Multipex for papillomavirus

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

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  • The causal role of human papillomavirus in all cancers of the cervix has been firmly established biologically and epidemiologically. Reference: Munoz, N. et al. “HPV in the etiology of human cancer.” Vaccine 24S3 2006;24S3: 1-10.
  • Genital HPV is transmitted sexually. Transmission occurs through contact with infected genital skin, mucous membranes, or body fluids from a partner with either overt or subclinical HPV infection. Other modes include oro-genital, manual-genital, and nonpenetrative genital-genital contact. Covering infected areas with a latex condom provides theoretical protection from infection. Areas not covered by condom can transmit HPV infection. Infrequently transmitted in the neonatal period, although published studies are conflicting. Most HPV infections are transient and are cleared by the immune system 70-90% will clear within 1-2 years Persistent HPV viral infections may lead to cancer and its precursors No treatment for HPV infection but cervical changes and warts CAN be treated Reference: Human Papillomavirus. ACOG Practice Bulletin No. 61. American College of Obstetricians and Gynecologists. Obstet Gynecol 2005; 105: 905-18.
  • Furumoto et al., 2002.
  • Munoz et al. Vaccine 24S3 (2006) S3/1-S3/10
  • Munoz et al. Vaccine 24S3 (2006) S3/1-S3/10.
  • Low-risk HPV types, such as types 6 and 11, most commonly cause benign low-grade cervical changes and genital warts. Types 16 and 18 and other “high-risk” HPV types are the most common cause of low-grade cervical cell abnormalities, and almost exclusively cause high-grade cervical cell abnormalities that are precursors to invasive cervical cancer and other lower genital tract malignancies including vulvar, vaginal, penile and anal cancer. The large IARC study of cervical cancers around the world demonstrated that over 90% of all of cervical cancers were associated with high-risk types of HPV Irrespective of geographical area 43% to 65% of the cancers were associated with HPV 16 8% to 31% were associated with HPV 18. Taken together, HPV 16 and 18 accounted for approximately two-thirds of all invasive cancers from all geographic areas. References: 1. Cox. Baillière’s Clin Obstet Gynaecol. 1995;9:1. 2. Munoz et al. N Engl J Med . 2003;348:518.
  • HPV is implicated as the cause of cervical cancer and many other ano-genital cancers. Reference: Parkin DM, Bray F, Ferlay J, Pisani P. “Global cancer statistics 2002.” CA Cancer J Clin 2005; 55:74-108.
  • Co-factors to HPV in cervical carcinogenesis may act in at least 3 ways: By influencing the acquisition of HPV infection (OCPs, multiparity) By increasing the risk of HPV persistence (HIV, immunosuppresion) By increasing the risk of progression from HPV infection to CIN 2,3 and cancer (smoking, multiparity) Cigarette smoking is a significant and independent risk factor for the development of CIN3 and SCC of cervix, increasing RR 2-5 fold. Risk increases with increased intensity and duration of smoking. Mechanism is likely that tobacco containing carcinogens promote neoplastic progression in HPV infected cells. HIV and other immunosupression cause an inability to clear HPV, increasing susceptibility to HPV and oncogenicity. Multiparity has been found to be associated with both cervical cancer and CIN3 with risk rising with increased number of pregnancies. Effect is independent of sexual behavior and socioeconomic variables. Pregnancy-induced alterations in nutritional status, the effects of hormones on the cervix or on HPV expression, increased susceptibility to potential mutagens or effect of trauma at delivery are proposed mechanisms. OCP use conveys a measurable increase in risk for SCC. The strongest evidence comes from an IARC multicenter case-control study demonstrating only a moderate association with cancer risk. Mechanism appears to be the physiologic effects such as eversion of the columnar epithelium, thus activating HPV-vulnerable immature squamous metaplasia. Reference: Ferris et al. Modern Colposcopy. 2004: 2-4, 78-82..
  • In summary Most will get HPV at some time during their lifetime Most, even with high-risk HPV, will clear or permanently suppress the virus. However, some do not. It is persistence of high-risk HPV that can lead to true pre-cancer Long persistence of high-risk HPV and HPV-induced CIN3 are necessary for the accumulation of random mutations that lead to cancer.
  • Key Point Incidence of cervical cancer cases in India is highest as compared to other cancers in women 15-44 yrs of age Reference 1) 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
  • Key Point Mortality in India due to cervical cancer is highest as compared to other cancers in women of all ages. Second most common cause is Breast Cancer. Reference 1) 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
  • Key Point Difference between incidence of Cervical cancer vs mortality due to cervical cancer narrows for the females between age group 15-44yrs. Reference 1) 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
  • Key Point HPV 16 and 18 cause approximately 77% of Invasive cervical cancer . Reference 1) 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
  • Primary cervical cancer screening essentially began with the introduction of the Pap Smear. Introduced in the 1940’s, by Dr. George N. Papanicolaou, the pap smear eventually became the standard screening test for cervical cancer and pre-malignant lesions. The Pap test is based on a relatively simple principle. Cells from squamous epithelium exfoliate over time. Thus, the cells removed for cytologic examination represent epithelial cells, normal or abnormal, found at the surface. Widespread use of the pap smear has decreased cervical cancer deaths by 70%. Reference: Ferris et al. Modern Colposcopy. 2004: 2-4, 49.
  • Cervical cytology screening is, in many respects, the ideal screening test. Cervical cancer has a defined premalignant phase of many years, which allows repeated tests to significantly reduce the impact of individual false-negative test results. Cervical cytology is inexpensive and is readily accepted among American women. However, cervical cytology, is not a diagnostic test. The sensitivity of the pap smear is low (ranges from 47-85%) and the specificity is high (95-98%). Reference: Cervical Cytology Screening. ACOG Practice Bulletin No. 45. 2003; 102:417-27.
  • In 1996, the FDA approved the first of two currently available liquid-based thin-layer cytology preparations for cervical screening. Liquid-based thin-layer cervical cytology was introduced to help reduce the potential sampling errors. The Thin-Prep method appears to have increased sensitivity for detecting cancer precursor lesions over the conventional method, but the degree to which sensitivity is increased is unknown. The reported increase in sensitivity may make this method especially useful in women who are screened infrequently (fewer false negatives). The difference in specificity between the liquid-based and conventional tests has not been determined. Although an increase in sensitivity will permit earlier detection of cancer precursor lesions, any decrease in specificity can result in increased cost and morbidity from false-positive diagnoses. Both the conventional test and the liquid-based thin-layer test can be effective in population screening. Providers selecting a cervical cytology method should consider the screening history of their patient, the cost of the test, and the possible effects of false-negative or false-positive results. Reference: ACOG Practice Bulletin. Cervical Cytology Screening. 2003; 45:1-11.
  • Cervarix ® , the candidate cervical cancer vaccine is composed of DNA-free virus-like particles of the capsid protein L1. 1 Virus-like particles of L1 alone have been shown to be similar in both structure and immunogenicity to authentic papilloma virions and to be strong inducers of neutralizing antibody responses. 1–3 References 1. Giannini S, et al. Vaccine 2006; 24: 5937–5949. 2. Christensen N, et al. J Gen Virol 1994; 75: 2271–2276. 3. Rose R, et al. J Gen Virol 1994; 75: 2075–2079.
  • Multipex for papillomavirus

    1. 1. Multiplex PCR:Multiplex PCR: A Game changerA Game changer inin infectious disease diagnosticsinfectious disease diagnostics
    2. 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. 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. 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. 5. HPV causes Cx Ca
    6. 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. 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.
    8. 8. Early Carcinoma Advanced Carcinoma
    9. 9. 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)
    10. 10. 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.
    11. 11. 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)
    12. 12. 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.
    13. 13. 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.
    14. 14. 22 Co-factors for HPV Infection •Smoking •HIV infection •Other immune system defect •Pregnancy •Oral contraceptive use Ferris et al. Modern Colposcopy. 2004.
    15. 15. 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
    16. 16. 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
    17. 17. 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
    18. 18. 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
    19. 19. 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
    20. 20. 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%
    21. 21. 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
    22. 22. 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
    23. 23. Diagnosis • 3% Acetic acid application • Lugol’s Iodine application • PAP smear • Liquid Based cytology • PCR • RT PCR • Multiplex PCR
    24. 24. 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.
    25. 25. 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.
    26. 26. • 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
    27. 27. 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
    28. 28. 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
    29. 29. 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.
    30. 30. 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.
    31. 31. 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.
    32. 32. 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
    33. 33. 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
    34. 34. DPO TM Dual Primer Oligonucleotide TOCE TM Tagging Oligonucleotide Cleavage & Extension
    35. 35. • 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™
    36. 36. NexGen MoDx Solutions
    37. 37. DPO TM Dual Primer Oligonucleotide TOCE TM Tagging Oligonucleotide Cleavage & Extension
    38. 38. Principles of TOCE™
    39. 39. NexGen MoDx Solutions
    40. 40. Key Features and Benefits of DPO™/TOCE™
    41. 41. Key Features and Benefits of DPO™/TOCE™
    42. 42. What is the Paradigm Shift ?
    43. 43. Current Offering • Respiratory: – RV 16 – RB 5 • Genital – STI 7 – HPV 28
    44. 44. • 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
    45. 45. 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)
    46. 46. Collection of Cx Swab
    47. 47. • 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
    48. 48. Current Offering • Respiratory: – RV 16 – RB 5 • Genital – STI 7 – HPV 28

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