Cryptosporidium in the post-genomic era

Stephen Hadfield

Cryptosporidium Reference Unit

Public Health Wales Microbiology Swansea

Cryptosporidiosis • Diarrhoea, abdominal cramps, loss of appetite, low-grade fever, nausea, and

vomiting

• Up to 6000 reported cases p.a. in UK

• Usually self-limiting (1-2 weeks)

• BUT the disease can be prolonged and life-threatening in severely immunocompromised persons. -Complications: pancreatitis, sclerosing cholangitis, biliary cirrhosis.

• Long term effects

Cryptosporidium

• Protozoan, obligate parasite

• Asexual AND sexual reproduction and autoinfection within the host

• Robust oocyst stage

Diagnosis of Cryptosporidiosis

• Mainly by microscopy

– Modified Zn

– Phenol auramine

• Increasingly: EIA

CRU reference typing

• Microscopy/EIA does not differentiate spp./genotypes

• CRU tests ~3000 samples/year

• Requested samples

• Referred samples

• Confirmation

• Outbreak investigation

• Primary screening

• Environmental samples

• Animal samples

DNA preparation from clinical samples

• Semi-purification of oocysts

• DNA extraction

Salt-flotation centrifugation

Boil/freeze-thaw DNA extraction

Molecular typing by PCR-RFLP

• Cryptosporidium Oocyst Wall Protein (COWP) gene

-RsaI digest

• 18S rRNA gene (Nested PCR)

-SspI & VspI digests

Worldwide diversity of Cryptosporidium spp. in human infection

Cryptosporidium spp.

C. parvum

C. hominis

Other species &

genotypes

C. meleagridis C. felis C.

cuniculus C. ubiquitum C. canis C. andersoni,

C. muris, C. suis, C. fayeri C. bovis

skunk, horse,

monkey, chipmunk, pig

genotypes

UK yearly trends

Chalmers et al., Eurosurveillance 2009 14(2) 15 January 2009; ii=19086; Chalmers RM et al., Epidemiology and Infection 2011; 139(5): 700-712; Elwin et al., Epidemiology & Infection 2011;140: 673-683; CRU unpublished data

• 2 peaks

• Spring – C. parvum

• Autumn – C. hominis

Sources of Cryptosporidium in human infection

C. hominis C. parvum

Anthroponotic and zoonotic cycles

Anthroponotic cycle

Typing and incident / outbreak management

• ~10% cases associated with outbreaks

• Identify clusters of cases

• Help identify source of infection or contamination

• Avoid inappropriate control measures

• With higher-resolution typing, link cases and suspected sources

Man

Farmed animals

and pets Wildlife

Early genomics – 1990’s early 2000’s

• Low resolution maps of Cryptosporidium genome

– Happy mapping

– Genome sequence surveys – Sanger sequencing of randomly cloned fragments.

– New assay targets

Molecular typing by real-time PCR

• PCR-RFLP time consuming and labour intensive

- COWP >6 hr run time >1.5 hr hands-on

• Real-time PCRs

• Species-specific targets

CRU real-time PCR v1.0 Tube 1 Tube 2

• VIC

• FAM

Cryptosporidium 18S rRNA Internal control - optional

C. parvum LIB13 C. hominis LIB13

Hadfield et al. 2011 J. Clin. Micro. 49: 918–924

Cycle

5 10 15 20 25 30 35 40 45 50 55

Norm

. F

luoro

.

0.60

0.55

0.50

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Threshold

Cycle

5 10 15 20 25 30 35 40 45 50 55

Norm

. F

luoro

.

0.60

0.55

0.50

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Threshold

Cycle

5 10 15 20 25 30 35 40 45 50 55

Norm

. F

luoro

.

0.55

0.50

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Threshold

Cycle

5 10 15 20 25 30 35 40 45 50 55

Norm

. F

luoro

.

0.25

0.20

0.15

0.10

0.05

0.00

Threshold

Analytical specificity

Cycle

5 10 15 20 25 30 35 40 45 50 55

Norm

. F

luoro

.

0.65

0.60

0.55

0.50

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Threshold

C. cuniculus most common non-C. hominis/C. parvum sp. in UK1

Waterborne outbreak – Northamptonshire 20082

1Chalmers et al. (2011) EID 17(3): 536 – 538 2Chalmers et al. (2009) EID 15(5): 829 - 830

Whole Genome Sequencing (WGS)

• 3 genomes on GenBank:

C. hominis

C. parvum

C. muris

• Allows development of new assay targets.

CRU real-time PCR v2.0

FAM: C. hominis A135

Cycle

5 10 15 20 25 30 35 40 45 50 55

Norm

. F

luoro

.

0.60

0.55

0.50

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Threshold

Cryptosporidium 18S rRNA

Cycle

5 10 15 20 25 30 35 40 45 50 55

Norm

. F

luoro

.

0.55

0.50

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Threshold

Negatives 18S rRNA gene sequencing

Cycle

5 10 15 20 25 30 35 40 45 50 55

Norm

. F

luoro

.

0.55

0.50

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Threshold

VIC: C. parvum LIB13

Single-tube

A135 C. hominis-specific

Cryptosporidium species distribution

• England and Wales positives: 2012-13 (n = 4,071)

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

C. hominis C. parvum Mixed C. hominis/C.

parvum

Other spp./gt

Not typable

0.00

0.10

0.20

0.30

0.40

0.50

0.60

% %

All Non C. hominis/C. parvum

WGS revolution

Sanger sequencing Next generation sequencing -Multiplex parallel reactions -Imaging -Computing

-Individual reactions

-Electrophoresis

Falling cost of WGS

Sanger sequencing Next generation sequencing

WGS from clinical faecal samples

• Potential – Identification of improved diagnostic targets

– Identification of virulence markers

– Identification of improved subtyping markers

• Difficulties – Complex matrix

– Low parasite numbers

– Animal amplification usual

DNA preparation from clinical samples at CRU

Semi-purified oocysts from National Collection

Concentration and further purification by IMS

Oocysts bleach treated (0.6% NaClO)

Freeze-thaw - DNA extraction

Nextera XT library

Direct WGS progress and goals

• Completed: Whole genomes sequenced for

C. hominis

C. parvum

C. viatorum – de novo

• Underway: – C. cuniculus –oocysts successfully prepared

• Planned: – Different subtypes of C. hominis and C. parvum

– Non-human infectious species

– Annotation and analysis!!

C. hominis & C. parvum subtyping

• Outbreak investigation

• Epidemiological studies

• No standardised method

– GP60 sequencing – Single locus

– Lacks discrimination for C. hominis in UK

• Multi-locus methods preferable

Multi-locus fragment typing (MLFT)

•MLFT - More rapid turnaround for OB investigations - Cost effective

•Meta-analysis of published studies1

8 C. hominis VNTR loci 8 C. parvum VNTR loci •Re-design of PCRs to allow standardised single-round PCR profile

PCR VNTR loci

Capillary electrophoresis (QIAxcel)

Amplicon size

Combine sizes to give MLFT

Marker

Marker

PCR fragment

1Robinson & Chalmers (2012) Experimental Parasitology 132 (2012) 200–215

MLFT subtyping – preliminary investigation

Locus- fragment size (bp)

Sample MM19 MSF MM18 MS9 MSD GP60 MSA MM5 MLFT

GP60

subtype 1

Outbreak 1

310 159 285 438 271 338 225 257 1 IIaA19G2R1

2 309 159 285 438 272 337 225 257 1 IIaA19G2R1

3 309 159 285 439 272 337 225 258 1 IIaA19G2R1

4 309 159 286 442 273 339 226 259 1 IIaA19G2R1

5 309 159 286 441 272 338 226 259 1 IIaA19G2R1

6

Outbreak 2 291 159 286 441 272 344 225 258 2 IIaA20G3R1

7 291 159 285 440 272 343 225 258 2 IIaA20G3R1

8 291 159 285 442 272 344 225 258 2 IIaA20G3R1

9

Outbreak 3

286 159 285 441 272 326 225 259 3 IIaA15G2R1

10 286 159 285 440 272 327 225 258 3 IIaA15G2R1

11 286 159 288 438 272 329 226 259 3 IIaA15G2R1

12 287 159 290 441 273 330 226 259 3 IIaA15G2R1

13 286 159 286 438 272 325 225 257 3 IIaA15G2R1

14 286 159 285 439 272 326 225 258 3 IIaA15G2R1

15 286 159 286 441 273 327 225 259 3 IIaA15G2R1

16 286 159 286 442 273 327 226 259 3 IIaA15G2R1

17 286 159 285 440 272 326 225 258 3 IIaA15G2R1

18

Outbreak 4

292 159 286 440 272 329 225 258 4 IIaA17G1R1

19 292 159 286 442 273 330 225 259 4 IIaA17G1R1

20 292 159 286 443 273 330 225 258 4 Neg

21 292 159 285 438 272 328 225 257 4 IIaA17G1R1

22

Outbreak 5

292 159 292 439 272 330 225 257 5 IIaA17G1R1

23 292 159 295 440 272 332 225 259 5 IIaA17G1R1

24 292 159 295 440 272 333 226 259 5 IIaA17G1R1

25 292 159 292 438 271 328 225 257 5 IIaA17G1R1

26

Selected

sporadics

274 159 285 448 271 325 225 230 6 IIaA15G2R1

27 245 159 292 441 273 327 226 259 7 IIaA15G2R1

28 291 159 285 439 272 326 225 258 4 IIaA15G2R1

29 292 159 290 438 272 328 225 257 5 IIaA16G2R1

30 286 159 285 439 272 332 225 258 3 IIaA16G3R1

31 286 159 285 438 271 331 225 231 8 IIaA16G3R1

32 425 171 239 442 258 323 196 231 9 IIaA16R1

33 292 159 285 437 271 327 225 257 4 IIaA17G1R1

34 292 159 286 439 272 329 225 257 4 IIaA17G1R1

35 286 159 289 440 272 357 226 259 10 IIaA17G1R1a

36 298 159 289 441 273 336 226 260 19 IIaA17G2R1

37 286 159 292 440 272 333 225 258 11 IIaA18G1R1

38 286 159 285 440 272 335 225 258 20 IIaA18G2R1

39 292 159 291 439 272 339 225 258 18 IIaA19G3R1

40 292 183 285 440 272 343 225 258 12 IIaA20G3R1

41 385 171 239 450 242 300 196 246 13 IIcA5G3

42 362 171 308 425 258 315 232 232 17 IIdA17G1

43 321 182 238 434 242 316 196 246 14 IIdA18G1

44 327 183 239 424 258 331 237 231 18 IIdA23G1

45 320 134 238 420 242 333 243 230 15 IIdA24G1

C. parvum C. hominis Locus- fragment size (bp)

Sample MSG MSE CP47 MS5 MSB TP14 MSC6-7 GP60 MLFT

GP60

subtype 1 272 254 485 330 280 177 533 348 13 IaA12R3

2 282 286 461 304 267 175 539 358 12 IgA24

7 280 284 460 303 265 173 536 356 12 IgA24

3 279 218 515 329 Neg 174 525 393 IaA30R3

4 302 240 486 280 277 174 526 380 15 IaA25

5 297 273 466 303 277 174 525 360 16 IfA12G1

6 319 264 483 329 278 174 516 373 17 IaA22R1

8 297 274 466 304 277 174 527 360 10 IfA12G1

9 279 269 486 329 289 175 524 Neg IdA14

10 266 270 474 382 280 176 545 405 8 IeA11G3T3

11 283 293 489 332 294 178 533 423 9 IdA14

12 280 260 489 332 284 178 532 Neg IaA24

17 304 267 493 231 271 167 535 644 6 IaA24

13 269 259 483 330 281 176 498 392 7 IaA23R4

14 278 247 446 330 262 169 541 359 1 IbA10G2

15 277 235 446 329 261 168 542 359 3 IbA10G2

16 276 245 445 329 259 168 539 358 1 IbA10G2

18 277 246 445 329 259 168 537 357 1 IbA10G2

19 276 244 446 329 259 168 543 358 1 IbA10G2

20 277 240 444 329 271 168 535 357 11 IbA10G2

21 283 240 447 330 273 169 541 359 2 IbA10G2

22 283 242 449 331 275 170 543 362 2 IbA10G2

23 285 243 450 332 277 172 546 362 2 IbA10G2

24 285 243 450 333 278 172 546 363 2 IbA10G2

25 284 242 449 331 275 171 543 361 2 IbA10G2

26 283 240 445 330 273 169 537 358 2 IbA10G2

27 282 240 446 330 272 169 538 358 2 IbA10G2

28 276 245 444 328 259 168 538 357 1 IbA10G2

29 276 245 446 328 259 167 533 356 1 IbA10G2

30 276 245 444 329 259 168 535 356 1 IbA10G2

31 277 242 444 329 259 168 537 357 4 IbA10G2

32 277 246 446 329 260 168 539 358 1 IbA10G2

33 278 247 447 330 261 169 542 360 1 IbA10G2

34 278 247 447 306 263 170 542 361 5 IbA10G2

35 Neg Neg Neg 333 267 173 Neg Neg IbA10G2

37 279 247 447 330 Neg 171 542 360 1 IbA10G2

38 278 246 446 328 262 169 542 360 1 IbA10G2

39 277 246 446 329 261 169 540 359 1 IbA10G2

40 276 246 446 329 260 168 542 359 1 IbA10G2

41 276 245 444 328 259 168 535 357 1 IbA10G2

42 277 245 445 329 Neg 168 537 357 1 IbA10G2

43 276 241 444 328 259 168 534 356 4 IbA10G2

44 277 246 446 329 260 169 541 358 1 IbA10G2

36 280 248 449 332 260 172 544 363 1 IbA10G2

45 277 246 446 329 261 169 538 358 1 IbA10G2

Acknowledgements CRU past and present: Dr Rachel Chalmers (Head of Unit), Dr David Casemore, Dr Angharad Davies, Dr Kristin Elwin, Dr Guy Robinson, Nigel Crouch,

Jonathan Goss, Anne Thomas, Rachael Seymore, David Gomez Sending labs IBERS, Aberystwyth University: Dr Justin Pachebat, Dr Simon Cameron Core work: DH via WAG and NHS Scotland Aquavalens project: European Commission