multipex for papillomavirus
DESCRIPTION
28 different genotypes of HPV can be simultaneously tested and reported using multiplex pcr, this includes 19 high risk and 9 low risk genotypesTRANSCRIPT
Multiplex PCR:Multiplex PCR:A Game changer A Game changer
in in infectious disease diagnosticsinfectious disease diagnostics
Multiplex Real Time PCRfor diagnosis of
Human Papilloma Virus Infections
Dr Ashok Rattan,MBBS, MD, MAMS
COO & MDStar Metropolis Clinical Laboratories
Non enveloped double stranded DNA virus: over 100 types15–20 oncogenic30–40 anogenital
L1: major viral capsid protein – immunogenic L2: minor viral capsid protein – immunogenic
Human Papilloma Viruses
4
HPV & Cervical Cancer• HPV recognized as the underlying cause of HPV 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
HPV causes Cx Ca
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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.
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, 56Furumoto et al., 2002.
Early Carcinoma Advanced Carcinoma
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 Cancer590% 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)
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 phenotypeMunoz et al. 2006.
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
Mechanism1. E7 binds & phosphorylates pRB, activating E2F
transcription factor2. DNA replication proteins of host cell are then expressed;
unchecked S-phase occurs3. E6 marks p53 for proteolytic degradation so it cannot
activate apoptosis (note: absence of p53 is not necessary for E6 to cause immortalization)
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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.
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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.
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Co-factors for HPV Infection
•Smoking•HIV infection •Other immune system defect•Pregnancy•Oral contraceptive use
Ferris et al. Modern Colposcopy. 2004.
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
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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
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
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
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
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
%
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
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.
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19
18
Largest single cause of years of life lost to cancer in the developing world
Diagnosis
• 3% Acetic acid application• Lugol’s Iodine application• PAP smear• Liquid Based cytology• PCR• RT PCR• Multiplex PCR
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.
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.
• 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 beinaccessible to the cervical
smear brush
Squamous cell carcinoma:usually accessible to the
cervical smear brush
Cervical smear brush
Cervix
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
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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
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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.
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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
• ExpensiveLinder J. et al. Arch Pathol Lab Med. 1998; 122: 139-144.
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.
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
NexGen MoDxsEnd 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
DPO TM
Dual Primer Oligonucleotide
TOCE TM
Tagging Oligonucleotide Cleavage & Extension
• 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™
NexGen MoDx Solutions
DPO TM
Dual Primer Oligonucleotide
TOCE TM
Tagging Oligonucleotide Cleavage & Extension
Principles of TOCE™
NexGen MoDx Solutions
Key Features and Benefits of DPO™/TOCE™
Key Features and Benefits of DPO™/TOCE™
What is the Paradigm Shift ?
Current Offering• Respiratory:
– RV 16– RB 5
• Genital– STI 7– HPV 28
• 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
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)
Collection of Cx Swab
• 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
Current Offering• Respiratory:
– RV 16– RB 5
• Genital– STI 7– HPV 28