28 different genotypes of HPV can be simultaneously tested and reported using multiplex pcr, this includes 19 high risk and 9 low risk genotypes

1. Multiplex PCR:Multiplex PCR:
A Game changerA Game changer
inin
infectious disease diagnosticsinfectious disease diagnostics

2. Multiplex Real Time PCR
for diagnosis of
Human Papilloma Virus Infections
Dr Ashok Rattan,
MBBS, MD, MAMS
COO & MD
Star Metropolis Clinical Laboratories

3. Non enveloped double stranded DNA virus: over 100 types
15–20 oncogenic
30–40 anogenital
L1: major viral capsid protein – immunogenic
L2: minor viral capsid protein – immunogenic
Human Papilloma Viruses

4. 4
HPV & Cervical Cancer
• HPV recognized as the underlying cause ofHPV recognized as the underlying cause of
cervical cancer since 1996cervical cancer since 1996
– NIH Consensus Conference on Cervical Cancer, 1996
– World Health Organization/European Research
Organization on Genital Infection and Neoplasia, 1996

5. HPV causes Cx Ca

6. 6
Natural History of HPV Infections
• Sexually transmitted
• Usually no symptoms
• No treatment for HPV infection before symptoms
• Immune system clears most cases; some persist
• HPV present in >99% of cervical cancers
• High risk types (16, 18) associated with cancer
• Low risk types (6, 11) are associated with genital warts
• All can cause abnormal Pap tests
Human Papillomavirus. ACOG Practice Bulletin No. 61. 2005; 105: 905-18.

7. HPV Classification:
Carcinogenic Risk
• Over 100 HPV strains
identified
• Risk assessment based
on transformative
potential of a strain’s
E proteins
• Low found in benign
lesions only
• Intermediate found in
benign lesions &
invasive cancers
• High usually found in
carcinomas;
occasionally seen in
benign lesions
Low Risk 60, 11, 42, 43, 44
Intermediate
Risk
31, 33, 35, 51,
52, 58
High Risk 16, 18, 45, 56
Furumoto et al., 2002.

9. Early Carcinoma Advanced Carcinoma

10. HPV 16
HPV 18
HPV 6
HPV 11
Cancer causing Types1,2,4 Non-cancer causing types1,2
• >75% of Cervical Cancer5,6
• ~50% of Vaginal & Vulvar Cancer5
90% of Anogenital warts5
HPV is a necessary cause of cervical cancer - 99.7%4
HPV
1.Schiffman M, Castle PE. Arch Pathol Lab Med. 2003;127:930–934. 2. Wiley DJ, Douglas J, Beutner K, et al. Clin Infect Dis. 2002;35(suppl 2):S210–S224. 3. Muñoz N, Bosch FX, Castellsagué X, et al. Int J
Cancer. 2004;111:278–285. Reprinted from J Virol. 1994;68:4503–4505 with permission from the American Society for Microbiology Journals Department. 4. Walboomers JM, Jacobs MV, Manos MM, et al. J
Pathol. 1999;189:12–19. 5. X. Castellsagué, S. de Sanjose, T. Aguado, K. S. Louie, L. Bruni, J.Muñoz, M. Diaz, K. Irwin, M. Gacic, O. Beauvais, G. Albero, E. Ferrer, S. Byrne,F. X. Bosch. HPV and Cervical
Cancer in the World. 2007 Report. WHO/ICO Information Centre on HPV and Cervical Cancer (HPV Information Centre). Available at: www.who.int/hpvcentre6. Bhatla N et al.Vaccine (2008;26; 2811-17
Human Papillomavirus (HPV)
Human Papilloma Virus (HPV)

11. HPV Genome
• E1-E7 = “Early” genes
(nonstructural)
• L1, L2 = Capsid genes
• URR = upstream regulatory
region
• E6 & E7 proteins play major
role in immortality &
malignant transformation of
infected cells
• E5 has role, but not required
to maintain cancer
phenotype
Munoz et al. 2006.

12. Early Genes Hijack Cell Cycle Checkpoint
• HPV’s E6 & E7 proteins interact with key cell cycle
proteins including pRB & p53, effectively over-riding the
G1/S-phase checkpoint
Mechanism
1. E7 binds & phosphorylates pRB, activating E2F
transcription factor
2. DNA replication proteins of host cell are then expressed;
unchecked S-phase occurs
3. E6 marks p53 for proteolytic degradation so it cannot
activate apoptosis (note: absence of p53 is not necessary
for E6 to cause immortalization)

19. 19
Common HPV Types and their effects
HPV Types Lead to:
Low-Risk
High-Risk
HPV 6, 11,
40,, 42, 43, 44,
54, 61, 70, 72, 81
HPV 16, 18,
31, 33, 35, 39, 45,
51, 52, 56, 58, 59,
68, 73, 82
Benign cervical changes
Genital warts
Precancer cervical changes
Cervical cancer
Anal and other cancers
1. Cox. Baillière’s Clin Obstet Gynaecol. 1995;9:1. 2. Munoz et al. N Engl J Med. 2003;348:518.

20. 20
Human Papillomavirus
Cancer of cervix 100%
Cancer of esophagus .
Cancer of skin .
Cancer of X,Y,Z…. .
Cancer of mouth 3%
Cancer of throat 12%
Cancer of penis 40%
Cancer of vulva, vagina 40%
Cancer of anus 90%
Parkin DM et al. CA Cancer J Clin 2005; 55:74-108.

22. 22
Co-factors for HPV Infection
•Smoking
•HIV infection
•Other immune system defect
•Pregnancy
•Oral contraceptive use
Ferris et al. Modern Colposcopy. 2004.

23. HPV
Infection
Low
Grade
Lesions
High
Grade
Lesions
Invasive
Cancer
0–1 Year 0–5 Years 1–20 Years
HPV Infection may clear
Adapted from Pinto AP et al. Clin Obstet Gynecol. 2000;43:352–362.
Facts about HPV Infection
Facts about HPV Infection

24. 24
HPV Infections: Summary
• HPV can lead to genital warts
• Persistent high-risk HP Most people are infected by HPV at some time
• Immune system usually clears HPV, but not always
• Persistent low-risk V can lead to pre-cancer
HPV
Long persistence of HPV
can lead to cancer

25. Incidence (Women: all ages) - Cervical Cancer
1. X. Castellsagué, S. de Sanjose, T. Aguado, K. S. Louie, L. Bruni, J.Muñoz, M. Diaz, K. Irwin, M. Gacic, O. Beauvais, G. Albero, E. Ferrer, S. Byrne, F. X. Bosch. HPV and Cervical Cancer in the World. 2007
Report. WHO/ICO Information Centre on HPV and Cervical Cancer (HPV Information Centre). Available at: www.who.int/hpvcentre

26. 1. X. Castellsagué, S. de Sanjose, T. Aguado, K. S. Louie, L. Bruni, J.Muñoz, M. Diaz, K. Irwin, M. Gacic, O. Beauvais, G. Albero, E. Ferrer, S. Byrne, F. X. Bosch. HPV and Cervical Cancer in
the World. 2007 Report. WHO/ICO Information Centre on HPV and Cervical Cancer (HPV Information Centre). Available at: www.who.int/hpvcentre
Mortality (Women: all ages) - Cervical Cancer

27. Age Specific Incidence vs Mortality
2. WHO/ICO Information Centre on HPV and Cervical Cancer (HPV Information Centre). Summary report on HPV and cervical cancer statistics in India. 2007.
[Accessed on 18th
March 2008. Available at www. who. int/ hpvcentre c WHO/ICO Information Centre on HPV and Cervical Cancer

28. HPV Type Distribution - Invasive cervical cancer
1. X. Castellsagué, S. de Sanjose, T. Aguado, K. S. Louie, L. Bruni, J.Muñoz, M. Diaz, K. Irwin, M. Gacic, O. Beauvais, G. Albero, E. Ferrer, S. Byrne, F. X. Bosch. HPV and Cervical Cancer in the World.
2007 Report. WHO/ICO Information Centre on HPV and Cervical Cancer (HPV Information Centre). Available at: www.who.int/hpvcentre
HPV
16+18=76.7%

29. HPV types and cervical cancer
1. Bosch FX et al. Vaccine 2008; 26S: K1–16. 2. Bhatla N et al. Vaccine 2008; 26(23):2811-2817.
Five most frequent and aggressive HPV types that cause
cervical cancer worldwide
+ + +
HPV 16 HPV 18 HPV 45 HPV 31 HPV 33
+
These 5 HPV types are responsible for
up to 92% of Cervical Cancer in India2

30. Years of Life Lost to Cervical Cancer*
*In women in the United States (2003), 1. Ries LAG, Harkins D, Krapcho M, et al. (eds). SEER Cancer Statistics Review, 1975–2003,
National Cancer Institute. Bethesda, MD; 2006.
26
19
18
Largest single cause of years of life lost to cancer in the developing world

31. Diagnosis
• 3% Acetic acid application
• Lugol’s Iodine application
• PAP smear
• Liquid Based cytology
• PCR
• RT PCR
• Multiplex PCR

34. Women were examined visually by simple
speculum and colposcopically after
application of 3 % acetic acid to cervix.
Equal detection rates of cervical
abnormalities by both techniques.

35. WHEN LUGOL’S IODINE IS APPLIED TO THE
CERVIX, THE NORMAL CELLS CONTAINING
GLYCOGEN STAIN DARK BROWN. THE
ABNORMAL CELLS ARE RAPIDLY DIVIDING
AND ARE DEFICIENT IN GLYCOGEN HENCE,
REMAIN UNSTAINED WHICH ARE FURTHER
EVALUATED BY COLPOSCOPY & BIOPSY.

38. • Adenocarcinoma is difficult to
detect with routine screening
methods1
– The cervical smear brush
cannot access the endocervical
canal as easily as the outer
surface of the cervix1
Adenocarcinoma is difficult to detect
Adenocarcinoma: may be
inaccessible to the cervical
smear brush
Squamous cell carcinoma:
usually accessible to the cervical smear
brush
Cervical smear brush
Cervix

39. Adenocarcinoma of the cervix-
An Emerging concern
• Incidence increasing (20–25% of all
cervical cancers), not prevented with traditional pap
screening
• More aggressive and occurs in younger women
• > 90% of adenocarcinomas result from HPV 16, 18, 45,
33 and 311
• HPV 18 confers the highest risk

40. 40
Conventional Pap Smear
• Developed by Dr. George N.
Papanicolaou in 1940’s
• Most common cancer screening
test
• Key part of annual gynecologic
examination
• Has greatly reduced cervical
cancer mortality in U.S.Ferris et al. Modern Colposcopy. 2004: 2-4, 49.
Photo accessed from http://www.cytology-iac.org/Cytopaths/1998/cytoFall98.htm

41. 41
Screening with the
Conventional Pap Smear
• Widely available
• Inexpensive
• But not perfect
– Screening test – not diagnostic
– 7-10% of women need further evaluation
– Low sensitivity – need regular repeats
Cervical Cytology Screening. ACOG Practice Bulletin No. 45. 2003; 102:417-27.

42. 42
New Liquid Pap Tests
• More accurate test
– Thin, uniform layer of cells
– Screening errors reduced by half
• Screening needed less often
• Can test for HPV with same
specimen if abnormal cells
found
• Expensive
Linder J. et al. Arch Pathol Lab Med. 1998; 122: 139-144.

43. HPV-containing double
stranded DNA
‘Empty’ non-infectious virus-
like particle (VLP) mimics the
virus
Virus-like particles (VLPs) as HPV vaccine antigens
mimic the virus structure
Stanley M, et al. Vaccine 2006; 24(suppl 3):S3/106–113.

44. Vaccines
• Gardasil (2006) : Contains HPV 16, 18, 6, 11
• Cerverix (2009): Contains HPV 16, 18
– Females 10 through 25
– For:
• Ca Cx
• CIN
• Adenocarcinoma in situ
– Gardasil for males too and for Ca Anus, warts etc

45. NexGen MoDxs
End point PCR:
• 1st
Generation : PCR
• 2nd
Generation: Isothermal PCR
» NASBA
» SDA
• 3rd
Generation:
Real Time PCR (21st
Century):
• Next Generation Multiplex Real Time PCR
• DPO technology
• TOCE technology

46. DPO TM
Dual Primer Oligonucleotide
TOCE TM
Tagging Oligonucleotide Cleavage & Extension

47. • How to increase specificity with out changing the basic thermodynamics and kinetics of PCR?
• Increased primer length increases specificity
• BUT increased primer length increases the Tm
Principle of DPO™

48. NexGen MoDx Solutions

49. DPO TM
Dual Primer Oligonucleotide
TOCE TM
Tagging Oligonucleotide Cleavage & Extension

50. Principles of TOCE™

51. NexGen MoDx Solutions

52. Key Features and Benefits of DPO™/TOCE™

53. Key Features and Benefits of DPO™/TOCE™

54. What is the Paradigm Shift ?

57. Current Offering
• Respiratory:
– RV 16
– RB 5
• Genital
– STI 7
– HPV 28

59. • Each swab contains
– Specimen collection
swab with a tip flocked
with soft nylon fibre
– Polypropylene screw cap
tube with 2 ml of eNAT
transport medium
– Each swab has a molded
breakpoint in the shaft

60. Sample collection & Transportation
• All sample must be collected using eNAT Swabs (COPAN)
• Three different swabs are available
– eNAT Regular applicator (606CS01 R)
– eNAT L Shaped Applicator (606CS01 L)
– eNAT Pernasal applicator (606CS01 P)
• eNAT medium stabilizes & preserves RNA/DNA for
prolonged time periods
• eNAT medium contains detergent & protein denaturant,
so not suitable for culture based tests
• Transport at 5 to 25 C (in cold)

62. Collection of Cx Swab

65. • Each swab contains
– Specimen collection
swab with a tip flocked
with soft nylon fibre
– Polypropylene screw cap
tube with 2 ml of eNAT
transport medium
– Each swab has a molded
breakpoint in the shaft

77. Current Offering
• Respiratory:
– RV 16
– RB 5
• Genital
– STI 7
– HPV 28