HPV

From the virological point of view, papillomaviruses form now a separate family called Papillomaviridae. More than a hundred types of them have been described to date. They include the relatively small viruses called capsids, with a diameter of about 55 nm, consisting of 72 pentavalent or hexavalent capsomers, which are formed in the icosahedral capsid. Their genome consists of circular dsDNA which contains genes for early (E) and late (L) proteins. The dsDNA also comprises of non-coding region (LCR, URR) formed by regulatory sequences. Proteins expressed from early genes initiate the origin of replication of the viral genome in the cell (E1, E2), and may be involved in the malignant transformation of cell (E6, E7). L1 and L2 genes encode the major and minor capsid protein.

Human papillomaviruses are known to be potentially oncogenic viruses in connection with numerous diseases of the skin and mucous membranes. The best known is their association with cervical cancer and precancerous states, which are proven to be major, although not the only etiological factors. Their diagnosis is part of preventive screening programs, as necessary as the cytology.

There are various forms of skin diseases associated with HPV. For example, verrucous lesions, gene dermatosis called epidermodysplasia veruciformis. In the area of mucosal lesions, except precancerosis and cervical cancer, vulvar and vaginal lesions, male genitourinary tract lesions and, oropharyngeal lesions are associated with the papillomavirus. The presence of human papillomaviruses have been proven also in bronchial lesions, conjunctiva lesions and esophagus lesions. According to the ability to induce an oncogenic transformation of infected cells, the HPVs are divided into so-called low risk (LR) and high risk (HR) types.

GYNAECOLOGICAL SAMPLES:

Detection of high-risk HPV for the purpose of cervical cancer screening

HPV testing is recommended as a supplementary examination of cytological findings of the type ASC-US (preferably above 30 years of age), AGC-NOS and postmenopausal women with the finding of LSIL. Furthermore, this test is indicated as a highly sensitive check after the implementation of an excision for lesions CIN2+, namely, preferably 12 months after the implementation of an excision and before the decommissioning from active monitoring following surgery on the cervix. HPV test is paid for by a health insurance company, 5 times in the life of a patient, with a minimum interval of 12 months between examinations (in most insurance companies, the cost of laboratory testing do not fall under induced care, see Reimbursement appendix of VZP for the 2015, Art. 2).

Method:

  1. Digene Hybrid Capture 2 system detects the DNA of 13 types of HR HPV. This method is based on the chemiluminescence determination of nucleic acid using hybridization on a microplate with amplification of the signal. It detects high risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. For positive results, a further test is performed to diversify the presence of types 16, 18 and 45, and other HR-HPV types.
    Material: mucosal smear into a collection tube with the medium (Qiagen / Digene)
  2. Aptima HPV assay, which detects 14 HR HPV types of mRNA. This method is based on the capture of the target mRNA, its amplification using a transcription-mediated amplification and detection of amplification products using a hybridization protection assay. It detects HR HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 66 and 68.
    This assay is highly sensitive (99%) for verification of HPV infection and, simultaneously, very specific for verification of heavy CIN2+ lesions (HSIL).
    Material: mucosal smear into a collection tube with the medium (Aptima, LBC)

Precise identification of both, high-risk and low-risk types of HPV from a smear (HPV genotyping)

Method:

  1. LINEAR ARRAY® HPV Genotyping Test includes HPV LR, HPV HR and possible genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 70, 73, 82, HPV LR genotypes 6, 11, 40, 42, 54, 55, 61, 62, 64, 69, 71, 72, 81, 83, 84, IS39, and CP6108.
    This test also detects possible and probable HR HPV types that are not normally included in screening tests, but that can; in rare cases, cause severe lesions. The finding is always verified by an independent method that uses CPI / IIG primers for the amplification.
    Material: mucosal smear into the collection tube with the medium (LBC, Aptima, Qiagen/Digene)

Stratifying the risk of cervical cancer development with HRHPV positive women using analyzing methylation tumor suppressor genes:

See PreCursor-M (methylation analysis of promoters of CADM1, MAL and miR124-2 genes in cervical smears).

HISTOLOGICAL SAMPLES / TISSUE (paraffin blocks):

Detection and accurate genotyping of high-risk and low-risk HPVs for diagnostic purposes

Method:

  1. In vitro detection of HR / LR HPV using PCR in combination with direct sequencing or PCR in combination with reverse hybridization.
    • Detection using MY 09/11 primers, the target gene is L1
    • Detection using GP 5+/6+ primers, the target gene is L1
    • Detection using CPI/IIG primers, the target gene is E1
    • Detection using E6E7 primers, the target genes are E6, E7
    • Detection using type-specific primers for types 16, 18, 31, 33, 35, 42, 45 a 81, target genes are E6, E7
    • RHA Kit HPV SPF10-LiPA25, that includes HR and possible HR HPV genotypes 16, 18, 31, 33-35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, and LR HPV genotypes 6, 11, 34, 40, 42, 43, 44, 54 and 74
    • INNO-LiPA® HPV Genotyping Extra, which includes HR and possible HR HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82, and LR HPV genotypes 6, 11, 40, 43, 44, 54, 69, 71 and 74
    Material: tissue fixed in formalin and embedded in paraffin, small native biopsies
  2. In vitro detection and accurate genotyping of dermal HPV types of beta and gamma genuses (former EV types)
    • Detection using CPI/IIG primers, the target gene is E1
    • RHA Kit Skin (beta) HPV, which detects dermal HPV types belonging to the group of beta papillomaviruses, namely types 5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93, and 96
      Material: tissue fixed in formalin and embedded in paraffin, small native biopsies
    In situ detection of HPV in tissue using ISH (in situ hybridization)
    • Detection using probe INFORM HPV III Family Probe that detects HR HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 66 without the possibility of determining the exact type (for automatic imunostainer Ventana BenchMark ULTRA)
      Material: tissue fixed in formalin and embedded in paraffin

References

  1. Manos MM, TingY, Wright DK, Lewis AJ, Broker TR, Wolinsky SM. Use of polymerase chain reaction amplification for the detection of genital human papillomaviruses. Cancer Cells. 1989;7:209-214
  2. Van Ranst M, Kaplan JB, Burk RD.: Phylogenetic classification of human papillomaviruses: correlation with clinical manifestations. J Gen Virol. 1992;73:2653-60
  3. Tieben LM, Schegget J, Minnaar RP, et al. Detection of cutaneous and genital HPV types in clinical samples by PCR using consensus primers. J Virol Method 1993;42:265-80
  4. Gross GE, Barraso R (eds.): Human papilloma virus infection: a clinical atlas. Ullstein Mosby, 1997.
  5. Majewski S, Jablonska S. Human papillomavirus-associated tumors of the skin and mucosa. J Am Acad Dermatol. 1997;36(5 Pt 1):659-85; quiz 686-8. Review.
  6. Qu W, Jiang G, Cruz Y, Chang CJ, Ho GY, Klein RS, Burk RD. PCR detection of human papillomavirus: comparison between MY09/MY11 and GP5+/GP6+ primer systems. J Clin Microbiol. 1997;35(6):1304-10.
  7. Kleter B, Van Doorn LJ, Schrauven L, Molijn A, Sastrowijoto S, Ter Schegget J, Lindeman J, Ter Harmsel Bram, Burger Matthe, Quint W. Development and Clinical Evaluation of a Highly Sensitive PCR-Reverse Hybridization Line Probe Assay for Detection and Identification of Anogenital Human Papillomavirus. J Clin Microbiol. 1999;37:2508-2517
  8. Syrjänen K, Syrjänen S.: Papillomavirus infection in Human Pathology. John Wiley&Sons,LTD, England, 2000.
  9. Fields. Virology (fourth edition). Lippincot Williams & Wilkins, 2001
  10. Sterling JC, Tyring SK.: Human papillomaviruses - clinical and scientific advances. Arnold, 2001.
  11. Srinivasan M, Sedmak D, Scott J. Effect of Fixatives and Tissue Processing on the Content and Integrity of Nucleic Acids. Am J Pathol. 2002;161:1961-1971