Cryptosporidium and Giardia (Round 33) Proficiency Testing ...capeyork.myownserver.net/~ptaasnau/documents/865.pdf · Measurement Uncertainty ... TABLE B: Cryptosporidium and Giardia

ACKNOWLEDGMENTS

PTA wishes to provided for this program by included providing input into the design of the program and technical commentary for the report. Ms BSciences, University of New South Walesand preparation, distribution and ho

Cryptosporidium

Proficiency Testing Program

ACKNOWLEDGMENTS

PTA wishes to provided for this program by included providing input into the design of the program and technical commentary for the report. Ms B FerraSciences, University of New South Walesand to Ms C Devoy and preparation, distribution and ho

© COPYRIGHT PR

REPORT NO.

Cryptosporidium


ACKNOWLEDGMENTS

PTA wishes to gratefully provided for this program by included providing input into the design of the program and technical commentary for the report.

Ferrari of the School of Biotechnology and Biomolecular Sciences, University of New South Wales

Ms C Devoy and preparation, distribution and ho

© COPYRIGHT PR

PO Box 7507, SILVERWATER NSW 2128

REPORT NO.

Cryptosporidium

(Round


July

ACKNOWLEDGMENTS

gratefully acknowledge the technical assistance provided for this program by included providing input into the design of the program and technical commentary for the report.

the School of Biotechnology and Biomolecular Sciences, University of New South Wales

Ms C Devoy and the staff at BTF Pty Ltdpreparation, distribution and ho

© COPYRIGHT PROFICIENCY


REPORT NO.

Cryptosporidium

(Round


July 201

acknowledge the technical assistance provided for this program by Professorincluded providing input into the design of the program and technical commentary for the report. Further appreciation is extended to

the School of Biotechnology and Biomolecular Sciences, University of New South Wales

the staff at BTF Pty Ltdpreparation, distribution and homogeneity testing

OFICIENCY TESTING AUSTRALIA 20


REPORT NO. 865

Cryptosporidium and

(Round 33)


2014

acknowledge the technical assistance essor J Smith

included providing input into the design of the program and technical Further appreciation is extended to

the School of Biotechnology and Biomolecular Sciences, University of New South Wales, for the supply of QC

the staff at BTF Pty Ltdmogeneity testing.

TESTING AUSTRALIA 20


865

Giardia


acknowledge the technical assistance J Smith. This assistance


the School of Biotechnology and Biomolecular for the supply of QC

the staff at BTF Pty Ltd for

TESTING AUSTRALIA 20

, Australia

Giardia


acknowledge the technical assistance . This assistance


the School of Biotechnology and Biomolecular for the supply of QC mud

for sample

TESTING AUSTRALIA 20 14

acknowledge the technical assistance . This assistance


the School of Biotechnology and Biomolecular ud

sample

CONTENTS PAGE(S)

1. FOREWORD 1

2. FEATURES OF THE PROGRAM 1

3. DESIGN OF THE PROGRAM 2 TABLE A: Round 33 Sample Design 2 Sample preparation 2

Confounding materials 3 Quality assurance of QC mud 3

4. FORMAT OF APPENDICES 3

5. FALSE RESULTS 4

6. LOW/HIGH RECOVERIES 4

7. PTA AND TECHNICAL ADVISER'S COMMENTS 4 Percentage Recovery Rate 4 Figure 1A: Comparison of total average recovery rates for Cryptosporidium 4 Figure 1B: Comparison of total average recovery rates for Giardia 5 Measurement Uncertainty (MU) Estimation 6 TABLE B: Cryptosporidium and Giardia Round 33 (Oo)cyst Recovery - % Measurement Uncertainty 6 TABLE C: Cryptosporidium and Giardia Round 33 Recovery - % Measurement Uncertainty 6 TABLE D: Comparison of Cryptosporidium Oocyst Levels for Each Round 7 TABLE E: Comparison of Giardia Cyst Levels for Each Round 7 Method Commentary 8 Overall Laboratory Performance 8 Measurement Uncertainty (MU) 9 TABLE F: Overall Laboratory Performance 10 Conclusions 13

8. REFERENCE 14

APPENDIX A Summary of Results A1.1 Summary of Percentage Recovery Rates and Charts A1.5

APPENDIX B Homogeneity Testing B1.1 TABLE G: Relative Standard Deviation for Various Sample Doses (Round 33) B1.1 Trip Control B1.2

APPENDIX C Instructions to Participants C1.1 Results Sheet C1.3

GLOSSARY

1 1. FOREWORD This report summarises the results of the thirty-third round of a planned series

of proficiency testing rounds involving the analysis of water samples for the detection and enumeration of Cryptosporidium and Giardia.

The exercise was conducted in April 2014 by PTA. The Technical Adviser was

Professor J Smith. The Program Coordinator was Ms K Cividin. This report was authorised by Mrs F Watton, PTA Quality - Business Development Manager.

The program aim was to assess laboratories’ ability to competently detect and report levels of Cryptosporidium and Giardia (oo)cysts in water.

2. FEATURES OF THE PROGRAM (a) A total of seven laboratories (four Australia, three New Zealand) received

samples, of which all returned results for inclusion in the report. (b) Participating laboratories were requested to report both total and confirmed

count results. Participants were also requested to calculate and report an estimate of measurement uncertainty (MU) for each reported result.

(c) Results as reported by participants are presented in Appendix A. (d) In addition to the samples, laboratories were provided with the Instructions to

Participants and a Results Sheet (see Appendix C). Laboratories were instructed to perform the tests according to their routine methods (method most frequently employed). Laboratories were reminded that PTA is aware of the internal positive control ColorSeedTM, developed by BTF Pty Ltd. Although PTA can see the advantage of ColorSeedTM as an internal positive control, participants were instructed to note that it is not acceptable for laboratories to adjust results obtained with the PTA proficiency testing samples on the basis of recoveries obtained using ColorSeedTM. An exception to this would be if the respective laboratory routine practice/standard operating procedure routinely uses ColorSeedTM as a true internal standard, i.e. addition to every sample, and correction of observed count using internal standard recovery during routine sample reporting.

(e) The samples for Round 33 were produced in line with EasySeed batch number

506, which are certified reference samples. The preparation of these certified reference samples is considered to have satisfied the homogeneity testing requirements (see Appendix B).

(f) Each laboratory was randomly allocated a unique code number for the round

to ensure confidentiality of results. Reference to each laboratory in this report is by code number.

2 3. DESIGN OF THE PROGRAM

Participants were requested to provide quantitative results for the presence of Cryptosporidium and Giardia in five water concentrate samples. Sample design is presented below.

TABLE A: Round 33 Sample Design

Sample Cryptosporidium (Count)

Giardia (Count)

Amount of QC mud added

A 110 50 100 µL

B 50 120 100 µL

C 0 80 500 µL

D 170 130 50 µL

E 70 0 500 µL

F (Trip control) 70 80 500 µL

Notes for Table A: 1. QC mud was added to these samples to simulate an environmental sample. 2. One nominated laboratory (Code 3) was provided with a sixth sample, as a trip control.

All samples were added to Milli-QTM water to make a final volume of approximately 3.5 mL.

Sample preparation

BTF Pty Ltd, NSW, prepared different water concentrate samples for this program, using PTA in-house method PTPM 11.1 Sample Preparation – Cryptosporidium and Giardia (Version No. 4). Seed samples were prepared on 27 March 2014. Seed samples were dispensed in IsoFlowTM and the sterilisation method was gamma irradiation. Cryptosporidium parvum (Iowa strain) oocysts were of bovine origin, excreted on 6 March 2014. Oocysts were purified by discontinuous sucrose and caesium chloride gradient centrifugation.

Giardia lamblia (H3 strain) cysts were obtained from experimentally-infected gerbils and were excreted on 13 March 2014. Cysts were purified by sucrose and PercollTM density gradient centrifugation, followed by water washes.

3 The seed samples were prepared using flow cytometry and an automated dispensing method. Cryptosporidium and Giardia (suspended in IsoFlowTM solution) were dispensed into 4 mL tubes. Seed samples were then sealed, labelled and exposed to a controlled dose of gamma irradiation. The Cryptosporidium oocysts were also heat treated to prevent excystation. Quality Control was performed on the seed samples. On 14 April 2014 each of the seed samples were spiked with QC mud (see ‘Confounding materials’ below) and then made up to approximately 3.5 mL with Milli-QTM water to produce the water concentrate samples sent to participants on 14 April 2014. Participating laboratories were asked to add each of the water concentrate samples to 10 L of water of their choice prior to analysis. The laboratories were also instructed to take care to ensure that the water used did not contain any cysts or oocysts and could, for example, use reverse osmosis or membrane filtered (suggested pore size ≤ 45 µm) water.

Confounding materials QC mud: QC mud was added to all water concentrate samples at a concentration of 50, 100 or 500 µL QC mud per water concentrate sample (see Table A).

Quality assurance of QC mud

To ensure the QC mud did not contain Cryptosporidium oocysts or Giardia cysts, QC mud samples were analysed prior to addition to proficiency samples. Briefly, samples of QC mud were stained using FITC-labelled monoclonal antibodies (EasyStainTM – BTF Pty Ltd), screened and purified using flow cytometry then examined using epifluorescence microscopy. No Cryptosporidium oocysts or Giardia cysts were found in 10 mL of QC mud.

4. FORMAT OF APPENDICES

Appendix A (A1.1 - A1.8) contains the total count and confirmed count results reported by participating laboratories for each of the five water concentrate samples. Percentage recovery rates and charts are also presented. Please note that recovery rates are calculated using total counts only.

Appendix B contains details of homogeneity testing, quality control and trip control results (B1.1 - B1.2). Appendix C contains the Instructions to Participants and the Results Sheet (C1.1 – C1.3).

5. FALSE RESULTS

Rtesting methods pooGiardia

6. LOW/HIGH RECOVERIES

The acceptable range set for this program is a recovery between 10This has been determined to be an appropriate acceptability range by technical experts in this area of testing. low/high recoveries (recovery rates tha110%) with all testing methods pooled.

No low/high recoveries were seen regarding the analysis of or

7. PTA AND TECHNICAL ADVIS

A total of Percentage Recovery Rate

O Tto the previous round.Cryptosporidium

FALSE RESULTS

Results were examined for falsetesting methods pooGiardia.

LOW/HIGH RECOVERIES


No low/high recoveries were seen regarding the analysis of or Giardia PTA AND TECHNICAL ADVIS

A total of 6 Percentage Recovery Rate

Overall recovery Total average to the previous round.Cryptosporidium

FALSE RESULTS

esults were examined for falsetesting methods poo

LOW/HIGH RECOVERIES


No low/high recoveries were seen regarding the analysis of results.

PTA AND TECHNICAL ADVIS

68 Total Count results

Percentage Recovery Rate

verall recovery rates for

otal average Cryptosporidiumto the previous round.Cryptosporidium for each

esults were examined for falsetesting methods pooled. There were no false results for

LOW/HIGH RECOVERIES


No low/high recoveries were seen regarding the analysis of

PTA AND TECHNICAL ADVIS

Total Count results

Percentage Recovery Rate

rates for Cryptosporidium

Cryptosporidiumto the previous round. Figure 1A

for each round (

4

esults were examined for false-positive and falseled. There were no false results for

The acceptable range set for this program is a recovery between 10This has been determined to be an appropriate acceptability range by technical experts in this area of testing. low/high recoveries (recovery rates that lie outside the acceptable range of 10110%) with all testing methods pooled.


PTA AND TECHNICAL ADVIS ER'S COMMENTS

Total Count results were received for this program.

Cryptosporidium

Cryptosporidium recovery rateFigure 1A shows the average percent recovery rate for

round (refer to

positive and falseled. There were no false results for

The acceptable range set for this program is a recovery between 10This has been determined to be an appropriate acceptability range by technical experts in this area of testing. The results were examined for

t lie outside the acceptable range of 10


R'S COMMENTS

were received for this program.

Cryptosporidium were lower than for

recovery rate (50.4%)shows the average percent recovery rate for efer to notes on page

positive and false-negative results with all led. There were no false results for Cryptosporidium

The acceptable range set for this program is a recovery between 10This has been determined to be an appropriate acceptability range by

The results were examined for t lie outside the acceptable range of 10



were lower than for

%) has increasedshows the average percent recovery rate for

otes on page 5).

negative results with all Cryptosporidium

The acceptable range set for this program is a recovery between 10This has been determined to be an appropriate acceptability range by

The results were examined for t lie outside the acceptable range of 10

No low/high recoveries were seen regarding the analysis of Cryptosporidium


were lower than for Giardia.

creased compared shows the average percent recovery rate for

negative results with all Cryptosporidium or

The acceptable range set for this program is a recovery between 10-110%. This has been determined to be an appropriate acceptability range by

The results were examined for t lie outside the acceptable range of 10-

Cryptosporidium

.

compared shows the average percent recovery rate for

Total average compared to the previous round. The graph below displays this (below

Notes to Average Recovery Rates Graphs

1.2.

3.

otal average compared to the previous round. The graph below displays this (below figure

Notes to Average Recovery Rates Graphs1. The vertical bars in the graphs represent 95% confidence intervals.2. All rounds, except rounds 1, 2, 3 and 8, contain QC mud (see table on pages

Round 5, one sone sample (Sample D) out of the five samples analysed by eachmud.

3. From Rounds 14excluding Rounds 14average recovery rates presented in this table prior to Round 14confirmed counts. From

otal average Giardiacompared to the previous round. The graph below displays this (

figure).

Notes to Average Recovery Rates GraphsThe vertical bars in the graphs represent 95% confidence intervals.All rounds, except rounds 1, 2, 3 and 8, contain QC mud (see table on pages Round 5, one sample (Sample type 4)one sample (Sample D) out of the five samples analysed by each

From Rounds 14-21, average recovery rates are calculated on confirmed counts only. For rounds excluding Rounds 14-21, participants reported either total or confirmed counts, and therefore the average recovery rates presented in this table prior to Round 14confirmed counts. From

Giardia recovery ratecompared to the previous round. The graph below displays this (

Notes to Average Recovery Rates GraphsThe vertical bars in the graphs represent 95% confidence intervals.All rounds, except rounds 1, 2, 3 and 8, contain QC mud (see table on pages

ample (Sample type 4)one sample (Sample D) out of the five samples analysed by each

21, average recovery rates are calculated on confirmed counts only. For rounds 21, participants reported either total or confirmed counts, and therefore the

average recovery rates presented in this table prior to Round 14confirmed counts. From Round 22 onwards

5

recovery rate (59.1compared to the previous round. The graph below displays this (

Notes to Average Recovery Rates Graphs: The vertical bars in the graphs represent 95% confidence intervals.All rounds, except rounds 1, 2, 3 and 8, contain QC mud (see table on pages

ample (Sample type 4); for Round 14, one sample (Sample C)one sample (Sample D) out of the five samples analysed by each


average recovery rates presented in this table prior to Round 14Round 22 onwards, only total counts are presented.

59.1%) has incompared to the previous round. The graph below displays this (

The vertical bars in the graphs represent 95% confidence intervals.All rounds, except rounds 1, 2, 3 and 8, contain QC mud (see table on pages

for Round 14, one sample (Sample C)one sample (Sample D) out of the five samples analysed by each


average recovery rates presented in this table prior to Round 14, only total counts are presented.

creased in this roundcompared to the previous round. The graph below displays this (

The vertical bars in the graphs represent 95% confidence intervals. All rounds, except rounds 1, 2, 3 and 8, contain QC mud (see table on pages

for Round 14, one sample (Sample C)one sample (Sample D) out of the five samples analysed by each laboratory did not contain QC


average recovery rates presented in this table prior to Round 14 may include both total and , only total counts are presented.

in this roundcompared to the previous round. The graph below displays this (refer to notes

All rounds, except rounds 1, 2, 3 and 8, contain QC mud (see table on pages 11 through to 14for Round 14, one sample (Sample C); and for Round 15,

laboratory did not contain QC


may include both total and

in this round when efer to notes

to 14). For and for Round 15,

laboratory did not contain QC


may include both total and

6 Measurement Uncertainty (MU) Estimation Results including MU calculations are presented in two formats, including absolute oocyst numbers (so that participants can compare numeric recoveries against seed doses) and relative % recoveries, as seen in Tables B and C below. These tables and comments are provided for information purposes only, and do not affect the evaluation of participants’ results.

TABLE B: Cryptosporidium and Giardia Round 33 (Oo)cyst Recovery - % Measurement

Uncertainty

SAMPLE ORGANISM (OO)CYST MEDIAN

RECOVERY

STANDARD DEVIATION

RELATIVE STANDARD DEVIATION (RSD - %)

��MEASUREMENT UNCERTAINTY (± [OO]CYSTS)

REFERENCE COUNT

A Giardia Cryptosporidium

29 10 34 68 50

60 16 27 54 110

B Giardia Cryptosporidium

73 25 34 68 120

22 7 32 64 50

C Giardia

46 16 35 70 80

D Giardia Cryptosporidium

80 29 36 72 130 110 35 32 64 170

E Cryptosporidium

26 16 62 124 70

Notes for Table B: 1. � = All measurement uncertainty values are at the 95% level of confidence. 2. Sample C did not include Cryptosporidium. 3. Sample E did not contain Giardia.

TABLE C: Cryptosporidium and Giardia Round 33 Recovery - % Measurement Uncertainty

SAMPLE ORGANISM (OO)CYST MEDIAN

RECOVERY

STANDARD DEVIATION

RELATIVE STANDARD DEVIATION (RSD - %)

��MEASUREMENT UNCERTAINTY

(RSD - %)

REFERENCE COUNT

A Giardia Cryptosporidium

58 20 35 40 50

55 15 27 30 110

B Giardia Cryptosporidium

61 21 34 42 120

44 14 32 28 50

C Giardia

58 20 35 40 80

D Giardia Cryptosporidium

62 22 36 44 130

65 21 32 42 170

E Cryptosporidium

37 23 62 46 70

Notes for Table C: 1. � = All measurement uncertainty values are at the 95% level of confidence. 2. Sample C did not include Cryptosporidium. 3. Sample E did not contain Giardia.

7 The table below shows Cryptosporidium oocyst levels for each round.

TABLE D: Comparison of Cryptosporidium Oocyst Levels for Each Round

Round Cryptosporidium levels (Counts) Round Cryptosporidium levels (Counts) 1 50-200 18 50-300 2 50-200 19 50-300 3 50-300 20 50-200 4 110 21 100-200 5 50-200 22 50-300 6 25-75 23 50-250 7 50-100 24 50-200 8 65-140 25 50-250 9 125 26 50-200

10 110-235 27 50-200 11 50-200 28 50-250 12 110-235 29 50-140 13 90-205 30 80-135 14 55-135 31 70-140 15 55-135 32 50-130 16 55-120 33 50-170 17 90-310

The table below shows Giardia cyst levels for each round.

TABLE E: Comparison of Giardia Cyst Levels for Each Round

Round Giardia levels (Counts) Round Giardia levels (Counts) 1 50-200 18 150-300 2 50-200 19 150-300 3 50 20 50-120 4 40 21 90-200 5 50-200 22 50-250 6 75-120 23 50-300 7 50 24 50-200 8 65-140 25 50-250 9 55 26 50-250

10 70-85 27 50-200 11 50-200 28 50-200 12 110-125 29 50-150 13 90-145 30 85-150 14 55-200 31 50-140 15 55-200 32 50-170 16 120-255 33 50-130 17 135-310

8 Method Commentary

The generally lower Cryptosporidium recoveries obtained by laboratory code 6 may have resulted from one of several sources, however, this laboratory uses the most common procedures between participating laboratories (cartridge filtration, IMS, IFM + DAPI staining). Therefore, investigation should be conducted regarding potential causal factors of respectively low recoveries in comparison with other laboratories utilising essentially the same procedures. Failures to add or dissociate Cryptosporidium IMS beads, material transfer steps, or the status of associated reagents are possibilities. Laboratory code 4 generally obtained substantially lower confirmation of both Cryptosporidium oocysts and Giardia cysts than other participants. Laboratory codes 6 and 7 obtained lower confirmation of Giardia cysts than other participants for most samples. All participants used DAPI staining for confirmation, including laboratory codes 4, 6 and 7. Some laboratories experience issues with low percentages of DAPI stained cysts and oocysts. Several methods indicate use of heat or acid for both dissociation of (oo)cysts from IMS beads, as well as permeabilisation for subsequent DAPI staining. Laboratory codes 1 and 5 may find the following publication of interest: Ware, MW, Wymer, L Lindquist, and Schaefer, FW. (2003) Evaluation of an alternative IMS dissociation procedure for use with method 1622: detection of Cryptosporidium in water. J.Microbiol. Meth. 55:575-583. Overall Laboratory Performance

Overall, median recoveries of Giardia cysts and Cryptosporidium oocysts were higher than the previous round (32) with the percentage increase greater for Cryptosporidium than Giardia (see figures 1A and 1B). Overall performance was satisfactory for all sample doses and matrix amounts. Pooled laboratory data indicated a wider range of median recoveries for each measurand compared to round 32 (23-41% Cryptosporidium and 41-63% Giardia) at (17-86% Cryptosporidium and 30-91% Giardia) Lowest-range recoveries were generally lower than those obtained in round 32. These overall results are typical of recoveries obtained using the methods employed. Laboratory codes 6 and 7 did not report results for Cryptosporidium, sample C, and Giardia, sample E. These are zero amount samples, however, all fields, besides MU, must be completed.

9

Laboratory codes 3 and 4 demonstrated generally lower mean recoveries of Giardia, while laboratory code 6 demonstrated generally lower mean recoveries of Cryptosporidium compared to other laboratories. It is recommended that these laboratories review these results and take potential corrective actions to potentially increase recoveries of these measurands as needed. For equivalent matrix amounts (samples A and B, 0.1 mL) but different Cryptosporidium doses (110 and 50 respectively) median recoveries were similar at 51% and 46% respectively. For equivalent matrix amounts (samples A and B, 0.1 mL) but different Giardia doses (50 and 120 respectively) median recoveries were similar at 60% and 59% respectively. For similar Cryptosporidium oocyst doses (samples B and E, 50 and 70, respectively) but different matrix amounts (0.1 mL and 0.5 mL, respectively) median recoveries were 44% and 37%, respectively, with lower recovery with greater matrix amount. For similar Giardia cyst doses (samples B and D, 120 and 130, respectively) but different matrix amounts (0.1 mL and 0.05 mL, respectively) median recoveries were similar at 61% and 62% respectively. The highest variability in median recoveries were 36% RSD for Giardia in sample D and 62% RSD for Cryptosporidium in sample E (assessed using % RSD MU). This reflects a decrease in maximum variability from round 32 for Giardia (67%) and no substantial change for Cryptosporidium (61%).

Table F that appears on pages 10-13 illustrates the comparison of overall laboratory performance for rounds 1 - 33. There were no low recoveries for Giardia or Cryptosporidium.

Measurement Uncertainty (MU) All seven participating laboratories provided MU information with their results.

Laboratory code 2 has reported uncertainty as a number but not indicated whether it is a range, plus/minus or relative standard deviation. Uncertainties between recoveries between laboratories were not atypical for this measurand.

10

TABLE F: Overall Laboratory Performance

Rou

nd

Sam

ple

Typ

e

Per

cent

age

fals

e po

sitiv

e an

d fa

lse

nega

tive

resu

lts

repo

rted

Num

ber

of

labo

rato

ries

repo

rtin

g fa

lse

resu

lts

Per

cent

age

low

/hig

h re

cove

ry r

esul

ts

repo

rted

Num

ber

of

labo

rato

ries

repo

rtin

g lo

w/h

igh

perc

enta

ge

reco

very

rat

es

1 10 Litres - tap water

11.0% 6 11.0% 7

2 10 Litres - tap water

6.7% 1 7.8% 3

3 10 Litres -Milli-Q water 3.8% 3 4.7% 3

4 10 Litres -

RO water + QC mud + confounding organisms

10.3% 3 11.8% 4

5 10 Litres - RO water + QC mud*

7.0% 4 11.0% 5

6 10 Litres - RO water + QC mud 8.3% 4 8.3% 5

7

Concentrate samples - QC mud -

Labs. add to 10 Litres distilled water

8.2% 4 6.4% 5

8 10 Litres - RO water 1.2% 1 1.2% 1

9 10 Litres -

RO water + QC mud 2.7% 1 7.3% 4

10


Labs. add to 10 Litres distilled water & 10 Litres -

RO water + QC mud

2.3% 1 3.5% 2

11

Rou

nd

Sam

ple

Typ

e

Per

cent

age

fals

e po

sitiv

e an

d fa

lse

nega

tive

resu

lts

repo

rted

Num

ber

of

labo

rato

ries

repo

rtin

g fa

lse

resu

lts

Per

cent

age

low

/hig

h re

cove

ry

resu

lts r

epor

ted

Num

ber

of

labo

rato

ries

repo

rtin

g lo

w/h

igh

perc

enta

ge

reco

very

rat

es

11 10 Litres - RO water + QC mud

0.0% 0 6.8% 4


5.5% 2 17.5% 6


0.0% 0 10.0% 4

14 10 Litres - RO water + QC mud*

2.6% 1 2.6% 1

15

Concentrate samples - QC mud* -


1.3% 1 5.0% 2

16



0.0% 0 3.3% 2

17



1.5% 1 2.9% 1

18


Labs. add to 10 Litres water

0.0% 0 0.0% 0

19



6.0% 1 11.4% 1

20



10.0% 4 7.1% 3

12

Rou

nd

Sam

ple

Typ

e

Per

cent

age

fals

e po

sitiv

e an

d fa

lse

nega

tive

resu

lts

repo

rted

Num

ber

of

labo

rato

ries

repo

rtin

g fa

lse

resu

lts

Per

cent

age

low

/hig

h re

cove

ry

resu

lts r

epor

ted

Num

ber

of

labo

rato

ries

repo

rtin

g lo

w/h

igh

perc

enta

ge

reco

very

rat

es

21



5.4% 1 10.7% 2

22



1.4% 1 1.4% 1

23



0.0% 0 1.7% 1

24



1.4% 1 0.0% 0

25



0.0% 0 0.0% 0

26



1.4% 1 4.3% 2

27



0.0% 0 0.0% 0

28



0.0% 0 3.3% 1

13

13R

ound

Sam

ple

Typ

e

Per

cent

age

fals

e po

sitiv

e an

d fa

lse

nega

tive

resu

lts

repo

rted

Num

ber

of

labo

rato

ries

repo

rtin

g fa

lse

resu

lts

Per

cent

age

low

/hig

h re

cove

ry

resu

lts r

epor

ted

Num

ber

of

labo

rato

ries

repo

rtin

g lo

w/h

igh

perc

enta

ge

reco

very

rat

es

29



10.0%

2

18.8%

3

30



2.5% 1 3.75% 3

31



0.0% 0 1.4% 1

32



0.0% 0 3.5% 1

33



0.0% 0 0.0% 0

Notes for Table F: 1. RO = reverse osmosis. 2. * = For Round 5, QC mud was only added to Sample types 1, 2, 3 and 5. For Round 14, QC mud was only

added to Samples A, B, D and E. For Round 15, QC mud was only added to Samples A, B, C and E.

Conclusions In this round the performance of all laboratories was satisfactory. Laboratory codes 4, 6 and 7 may wish to review their relative performance in terms of DAPI staining and take corrective action as necessary. Variable results were reported for Round 33, with a decreased number of low recoveries and false results reported when compared to previous rounds. Laboratories reporting low recoveries should undertake corrective actions to investigate sources of error and possible cyst/oocyst losses/failure to stain during sample processing; including filter elution, IMS and IFA staining (if on slides, particularly associated wash steps), as needed and applicable.

14

8. REFERENCE

[1] Guide to Proficiency Testing Australia, 2012 (this document can be found on the PTA website, www.pta.asn.au).

APPENDIX A

Summary of Results

A1.1

Results Cryptosporidium (total counts)

REFERENCE COUNTS

110 50 0 170 70

QC Mud per Vial

100 µL 100 µL 500 µL 50 µL 500 µL

Lab Code No. Sample A Sample B Sample C Sample D Sample E Method Codes

Code 1 - Total Count 80 28 0 147 57

3,7,8,9,10 Confirmed Count 78 26 0 141 54

MU ±24, n=15 ±8, n=15 - ±44, n=15 ±17, n=15


3,7,8,9

Confirmed Count 60 33 0 110 24

MU 10.4/25 10.4/25 10.4/25 10.4/25 10.4/25


5,7,8,9,10 Confirmed Count 60 17 0 68 26

MU 26.05% n=>500

26.05% n=>500

26.05% n=>500

26.05% n=>500

26.05% n=>500


3,7,8,9 Confirmed Count 35 12 0 94 18

MU 24-148/17 10-69/117 - 61-430/17 19-131/17


2,7,8,9 Confirmed Count 51 27 0 118 23

MU 51±19 oocysts at 95%CI (600)

27±10 oocysts at 95%CI (600 - 120±44 oocysts

at 95%CI (600 23±9 oocysts at 95%CI (600

Code 6 - Total Count 30 15 - 50 12

3,6,7,8,9,10 Confirmed Count 28 14 - 46 12

MU 25-36, n=100 13-18, n=100 - 42-60, n=100 10-14, n=100


1,6,7,8,9,10

Confirmed Count 57 15 - 62 39

MU ±44(6) ±41(7) - ±48(9) ±44(10)

Note: 1. A “-” indicates that no result was returned for this sample/test.

A1.2 Summary Statistics for Cryptosporidium (total counts)

Sample A Sample B Sample C Sample D Sample E

No. of Results s 7 7 7 7 7

Minimum 30 15 0 50 12

Maximum 80 33 0 147 57

Average 56 23 0 98 33

Median 60.0 22.0 0.0 110.0 26.0

SD 16.0 6.6 0.0 34.8 16.2

Method Codes

Analysis Method used to obtain results Code Concentration Filtration (Sponge) 1 Filtration (Flat Bed) 2 Filtration (Cartridge) 3 Filtration (Tangential Flow) 4 Flocculation 5 Centrifugation 6 Purification IMS 7 Enumeration Immunofluorescence Microscopy 8 Confirmation DAPI Staining 9 DIC Microscopy 10 Methods not defined - 11

A1.3

Results Giardia (total counts)

REFERENCE COUNTS

50 120 80 130 0

QC Mud per Vial

100 µL 100 µL 500 µL 50 µL 500 µL

Lab Code No. Sample A Sample B Sample C Sample D Sample E Method Codes

Code 1 - Total Count 44 109 61 104 0

3,7,8,9,10 Confirmed Count 35 97 55 93 0

MU ±13, n=15 ±33, n=15 ±18, n=15 ±31, n=15 -


3,7,8,9 Confirmed Count 35 88 59 80 0

MU 17.8/26 17.8-26 17.8/26 17.8/26 17.8/26


5,7,8,9,10

Confirmed Count 19 30 24 44 0

MU 25.88% N=>500

25.88% N=>500

25.88% N=>500

25.88% N=>500

25.88% N=>500


3,7,8,9 Confirmed Count 15 31 22 47 0

MU 11-50/17 33-144/17 16-72/17 33-146/17 -

Code 5 - Total Count

39 77 44 116 0

2,7,8,9 Confirmed Count 39 71 42 114 0

MU 39±16 cysts at 95%CI (600)

77±32 cysts at 95%CI (600)

44±18 oocysts at 95%CI (600)

116±48 oocysts at 95%CI (600)

Code 6 - Total Count 24 64 46 45 -

3,6,7,8,9,10 Confirmed Count 21 44 40 29 -

MU 21-28, n=103 55-74, n=103 40-53, n=103 39-52, n=103 -


Confirmed Count 24 61 48 70 - 1,6,7,8,9,10

MU ±35(6) ±45(7) ±45(8) ±50(9) -

Note: 1. A “-” indicates that no result was returned for this sample/test.

A1.4 Summary Statistics for Giardia (total counts)

Sample A Sample B Sample C Sample D Sample E

No. of Results s 7 7 7 7 7

Minimum 18 30 24 44 0

Maximum 44 109 61 116 0

Average 30 70 46 76 0

Median 29.0 73.0 46.0 80.0 0.0

SD 9.9 25.4 15.5 29.0 0.0

Method Codes

Analysis Method used to obtain results Code Concentration Filtration (Sponge) 1 Filtration (Flat Bed) 2 Filtration (Cartridge) 3 Filtration (Tangential Flow) 4 Flocculation 5 Centrifugation 6 Purification IMS 7 Enumeration Immunofluorescence Microscopy 8 Confirmation DAPI Staining 9 DIC Microscopy 10 Methods not defined - 11

Summary of Percentage Recovery

Rates and Charts

A1.5

Results Cryptosporidium (% Recovery Rate)

Calculated on TOTAL Counts only

Code No.

Sample A

Sample B

Sample D

Sample E

1 73% 56% 86% 81%

2 55% 66% 65% 34%

3 55% 34% 40% 37%

4 42% 38% 69% 51%

5 46% 54% 71% 33%

6 27% 30% 29% 17%

7 61% 44% 43% 73%

Minimum 27% 30% 29% 17% Maximum 73% 66% 86% 81% Average 51% 46% 58% 47% Median

53%

44%

65%

37%

Notes:

1. The acceptable percentage recovery rate range is 10-110%. 2. The median is provided for information only. It is the middle result. It is a measure of the centre

of the data and is similar to the mean (or average), however, is less subject to outlier results.

Note: 1. Cryptosporidium

Cryptosporidium reference count included in brackets alongside corresponding sample name.reference count included in brackets alongside corresponding sample name.reference count included in brackets alongside corresponding sample name.

A1.6

reference count included in brackets alongside corresponding sample name.reference count included in brackets alongside corresponding sample name.reference count included in brackets alongside corresponding sample name.reference count included in brackets alongside corresponding sample name.

A1.7

Results Giardia (% Recovery Rate)

Calculated on TOTAL Counts only

Code No.

Sample A

Sample B

Sample C

Sample D

1 80% 91% 76% 80%

2 70% 73% 74% 62%

3 40% 25% 30% 34%

4 36% 43% 33% 41%

5 78% 64% 55% 89%

6 48% 53% 58% 67%

7 58% 61% 74% 54%

Minimum 36% 25% 30% 34% Maximum 88% 91% 76% 89% Average 60% 59% 57% 61% Median

58%

61%

58%

62%

Notes:

1. The acceptable percentage recovery rate range is 10-110%. 2. The median is provided for information only. It is the middle result. It is a measure of the centre

of the data and is similar to the mean (or average), however, is less subject to outlier results.

Note: 1. Giardia

Giardia reference count included in brackets alongside corresponding sample name.reference count included in brackets alongside corresponding sample name.reference count included in brackets alongside corresponding sample name.

A1.8

reference count included in brackets alongside corresponding sample name.reference count included in brackets alongside corresponding sample name.reference count included in brackets alongside corresponding sample name.reference count included in brackets alongside corresponding sample name.

APPENDIX B

Homogeneity Testing and

Trip Control

B1.1 Homogeneity Testing The samples were produced in line with EasySeed batch number 506, which are certified reference samples. The preparation of these certified reference samples is considered to have satisfied the homogeneity testing requirements. An estimate of uncertainty, expressed as Relative Standard Deviation (RSD), for each organism for the Cryptosporidium and Giardia proficiency testing program was calculated for each dose within the sample set. These are presented in the table below: TABLE G: Relative Standard Deviation for Various Sa mple Doses (Round 33)

ORGANISM DOSE RSD (%) MU as RSD (absolute)

Resultant dose with absolute

uncertainty Cryptosporidium 50 1.2 1 50 ± 1

Cryptosporidium 70 1.2 1 70 ± 1



Giardia 50 1 1 50 ± 1

Giardia 80 1.4 2 80 ± 2

Giardia 120 1.8 2 120 ± 2

Giardia 130 1.8 2 130 ± 2 Notes for Table G: 1. Historical QC data and homogeneity testing data have been used to calculate the information in the above table. 2. All measurement uncertainty estimates are at the 95% level of confidence. 3. All numbers have been rounded to whole numbers. Although it may appear that the “MU as RSD is always

2 x RSD%” rule has been ignored, the rule itself ignores the impact of the continuous data used to calculate each value (the impact of rounding up/down).

B1.2 Trip Control Water concentrate sample F, spiked with 70 Cryptosporidium oocysts and 80 Giardia cysts was used as the trip control. BTF Pty Ltd retained a 3.5 mL water concentrate sample F (FNoT), on their premises after preparation. Sample FNoT was added to 10 L of distilled water, concentrated and analysed on 30 April 2014. One nominated laboratory (Code 3) was provided with a 3.5 mL water concentrate sample F (FT) and was requested to return the sample to BTF Pty Ltd immediately upon receipt. Sample FT was subsequently added to 10 L of distilled water and analysed by BTF Pty Ltd on 30 April 2014. Trip control samples were concentrated using membrane filtration, and then analysed using the Dynal IMS system and epifluorescence microscopy.

Results for Control Samples F NoT , FT

Date Analysed Crypto. Counts

No. DAPI

positive

Giardia Counts

No. DAPI

positive

30 April 2014 (Sample kept on premises) 46 98% 72 84%

30 April 2014 (Sample sent to laboratory and returned) 30 100% 73 98%

Actual counts 70 80

FNoT % Recovery Rate 66% 90%

FT % Recovery Rate 43% 91%

The trip control sent to the laboratory indicated sample stability during transport. Percentage recovery rates for trip control samples lie within the acceptable range of 10% - 110%.

APPENDIX C

Instructions to Participants

and

Results Sheet

To ensure that results from this program can be properly analysed, participants are asked to carefully adhere to the following instructions. 1. For this round each participant will b

water concentrate samples. Each sample contains reverse osmosis water that may contain matrix materials from reservoir water (added to simulate an environmental water sample). Samples may have beeYour laboratory utilised as the proficiency testing program trip control. If you receive this sample (labelSample F”), refer to the included associated covering letter for further instructions.

2. On receipt at your facility, samples should be refrigerated at 1

sample receipt 3. Mix the 3.5 mL tube by inversion then immediately place the bottom of the tube on a vortex and

mix such that the vortex extends to the bottom of the tube. Add each of the 3.5 mL bulk water concentrates to individual, respective 10 L water samples of your cmix-up the order of the sample vials in relation to their respective 10 L water samples. Ensure the water used does not contain any use reverse osmosis or membranebulk water concentrate sample vial is effectively rinsed and thoroughly dispersed into the 10 L bulk water

i)

ii)

iii)

iv)

*Laurethfilters may be used to rinse tubes if

4. A Senior QA/QC Officer (or similar) must sign the results sheet to declare your laboratory has diluted the concentrate samples to 10 L.

5. Laboratories must then proceed to analyse the 10 L samples using their

(method most frequentResults SheetParticipants are advised that analytical methods used will be noted in the final report.for confidential treatment of your results in the final report, your facility has been allocated a code number, which appears on your

To ensure that results from this program can be properly analysed, participants are asked to carefully adhere to the following instructions.

For this round each participant will bwater concentrate samples. Each sample contains reverse osmosis water that may contain matrix materials from reservoir water (added to simulate an environmental water sample). Samples may have beeYour laboratory mayutilised as the proficiency testing program trip control. If you receive this sample (labelSample F”), refer to the included associated covering letter for further instructions.

On receipt at your facility, samples should be refrigerated at 1sample receipt must

Mix the 3.5 mL tube by inversion then immediately place the bottom of the tube on a vortex and mix such that the vortex extends to the bottom of the tube. Add each of the 3.5 mL bulk water concentrates to individual, respective 10 L water samples of your c

up the order of the sample vials in relation to their respective 10 L water samples. Ensure the water used does not contain any use reverse osmosis or membranebulk water concentrate sample vial is effectively rinsed and thoroughly dispersed into the 10 L bulk water. The following rinse procedure is recommended to ensure optimal sample transfer:

Carefully add the conttube to respective 10 L water samples.

Add 3 mL 0.05% (v/v) Tween 20* to the empty sample tube, recap and vortex for 20 sec. Empty contents into the 10 L water sample.

Add 3 mL reagent grade wsec. Empty contents into 10 L water sample.

Repeat steps ii

*Laureth-12 Envirocheck® elution buffer or other Tweenfilters may be used to rinse tubes if

A Senior QA/QC Officer (or similar) must sign the results sheet to declare your laboratory has diluted the concentrate samples to 10 L.

Laboratories must then proceed to analyse the 10 L samples using their (method most frequentResults Sheet. One hundred percent (100%) of each sample supplied must be analysed. Participants are advised that analytical methods used will be noted in the final report.

confidential treatment of your results in the final report, your facility has been allocated a code number, which appears on your

Cryptosporidium

INSTRUCTIONS TO PARTICIPANTS


For this round each participant will bwater concentrate samples. Each sample contains reverse osmosis water that may contain matrix materials from reservoir water (added to simulate an environmental water sample). Samples may have been spiked with

may receive an additional 3.5 mL bulk water concentrate sample that will be utilised as the proficiency testing program trip control. If you receive this sample (labelSample F”), refer to the included associated covering letter for further instructions.

On receipt at your facility, samples should be refrigerated at 1must be recorded on the


up the order of the sample vials in relation to their respective 10 L water samples. Ensure the water used does not contain any use reverse osmosis or membranebulk water concentrate sample vial is effectively rinsed and thoroughly dispersed into the 10 L

. The following rinse procedure is recommended to ensure optimal sample transfer:Carefully add the conttube to respective 10 L water samples.Add 3 mL 0.05% (v/v) Tween 20* to the empty sample tube, recap and vortex for 20 sec. Empty contents into the 10 L water sample.Add 3 mL reagent grade wsec. Empty contents into 10 L water sample.Repeat steps ii-iii.

12 Envirocheck® elution buffer or other Tweenfilters may be used to rinse tubes if


Laboratories must then proceed to analyse the 10 L samples using their (method most frequently employed)

. One hundred percent (100%) of each sample supplied must be analysed. Participants are advised that analytical methods used will be noted in the final report.

confidential treatment of your results in the final report, your facility has been allocated a code number, which appears on your

Proficiency Testing Australia

Proficiency Testing

Cryptosporidium



For this round each participant will be supplied with a sample set consisting of five 3.5 mL bulk water concentrate samples. Each sample contains reverse osmosis water that may contain matrix materials from reservoir water (added to simulate an environmental water sample).

n spiked with Cryptosporidiumreceive an additional 3.5 mL bulk water concentrate sample that will be

utilised as the proficiency testing program trip control. If you receive this sample (labelSample F”), refer to the included associated covering letter for further instructions.

On receipt at your facility, samples should be refrigerated at 1be recorded on the


up the order of the sample vials in relation to their respective 10 L water samples. Ensure the water used does not contain any Giardiause reverse osmosis or membrane-filtered (sbulk water concentrate sample vial is effectively rinsed and thoroughly dispersed into the 10 L

. The following rinse procedure is recommended to ensure optimal sample transfer:Carefully add the contents of the proficiency testing sample (bulk water concentrate) tube to respective 10 L water samples.Add 3 mL 0.05% (v/v) Tween 20* to the empty sample tube, recap and vortex for 20 sec. Empty contents into the 10 L water sample.Add 3 mL reagent grade water to the empty sample vial, recap and vortex for 20 sec. Empty contents into 10 L water sample.

iii.

12 Envirocheck® elution buffer or other Tweenfilters may be used to rinse tubes if preferred.


Laboratories must then proceed to analyse the 10 L samples using their ly employed). Samples are to be tested in the respective order on the


confidential treatment of your results in the final report, your facility has been allocated a code number, which appears on your Results Sheet.

C1.1

roficiency Testing Australia

Proficiency Testing

Cryptosporidium and Giardia



e supplied with a sample set consisting of five 3.5 mL bulk water concentrate samples. Each sample contains reverse osmosis water that may contain matrix materials from reservoir water (added to simulate an environmental water sample).

Cryptosporidiumreceive an additional 3.5 mL bulk water concentrate sample that will be


On receipt at your facility, samples should be refrigerated at 1be recorded on the Results Sheet.


up the order of the sample vials in relation to their respective 10 L water samples. Ensure Giardia cysts or

filtered (suggested pore size bulk water concentrate sample vial is effectively rinsed and thoroughly dispersed into the 10 L

. The following rinse procedure is recommended to ensure optimal sample transfer:ents of the proficiency testing sample (bulk water concentrate)

tube to respective 10 L water samples. Add 3 mL 0.05% (v/v) Tween 20* to the empty sample tube, recap and vortex for 20 sec. Empty contents into the 10 L water sample.

ater to the empty sample vial, recap and vortex for 20 sec. Empty contents into 10 L water sample.

12 Envirocheck® elution buffer or other Tweenpreferred.


Laboratories must then proceed to analyse the 10 L samples using their . Samples are to be tested in the respective order on the


confidential treatment of your results in the final report, your facility has been allocated a Results Sheet.

roficiency Testing Australia


Giardia Round 33


To ensure that results from this program can be properly analysed, participants are asked to


Cryptosporidium and/or Giardia receive an additional 3.5 mL bulk water concentrate sample that will be


On receipt at your facility, samples should be refrigerated at 1-Results Sheet.


up the order of the sample vials in relation to their respective 10 L water samples. Ensure cysts or Cryptosporidiumuggested pore size

bulk water concentrate sample vial is effectively rinsed and thoroughly dispersed into the 10 L . The following rinse procedure is recommended to ensure optimal sample transfer:

ents of the proficiency testing sample (bulk water concentrate)

Add 3 mL 0.05% (v/v) Tween 20* to the empty sample tube, recap and vortex for 20 sec. Empty contents into the 10 L water sample.

ater to the empty sample vial, recap and vortex for 20 sec. Empty contents into 10 L water sample.

12 Envirocheck® elution buffer or other Tween-containing solutions for rinsing

A Senior QA/QC Officer (or similar) must sign the results sheet to declare your laboratory has

Laboratories must then proceed to analyse the 10 L samples using their . Samples are to be tested in the respective order on the


confidential treatment of your results in the final report, your facility has been allocated a Results Sheet.

Round 33




Giardia at various concentrations. receive an additional 3.5 mL bulk water concentrate sample that will be


-8ºC. The date and time of

Mix the 3.5 mL tube by inversion then immediately place the bottom of the tube on a vortex and mix such that the vortex extends to the bottom of the tube. Add each of the 3.5 mL bulk water concentrates to individual, respective 10 L water samples of your choice, taking care not to

up the order of the sample vials in relation to their respective 10 L water samples. Ensure Cryptosporidium oocysts. For example,

uggested pore size ≤ 45 µm) water. bulk water concentrate sample vial is effectively rinsed and thoroughly dispersed into the 10 L

. The following rinse procedure is recommended to ensure optimal sample transfer:ents of the proficiency testing sample (bulk water concentrate)

Add 3 mL 0.05% (v/v) Tween 20* to the empty sample tube, recap and vortex for 20

ater to the empty sample vial, recap and vortex for 20

containing solutions for rinsing


Laboratories must then proceed to analyse the 10 L samples using their routine method . Samples are to be tested in the respective order on the


confidential treatment of your results in the final report, your facility has been allocated a



at various concentrations. receive an additional 3.5 mL bulk water concentrate sample that will be


8ºC. The date and time of

Mix the 3.5 mL tube by inversion then immediately place the bottom of the tube on a vortex and mix such that the vortex extends to the bottom of the tube. Add each of the 3.5 mL bulk water

hoice, taking care not to up the order of the sample vials in relation to their respective 10 L water samples. Ensure

oocysts. For example, m) water. Ensure the







routine method . Samples are to be tested in the respective order on the





at various concentrations. receive an additional 3.5 mL bulk water concentrate sample that will be

utilised as the proficiency testing program trip control. If you receive this sample (labelled “PTA

8ºC. The date and time of

Mix the 3.5 mL tube by inversion then immediately place the bottom of the tube on a vortex and mix such that the vortex extends to the bottom of the tube. Add each of the 3.5 mL bulk water

hoice, taking care not to up the order of the sample vials in relation to their respective 10 L water samples. Ensure

oocysts. For example, Ensure the







routine method . Samples are to be tested in the respective order on the

. One hundred percent (100%) of each sample supplied must be analysed. Participants are advised that analytical methods used will be noted in the final report. To allow


at various concentrations.

led “PTA

Mix the 3.5 mL tube by inversion then immediately place the bottom of the tube on a vortex and



C1.2 PTA is aware of the internal positive control ColorSeedTM, developed by BTF Pty Ltd. Although PTA understands the advantage of ColorSeedTM use as an internal positive control, laboratories should note that it is not acceptable for laboratories to adjust results obtained with the PTA proficiency testing samples on the basis of recoveries obtained using ColorSeedTM unless the respective laboratory routine practice/standard operating procedure uses ColorSeedTM as a true internal standard, i.e. addition to every sample, and correction of observed count using internal standard recovery during routine sample reporting. 6. Record the results for each sample on the Results Sheet provided. Participants must report

both Total and Confirmed Counts on the PTA Results Sheet and specify the method(s) used for confirmation. Please be advised that PTA uses Total Counts (rather than Confirmed Counts) in data analysis. Participants must not report non-numerical values (i.e. less than/greater than values, presence/absence, detected/not detected etc) on the PTA Results Sheet. Actual counts observed under the microscope must be reported. Participants must not use conversion (recovery) factors derived from quality control to adjust raw data unless a true internal standard is employed for every routine sample as described above.

7. Participants are requested to calculate and report an estimate of measurement uncertainty (MU) for each reported Total Count result. All MU must be reported as a 95% confidence interval (coverage factor k ≈ 2). Estimates must be reported as either relative (% RSD – e.g. 100 +/- 10% [oo/cysts] at 95% CI) or absolute (e.g. 100 +/- 10 [oo/cysts] at 95% CI) including the number (n) of determinations used to generate the MU estimate.

8. Commence testing as soon as possible after samples are received. IMPORTANT: All participants must return Results Sheets no later than Friday 2 May 2014 to: Karen Cividin Proficiency Testing Australia PO Box 7507 SILVERWATER NSW 2128 phone: +61 2 9736 8397 fax: +61 2 9743 6664 email : [email protected]

Date / Time of Sample Receipt:

Condition of Shipment Upon Receipt:

Sample

A

B

C

D

E*n – number of determinations used to generate

Methods used:

Concentration (e.g. Flocculation Method)Filtration Method Purification (e.g. IMS)Enumeration (e.g. Microscopy) Details of

*Please be advised that methods used to obtain resu lts will be noted in the final report.*

Print Name:




Sample

Total Count

A

B

C

D

E number of determinations used to generate

Methods used:

Concentration (e.g. Flocculation Method)Filtration Method Purification (e.g. IMS)Enumeration (e.g. Microscopy) Details of confirmation methods


Print Name:

Signed:

I confirm that the concentrate was added to 10L of water prior to analysis.

Print Name:

Signed:Return no later than

Karen CividinProficiency Testing Australia, PO Box 7507, Silverwater NS W 2128Email:


Cryptosporidium



Cryptosporidium

Total Count

MU and

number of determinations used to generate

Methods used:

Concentration (e.g. Flocculation Method)Filtration Method (please tick)

Purification (e.g. IMS) Enumeration (e.g. Microscopy)

confirmation methods


Print Name:

Signed:


Print Name:

Signed:

Return no later than Friday 2Karen Cividin Proficiency Testing Australia, PO Box 7507, Silverwater NS W 2128Email: [email protected]


Cryptosporidium and



Cryptosporidium

MU and *n

number of determinations used to generate MU estimate.

Concentration (e.g. Flocculation Method)(please tick): Sponge

Enumeration (e.g. Microscopy)

confirmation methods



Friday 2 May 2014

Proficiency Testing Australia, PO Box 7507, Silverwater NS W [email protected] Phone: +61 2 9736 8397 Fax: +61 2 9743 666

and Giardia

Results Sheet


Cryptosporidium Counts

Confirmed Count

MU estimate.

Concentration (e.g. Flocculation Method) Sponge � Flat Bed



May 2014 to:

Proficiency Testing Australia, PO Box 7507, Silverwater NS W 2128Phone: +61 2 9736 8397 Fax: +61 2 9743 666

C1.3

Round 33

Results Sheet

Confirmed Count

Total Count

Flat Bed �



(Senior QA/QC Officer or similar)


Round 33 - Proficiency Testing Program

Results Sheet

Giardia

Total Count

Cartridge


(Analyst/s)


Date:




Laboratory Code:

Giardia Counts

MU and *n

Cartridge � Tangential Flow


Date:

(Analyst/s)



Proficiency Testing Australia, PO Box 7507, Silverwater NS W 2128 Phone: +61 2 9736 8397 Fax: +61 2 9743 666


Laboratory Code:

Counts

*n Confirmed Count

Tangential Flow




Phone: +61 2 9736 8397 Fax: +61 2 9743 666 4

Laboratory Code:

Date & time of testingConfirmed

Count

Tangential Flow �


Date & time of testing

GLOSSARY

Trip Control

A sample used to monitor the effect(s) of sample set transport. Sent to a nominated laboratory and returned.

Seed Sample

Sample containing Cryptosporidium oocysts and/or Giardia cysts in various doses, prior to dispensing into the PTA sample container.

Water Concentrate Sample

Final proficiency testing sample, containing Cryptosporidium oocysts and/or Giardia cysts, QC mud and Milli-QTM water.

IMS Immunomagnetic separation

DAPI 4’,6-diamidino-2-phenylindole

DIC Differential Interference Contrast (Microscopy)

IFA Immunofluorescent Antibody

FITC Fluorescein isothiocyanate

---------- End of report ---------

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Cryptosporidium and Giardia (Round 33) Proficiency Testing ...capeyork.myownserver.net/~ptaasnau/documents/865.pdf · Measurement Uncertainty ... TABLE B: Cryptosporidium and Giardia