research and diagnosis of leptospirosis. Julie Collins-Emerson*, - - PowerPoint PPT Presentation

Utility of diagnostic tests for research and diagnosis of leptospirosis.

OIE Collaborating Centre for Veterinary Epidemiology and Public Health

Julie Collins-Emerson*, Fang Fang, Jackie Benschop, Cord Heuer & Peter Wilson.

Key questions:

• 1. What tests are most successful for detecting Leptospira in:
• different specimen types
• different species (sheep/cattle)
• at different stages of the disease

Key questions:

• 1. What tests are most successful for detecting Leptospira in:
• different specimen types
• different species (sheep/cattle)
• at different stages of the disease
• 2. How well do the same diagnostic tests run under the “ideal”

conditions attainable in a research laboratory compare to those performed within the “commercial reality” of a veterinary diagnostic laboratory?

Key questions:

1. What tests are most successful for detecting Leptospira in:

• different specimen types
• different species (sheep/cattle)
• at different stages of the disease
• 2. How well do the same diagnostic tests run under the “ideal” conditions attainable in a

research laboratory compare to those performed within the “commercial reality” of a veterinary diagnostic laboratory?

• 3. What are the sources of variation with diagnostic test results?

Diagnostic tests utilised

• Microscopic agglutination test (MAT)

by Becca Chandler

• Dark field microscopy (DFM)
• Real-Time PCR (RT-PCR) (two chemistry types –

SYTO9 vs. TaqMan probe)

vs.

• culture

Sources of possible variation examined.

• host species
• serovar strain
• stage of disease
• diagnostic sample type
• kind of diagnostic test
• sample preparation e.g. DNA extraction
• technical operator both within and between labs

Strategy

Two main lines of enquiry: 1) Appraise test results:

• different diagnostic samples
• at different stages of disease
• in different host species

……under controlled experimental conditions

Strategy

Two main lines of enquiry:

1) Test result comparisons:

• diff. diagnostic samples
• at diff. stages of disease
• in diff. host species

……under controlled experimental conditions

2) Inter-lab comparisons of diagnostic tests i.e. research (HLRL) vs. commercial (GV) results on the same samples

4 Controlled trials

Note:

• Involvement in the 4 trials (A,B,C &D) was opportunistic
• As we “piggy-backed” on commercial trials, there was

little opportunity to influence the design of the trials.

• Accepted their design was not ideal but did allow access

to animals under controlled challenge conditions

All trials had animal ethics approval (Kaiawhina Animal Ethics Committee, Palmerston North, New Zealand)

The trials

• Trial A used to passage and “hot up” suitable lab-

stored field isolates for challenge trials i.e. selection for virulence.

• L. borpetersenii sv. Hardjobovis (H.bovis) and L.

interrogans sv. Pomona (Pom) used

• Trials to serve as pilots for later vaccination trials

Trial A – initial passage trial (pilot)

Cultures used:

H.bovisa = isolate from commercial vet. path. lab., (GV),

• rigin unknown

H.bovisb = isolated from a deer by Hopkirk Leptospirosis Research Laboratory (HLRL) Poma = isolate from commercial vet. path. Lab. (GV),

• rigin unknown

Pomb = isolated from a sheep by Hopkirk Leptospirosis Research Laboratory (HLRL)

2 x H.bovis strains 2 x Pom strains

Trial A – initial passage trial (Pilot)

A1 A3 A6 A7 A2 ‡ A4 A5 A8 H.bovisa H.bovisb Poma Pomb Pen 1 Pen 2 Pen 3 Pen 4

Challenged on 3 success days

Trial A – initial passage trial (Pilot)

Samples taken:

• Urine - (for culture, PCR, DFM & MAT)
• Kidney - (for culture, PCR & DFM)
• blood - (for serology)
• Length of trial = 42 days

Trial A – Results

Detection of leptospires in urine on each sampling day after challenge, and kidney on Day 42, by culture (C), PCR (P) and Dark Field Microscopy (D) and antibody titres against serovars Hardjobovis and Pomona by MAT for sheep Trial A.

Sheep ID Serovar inoculated Urine* Kidney MAT titre Day Day Day 19 22 27 30 33 42 1 2 3 19 † 42 A1 Hardjobovis a − − D − − − − − − − − − A2 Hardjobovis b P P,C C P − P,C,D − − − − 1:100 1:192 A3 Hardjobovis a − − − − − − − − − − − − A4 Hardjobovis b C C C C − P,C − − − − 1:1600 1:768 A5 Pomona b − − − − − P,D − 1:24 − − − 1:24 A6 Pomona a − − − − − − − − − − − − A7 Pomona a − − D − − − − − − − − − A8 Pomona b − − − − − − − − − − − − * N.B. Urine not collected on Day 0 ; † MAT performed by GV rather than HLRL

‡ This passage isolate was used for challenge in Trial B

‡

Trial A – Results

• HLRL H.bovisa most successful for PCR, DFM. Also
• nly isolate to be successfully cultured after

passage.

• HLRL marginally better results for PCR and DFM

detection than GV cultures

• Neither Pom strains successfully passaged

Trial A conclusions

• Either challenge method not optimal for Pom strains,
• r, not particularly virulent.
• H.bovisb & Pomb from HLRL more successful –

selected for further trialing.

• Use passaged culture from sheep A2 for following

H.bovis trials.

Trial B results

Sheep ID Urine Kidney MAT titre against serovar Hardjobovis Day Day Day 5 8 12 14 21 28 37 42/15a 5 8 12 14 21 28 37 B1 − − − − − P − − − 1:384 1:192 1:384 1:48 1:96 B2 − − − − − − − − D − − 1:96 1:1536 1:768 1:384 1:192 1:48 B3 − − − − − − D D − − 1:48 1:1536 1:768 1:96 1:48 1:24 B4a − − − − − − − − 1:96 1:768 1:768 B5 − − − − − − − D D − − 1:48 1:3072 1:1536 1:384 1:192 1:192 B6 P,C P,C P,C P,C P,C,D P,C,D P P,C,D P,D 1:24 1:48 1:192 1:96 − 1:192 1:96 1:24 B7 − − − − − D − − D − − 1:384 1:768 1:1536 1:384 1:384 1:96 B8 − − − − − − − D D − − 1:768 1:1536 1:768 1:192 1:96 1:96

Detection of Leptospira in urine and kidney and MAT results

N.B. B4a died on Day15 at which time the kidney was sampled. C = culture, P = PCR, D = dark field microscopy, - = neg. result

B: H.bovis isolated from sheep ‡ A2 into 8 x

Trial B results

• Not successful in establishing an infection.
• No Lepto detected in any blood samples
• 1 PCR pos. kidney
• All animals “saw” the antigen
• *Animal B6 – detected Lepto in urine and kidney each

sampling by PCR and culture – silent carriage!

• Therefore techniques successful

Trial B conclusions

• The MAT results suggested all animals “saw” the antigen
• However, not particularly successful at establishing infection
• *Animal B6 – detected Lepto in urine and kidney at each

sampling by PCR and culture – silent carriage!

• Established the PCR and culture as consistent and reliable in

this case.

Trial C results

Inoculated strains Sheep ID Blood Serum Urine Kidney MATa titre against serovar Pomona Day Day Day Day Day 3 5 7 14b 3 5 7 14 7 14 21 28 35 42 42 5 7 14 28 42 LPC 04/08

C1

− − − − − − P P − − − P P,C C P − − − 1:96 1:3072 1:800 1:50 −

C2

− − P P − − P P − − − − − − P − − − 1:96 1:192 1:50 1:25 −

C3

− − − − − − − − − − D P C − P,D D − − 1:24 1:768 1:100 1:50 −

C4

− C − − − − P P − − − P,C P,C P,C,D P,C,D P,C P,C,D − 1:48 1:3072 1:800 1:25 −

C5

− C P − − − P P − − − P,C P,C,D P,C P,C,D P,C P,C − 1:24 1:1536 1:800 1:50 −

C6

− C C − − − P P − − P C C P,C P,C − P,C − − 1:1536 1:800 1:25 −

C7

− P,C P − − − P − − − D P,C P,C P,C,D P,C,D P,C P,C − 1:48 1:3072 1:800 1:100 −

C8

− C P − − − P P − − − C P,C P,C P,C P,C P,C − 1:48 1:3072 1:200 1:100 − LPC 04/04

C9

− P,C − − − − P P P − − C C P,C P,C P,C P,C − 1:24 1:384 1:400 1:50 −

C10 − C

C P − − P P − − − P,C P,C P,C,D P,C P,C P,C − − 1:384 1:400 1:25 −

C11 − C

P − − − P P − − − − P,C,D P,C P,C P,C P,C − 1:48 1:1536 1:400 1:25 −

C12 −

P,C − − − − P − − − − C P,C P,C,D P,C P,C P,C − 1:24 1:768 1:800 1:100 1:50

C13 −

P,C P − − − P P − − − P,C P,C P,C P,C,D P,C P − 1:48 1:1536 1:400 1:100 −

C14 − −

− − − − P − − − P D C P,C P,C,D C C − 1:48 1:768 1:1600 1:100 1:100

C15 − C

− − − − P P − − − C P,C P,C P,C P,C P,C,D − − 1:384 1:800 1:50 −

C16 −

− − − − − − − − − − − − C C − C − 1:48 1:768 1:400 1:25 −

Pom human NZ isolate into 16 x

a = MAT performed by NZVP Days 14-42, b = blood culture not performed on Day 14

Trial C conclusions recap.

• Successful challenge with the human Pom isolates
• Lepto first detected by both PCR and DFM on Day 7

and still on Day 42, culture by Day 14

• RT-PCR in serum slightly more successful than in

whole blood in this study (but no.s. small). (Covered in detail by Jackie in the previous talk.)

• MAT peaking ~ Day 7-Day 14

Trial D results

Sample ID Serovar Inoculated Urine a MAT titres Day Day 20 23 26 28 35 42 7 9 14 20 23 42 D1 Hardjobovis − − − − − − − − − 1:192 1:3072 1:384 − D2 Hardjobovis − − − P P,C P − − 1:384 1:192 1:1536 1:192 − D3 Hardjobovis − P P C,D − − − − 1:384 1:384 1:384 1:96 − D4 Pomona − − − − − − − − − − − − − D5 Pomona − − − − − − − − − − − − − D6 Pomona − − − − − − − − − − − − −

H.bovis NZ 3 x Same cultures as Trial A

Pom 3 x a = urine not collected on Day 0.

Trial D conclusions

• Limited success – only H.bovis successful for infection
• Blood samples taken throughout trial but no positives

by any diagnostic method

• Detected in urine from Day 23 through Day
• MAT peaked between Day 9 and Day 20

Intra-lab comparisons of tests for detection of Leptospira

Under our laboratory (HLRL)conditions:

• Leptospira detected in sheep blood Day 3-Day 7.
• RT-PCR of serum > blood culture ~ whole blood RT-PCR.

Comparison of tests for detection of Leptospira

• For sheep urine and kidney: Culture >RT-PCR> DFM more successful

at detecting Leptospira in sheep challenged with both H.bovis and Pom

• RT-PCR results for urine and kidney - almost perfect agreement (Kappa

= 0.84, 95% CI: 0.72-0.96) on urine

• Agreement between MAT and urine/kidney RT-PCR (not stratified by

animal species) Kappa = 0.46/0.50, 95%, CI: 0.35-0.57/0.42-0.58 for H.bovis

Inter-lab comparisons

Samples collected from small abattoir

Waikato Region

Details

• sheep (399) and cattle (146)
• Same urine and serum samples split between HLRL and GV
• MAT and RT-PCR results compared between labs.
• Diff. RT-PCR chemistries between labs
• HLRL used SYTO9; (target gyrB, 1 gene copy)
• GV TaqMan probe; (target 16s rRNA, 2 gene copies)
• 18 DNA samples exchanged between labs to eliminate the

extraction method variable.

Inter-lab comparisons

• RT-PCR demonstrated almost perfect agreement (0.93, 95%

CI: 0.82-1.00).

• MAT showed better agreement for H.bovis (Kappa = 0.94, 95%

CI: 0.85-1.00) than for Pom (Kappa= 0.53, 95% CI: 0.45-0.60).

• Higher H.bovis MAT titres and lower Pom titres were reported

by the commercial laboratory than those reported by the research laboratory – strain variation?

Additional results

• Better correlation between MAT urine/kidney RT-PCR

better than for Pom.

• Suggests H.bovis is shed for longer – possibly better

host adapted than Pom in NZ.

• Amongst H.bovis animals, more sheep than cattle

were detected to be shedding.

Summary of findings

• Study demonstrated agreement between the RT-PCRs from a

research and a vet. diagnostic laboratories (with different RT- PCR chemistries) on urine samples from sheep and cattle.

• Inter-lab variation in MAT known already (strain/operator

factors).

• What was novel? - we looked at operational affect e.g.

delaying for batch testing vs. “ideal” research lab conditions.

• Overall, found this not to unduly influence results

Additional points

• Different extraction methods could influence results at

sensitivity level.

• MAT results between labs compared better for H.bovis than

for Pom. – could be serovar strain used.

• Note of interest: Commercial PCR tests often validated for

human samples but not necessarily tested in animals and on the different sample types. Vet. Path. labs need to be mindful

• f assessing tests in diff. species and diff. samples.

Acknowledgements

Fang Fang, PhD candidate Neville Haack for technical support

• Prof. Nigel French for lab facilities

Estendart Ltd., Gribbles Vet. Path., New Zealand Vet. Path., AgResearch., Pfizer NZ. Funders: Rural Women NZ, Massey Uni.

Thank you.

OIE Collaborating Centre for Veterinary Epidemiology and Public Health

Challenge counts

Count of challenge cultures for all four challenge trials

Table 1. Count of challenge culture (organisms/ml) for challenge Trial A Organism 1st Challenge (Day 0) 2nd Challenge (Day 1) 3rd Challenge (Day 2) 10th March 2010 11th March 2010 12th March 2010 Pomona (HLRL) ~ 5.4 × 107 ~ 6.1 × 107 ~ 9 × 107 Hardjobovis (HLRL) ~ 4.4 × 107 ~ 5.4 × 107 ~ 5.3 × 107 Pomona (Gribbles) ~ 6.3 × 107 ~ 5.3 × 107 ~ 6.6 × 107 Hardjobovis (Gribbles) ~ 6.2 × 107 ~ 5.1 × 107 ~ 8.9 × 107 * 2 ml of culture were used for challenge Table 2. Count of challenge culture (organisms/ml) for challenge Trial B rganism 1st Challenge (Day 0) 2nd Challenge (Day 1) 3rd Challenge (Day 2) 7th July 2010 8th July 2010 9th July 2010 ardjobovis (HLRL, passaged from Trial A) ~ 5 × 108 ~ 5 × 108 ~ 4.5 × 108 * 2 ml of culture were used for Day 0 and 1, 2.3mls were used on Day 2 Table 3. Count of challenge culture (organisms/ml) for challenge Trial C Organism 1st Challenge (Day 0) 2nd Challenge (Day 1) 3rd Challenge (Day 2) 8th December 2010 9th December 2010 10th December 2010 Pomona (LPC04/4) ~ 6.6× 108 ~ 7.4 × 108 ~ 6.4 × 108 Pomona (LPC04/8) ~ 7.6× 108 ~ 7.6 × 108 ~ 6.2 × 108 * 2 ml of culture were used for challenge Table 4. Count of challenge culture (organisms/ml) for challenge Trial D Organism 1st Challenge (Day 0) 2nd Challenge (Day 1) 3rd Challenge (Day 2) 14th April 2010 15th April 2010 16th April 2010 Pomona (HLRL) a ~5× 108 ~5 × 108 ~5 × 108 Hardjobovis (HLRL) a ~5× 108 ~5× 108 ~5× 108 * 2 ml of culture were used for challenge

a Same culture as Trial A were applied in this Trial for challenge

Trial C

• Pom human NZ isolate into 16 x
• This trial was covered in detail by Jackie in the last

presentation