1000X A World of Solutions TM 20

21 Laboratory Support 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 A World of Solutions TM 21

22 Analyst Training Workshops and Forums Skill sharing forums with experienced analysts Hands-on workshops for newer analysts Microscopy technique IMS tips and tricks Productivity Recovery Workshops offered A World of Solutions TM 22

23 Percentage of the Total Cryptosporidium and Giardia Recoveries can be matrix dependent Matrix Spike Recovery Range 23

24 Matrix Effects Can Impact IMS Bead Steps N.J. Shaw et al. / Journal of Microbiological Methods 75 (2008)

25 Cryptosporidium Percent Recovery Lot to lot variation has been reported Suspect Lot (12) New Lot (17) Suspect Lot (11) New Lot (13) IMS Control Full Method 25

26 Three Beads Evaluated Dynabeads Alternate B Alternate C *Scale bar is 5 microns for each image 26

27 Percent recovery Reagent Water IMS Control Recoveries Alternative IMS Beads n 4 Cryptosporidium Giardia * * Giardia IMS beads are not available with this product 27

28 Change from IMS Controls to Full Method

29 Percent recovery Method Recovery in Reagent Water Cryptosporidium Giardia n

30 Percent recovery Method Recovery in Ohio River Water Cryptosporidium Giardia n =

31 Result of Laboratory Support Study 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 Giardia bead. Overall, tests of the alternate IMS beads did not demonstrate that they were comparable to results obtained using Dynabeads 31

32 Continuing Certification Officer and Laboratory Support 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 32

33 Thank You!! For more information contact: