Improvement to Method 1623 to Detect Cryptosporidium and Giardia

Transcription

1 Improvement to Method 1623 to Detect Cryptosporidium and Giardia Leah Fohl Villegas Ph.D. CB&I Federal Services A World of SolutionsTM

2 More than 400,000 people sick from 1993 Milwaukee Cryptosporidium outbreak A World of Solutions TM 1

3 Cryptosporidium Detection Method Overview Sample Filter Centrifuge Tubes Immunomagnetic Separation 10 L 100 ml Slide A World of Solutions TM 2

4 Enhancements to Method 1623: Can we improve data quality and consistency for laboratories and water sources? A World of Solutions TM 3

5 Start with collecting the oocysts Filtration Elution Centrifugation IMS Sodium hexametaphosphate dispersant is used to facilitate recoveries for finished water and waste water Detection A World of Solutions TM 4

6 Cryptosporidium recovery in clay matrix with NaHMP treatments X X X Sample Filter Centrifuge Tubes IMS NaHMP addition Cryptosporidium % Recovery Untreated % to sample % pre-elute % pre-elute 48.2 A World of Solutions TM 5

7 Percent Recovery Cryptosporidium recovery in artificial matrices using 5.0% NaHMP pre-elution treatment Untreated Treated Reagent Water DE Clay TNRS A World of Solutions TM 6

8 What about the secondary concentration steps? Filtration Elution Centrifugation Secondary concentration requires reducing the 250 ml eluate containing debris and oocysts to microliter volumes IMS Detection A World of Solutions TM 7

9 IMS rinse No Wash Wash Performed on over 1000 samples with no adverse impact on recovery Reduced scope time an average of 10 min/slide (oo)cysts 200X Total magnification A World of Solutions TM 8

10 Testing the Concept: Side-by-side analysis Filtration Elution with & without NaHMP Centrifugation IMS Rinse all samples Detection A World of Solutions TM 9

11 Side-by-side studies on raw surface water samples Sites had reported low recoveries Reservoir River Ground water under the influence A World of Solutions TM 10

12 Percent Recovery Improved Recovery in Challenging Surface Waters Method 1623 Method % Improvement ntu Reservoir Reservoir Reservoir River River Reservoir River GWUI Reservoir A World of Solutions TM 11

13 Multiple labs 3 LT2 labs & EPA lab Utility collection 4 reagent water samples EPA spike, filter Same 3 raw waters Same reagents Side by side samples Lab 1 Method Lab 2 Method Lab 3 Method 3 w/ NaHMP 3 w/o NaHMP 3 w/ NaHMP 3 w/o NaHMP 3 w/ NaHMP 3 w/o NaHMP

14 Percent Recovery Multi-Laboratory Improvement with Challenging Matrix 100 Method 1623 Method ntu GWUI Reservoir River A World of Solutions TM 13

15 Summary of side-by-side studies A pre-elution step using 5% NaHMP was practical and effective In those waters where recoveries were low, Cryptosporidium recoveries improved with dispersant addition In those waters where recoveries were high, the dispersant did not interfere NaHMP treatment did not affect reagent water or Giardia recoveries A World of Solutions TM 14

16 Building Method : The validation process A World of Solutions TM 15

17 Small changes that improve Filtration Elution Centrifugation IMS oocyst recovery Pre-elution treatment with NaHMP IMS rinse to reduce analyst fatigue More rigorous QA/QC Detection A World of Solutions TM 16

18 Method Validation: study plan 14 laboratories 14 public water utilities 4 reagent water & 4 source water for each lab Variation in source water samples Turbidity: 1 to 53 NTU Conductivity: 33 to 885 µs ph: 6.5 to 8.5 River Reservoir/Lake Ground

19 Reagent Water Results 20 percentage point improvement in Cryptosporidium recovery Statistic Method 1623 Method Mean % Recovery Standard deviation Mean RSD n A World of Solutions TM 18

20 Source Water Results 23 percentage point improvement in Cryptosporidium recovery Statistic Method 1623 Method Mean % Recovery Standard deviation Mean RSD n A World of Solutions TM 19

21 Method Cryptosporidium Acceptance Criteria Method 1623 Method Reagent Water * Raw Source Water (61) (46) *Same criteria as an update based on 5 reagent water PT rounds A World of Solutions TM 20

22 Advantages of Method A World of Solutions TM 21

23 Method 1623 vs. Method A World of Solutions TM 22

24 Improved Accuracy in River Matrix Method 1623 Method /14/2009 9/21/2009 4/25/2011 9/14/2009 9/21/2009 4/25/2011 Independent Samples A World of Solutions TM 23

25 Method improves Cryptosporidium recoveries in challenging matrices and Improves precision Strengthens quality control criteria Improvements cost less than 25 Processing adds 10 minutes Saves time at the microscope, allowing for a net time savings A World of Solutions TM 24

26 Which Method is Right for My Lab? A World of Solutions TM 25

27 Who Benefits by Switching to ? Labs testing any water source giving low recoveries in matrix spikes Labs testing multiple water sources A World of Solutions TM 26

28 Initial Demonstration of Capability 4 spiked reagent water samples with 38% recovery 3 matrix samples (2 spiked & 1 unspiked) One method blank Don t forget PT samples for A World of Solutions TM 27

29 Summary Method significantly improves recoveries for challenging water sources Only 8 samples are needed to demonstrate capability Everyone is encouraged to adopt the 33% acceptance criteria Everyone is encouraged to use the IMS rinse A World of Solutions TM 28

30 Thank You!! For more information contact: Contact Information, Method and video are found on LT2 Lab Quality Assurance Program page: t2/lab_home.cfm