Nigerian Biomedical Science Journal
kaede niiyama japan wife plays nasty along her hubby.fapfapita.com tubegalore jamie shows off her asshole.porn xxx

Overview of Molecular Analysis of Cervical Cancer.

TheTao

Ajileye A. B.

Department of Biomedical Laboratory Science, College of Medicine, University of Ibadan. ayobless05@gmail.com

Esan E. O.

Department of Medical Laboratory Services, State General Hospital, Okitipupa, Ondo State.

Adeyemi O. A.

Department of Medical Laboratory Science, College of Medicine and Health Sciences, Afe Babalola University,

Ado Ekiti, Ekiti State. olkems145@gmail.com

Alade D. T.,

Department of Medical Laboratory Services, Ladoke Akintola University of Technology Teaching Hospital, Osogbo, Osun State. dupe401@gmail.com

All correspondence to: Ajileye A. B. Department of Biomedical Laboratory Science, College of Medicine, University of Ibadan. ayobless05@gmail.com

ABSTRACT

Cervical cancer is a type of cancer that develops in a woman’s cervix, it’s characterized by the growth of abnormal cells in the cervix. The main cause of cervical cancer is Human Papillomavirus (HPV). Others include; smoking, multiple sexual partners, use of oral contraceptives etc. Signs and symptoms include; increased vaginal discharge, pelvic pain, bleeding or pain after sexual intercourse etc. Screening tests include; Pap test, HPV test, Visual inspection by lugol’s iodine and acetic acid. The sample for screening is collected from the cervix using a cytobrush, while the patient lies in a lithotomy position. Further cytological tests are carried out to diagnose cervical cancer after screening like cone biopsy, colposcopy and pap smears. However these cytological tests have low specificity and significant variability in the diagnosis of cervical dysplastic lesions. Recently, the use of molecular-biology methods to detect the presence of Human Papillomavirus (HPV) has been employed. The molecular methods are more specific than the cytological methods. After the samples are collected, the DNAs are extracted using QIAamp DNA kit. The methods for detecting HPV are – Nucleic acid hybridization assays, Signal amplification assay, Nucleic-acids amplification assay. Molecular techniques are most commonly used for HPV testing, and are the gold standard for diagnosing this viral infection. In spite of their value, molecular techniques still must become more rapid, automated, and low-cost to be of practical use in low-income populations and countries.

KEYWORDS: Cervical-Cancer, Papillomavirus, Lithotomy, Molecular, Hybridization.

INTRODUCTION

Cervical growth is a malignancy emerging from the cervix. It is because of the irregular development of cells that can attack or spread to different parts of the body. From the get-go, commonly no indications are seen1; later indications may incorporate unusual vaginal bleeding, pelvic pain, or torment amid sex. While bleeding after sex may not be serious, it may also indicate the presence of cervical cancer1. Human Papilloma Virus (HPV) as an important cause of cervical cancer, based on their association with cervical cancer and precursor lesions, HPVs can also be grouped to high-risk and low-risk HPV types. Low-risk HPV types include types 6, 11, 42, 43, and 44. High-risk HPV types include types 16, 18, 31, 33, 34, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 702. Included in the high-risk group are some HPV types that are less frequently found in cancers but are often found in squamous intraepithelial lesions (SILs). Some authors refer to these HPV types as intermediate-risk. Low-risk subtypes are also occasionally found in cervical carcinomas2&3. HPV DNA testing may reduce costs by triaging patients into appropriate management strategies and reducing unnecessary colposcopy and less frequent screening in low-risk patients5.

Fig 1. ANATOMY OF THE CERVIX3

“The cervix is the lower part of the uterus in the human female reproductive system. The opening into the uterus is called the internal OS, and the opening into the vagina is called external os. The lower part of the cervix is known as the vaginal portion of the cervix or ectocervix, bulges into the top of the vagina” 5.

The cervical canal is lined with a single layer column shaped cells, while the ectocervix is covered with multiple layers of cells topped with flat cells. The two types of epithelia meet at the squamocolumnar junction. Infection with the human papillomavirus (HPV) can cause changes in the epithelium, which can lead to cancer of the cervix5.

Risk factors for contracting cervical cancer include

  1. Human Papilloma Virus (HPV)
  2. Smoking
  3. Having a weakened immune system e.g HIV
  4. Having more than one sexual partner
  5. Using oral contraceptives for a long time6.

SIGNS AND SYMPTOMS OF CERVICAL CANCER

  1. Bleeding that occurs between regular menstrual periods.
  2. Bleeding after sexual intercourse.
  3. Menstrual periods that last longer and are heavier than before.
  4. Pelvic pain.
  5. Bleeding after going through menopause.
  6. Increased vaginal discharge7.

Cervical screening is the process of detecting and removing abnormal tissue or cells in the cervix before cervical cancer develops. By aiming to detect and treat cervical neoplasia early on, cervical cancer. Several screening methods for cervical cancer include:

  1. Pap test ( also known as Pap smear or conventional cytology)
  2. Liquid-based cytology
  3. The HPV DNA testing
  4. The visual inspection with acetic acid and lugols iodine8.

Pap test is a method of examining with a microscope a sample of superficial cells that line the inner wall of the uterine cervix to detect any abnormal cell for early diagnosis of uterine cancer9. The position for taking the pap. smear from the patient is known as lithotomy10. After the sample is collected from the patient, smears are made on clean slides, fixed immediately in 95% alcohol and then stain with Papanicolaou stain, mounted and then view under a microscope for analysis10.

Fig 2.0: A Lithotomy Position, Showing how Cervical Samples are Collected10

Colposcopy

A colposcopy is a special way of looking at the cervix. It uses a light and low powered microscope to make the cervix appear much larger11. It helps in the diagnosis of premalignant and early malignant changes in the cervix 12.

Cone Biopsy

A cone biopsy is a small operation to remove a cone shaped piece of tissue from the cervix and examined under a microscope11. Cone biopsy removes abnormal tissue that is high in the cervical canal. Pap test and liquid-based cytology have been effective in diminishing incidence and mortality rates of cervical cancer in developed countries but not developing countries11. Prospective screening methods that can be used in low-resource areas in the developing countries are the HPV DNA and the visual inspection8.

Conventional Cytology

In the conventional Pap smear, the cells collected are smeared on a microscopic slide and its fixed in a cytological fixative. The slide is then sent to a laboratory for evaluation. The report for the accuracy of conventional cytology shows 94% specificity and 72% sensitivity13.

Liquid Based Cytology

Liquid based cytology is a technique that enables cells to be suspended in a monolayer and thus making better morphological assessment possible with improved sensitivity and specificity10. Liquid-based cytology is a method of preparing samples for examination in cytopathology. The sample is collected, normally by a small brush, in the same way as for a conventional smear test, but rather than the smear being transferred directly to a microscope slide, the sample is deposited into a small bottle of preservative liquid. At the laboratory the liquid is treated to remove other elements such as mucus before a layer of cells is placed on a slide. The technique allows more accurate results13. For many years, efforts have been made to develop methods that would enhance the sensitivity and specificity of the Papanicolaou smear. Emphasis has been placed on creating automated screening machines whose success depends on a representative sampling of cells on standardized slides containing a monolayer of well-stained, well-preserved cells.

From recent research and development, liquid-based preparations outperform conventional smears because of improved fixation, decreased obscuring factors, and standardization of cell transfer. Proponents point out that, in direct smears, the cells are not transferred in a representative fashion and that up to 90% of the material scraped from the cervix may be discarded with the sampling device. With liquid-based collection, the sampling will be representative and operator-dependent variation will not occur since processing is controlled by the laboratory13.

Methods of Carrying Out liquid based cytology

There are several systems that are currently available. The most widely used are:

  1. Sure Path (autocyte, TriPath Imaging)
  2. Thin Prep (CYTYC)

The SurePath method, the sample is vortexed, strained, layered onto a density gradient, and centrifuged. Instruments required are a computer-controlled robotic pipette and a centrifuge. The cells form a circle 12.5 mm in diameter.

The ThinPrep method requires an instrument and special polycarbonate filters. After the instrument immerses the filter into the vial, the filter is rotated to homogenize the sample. Cells are collected on the surface of the filter when a vacuum is applied. The filter is then pressed against a slide to transfer the cells into a 20 mm diameter circle.

Both methods result in a well-preserved approximate monolayer of cells, with a background devoid of blood and mucus. Other methods not commonly being used in liquid based cytology include:

  1. The cytoscreen method
  2. The Labonard Easy Prep

The cytoscreen method is a manual process relying upon photometry to evaluate the cellularity of the cell suspension prior to centrifugation onto a glass slide.

The Labonard Easy Prep is another manual method, whereby an aliquot of sample fluid is loaded into a separation chamber attached to a glass slide, which contains absorbent Paper. The cells settle in a thin layer and the preparation is stained using normal laboratory procedures.

The Human Papilloma Virus (HPV) DNA Test

The test is done by looking for pieces of the DNA of the HPV carcinogenic genotypes in cervical cells. The test can be done at the same time as the Pap test, with the same swab or a second swab. The HPV DNA test is most often done and used in 2 situations:

Visual Inspection Methods

Visual inspection of the cervix after application of Lugol’s iodine, the first method used for cervical cancer screening, was introduced in the 1930s by Schiller. However, Schiller’s test has poor specificity and was almost replaced with the advent of cervical cytology.

Current cervical cancer screening protocols typically include a combination of cervical cytology and human papillomavirus testing. Visual inspection of the cervix has re-emerged as a screening tool for low-resource settings, despite its limited specificity, since it is economical and provides immediate results. Visual inspection can be performed with acetic acid (VIA) or Visual inspection with Lugol’s iodine (VILI).

Visual inspection is indicated for women for whom cervical cancer screening is recommended and for whom these methods are the best screening option i.e women who do not have access to cervical cytology and human papillomavirus testing.

There are no absolute contraindications to visual inspection of the cervix. Visual inspection with acetic acid (VIA), rather than visual inspection with Lugol’s iodine (VILI), should be performed in women with an allergy to iodine. Visual inspection can be performed during pregnancy, but cervical biopsies are relatively contraindicated in pregnant women unless invasive cancer is suspected16.

Molecular Methods over Cytology Methods in the Diagnosis Of Cervical Cancer

Although traditional cytology still has a place in the modern clinical laboratory, it is now starting to make way for techniques that utilizes the increased resolution, accuracy and speed offered by the molecular revolution16. Microarrays, next generation sequencing, and advances in automation all have the potential to further improve the accuracy and reliability of clinical research and diagnosis, and may eventually replace microscope-based methods16.

To date, HPV cannot be cultured in vitro, and immunological tests are inadequate to determine the presence of HPV cervical infection. Indirect evidence of anogenital HPV infection can be obtained through physical examination and by the presence of characteristic cellular changes associated with viral replication in Pap smear or biopsy specimens15. Alternatively, biopsies can be analyzed by nucleic acid hybridization to directly detect the presence of HPV DNA17.

Most adults have been infected with HPV at some time. An infection may go away on its own. But sometimes it can cause genital warts or lead to cervical cancer. That’s why it’s important for women to have regular Pap tests. Pap test can find changes in cervical cells before they turn malignant18. If you treat these cell changes, you may prevent cervical cancer.

Cytology-based nation-wide cervical screening has led to a substantial reduction of the incidence of cervical cancer in western countries. However, the sensitivity of cytology for the detection of high-grade precursor lesions or cervical

cancer is limited; therefore, repeated testing is necessary to achieve a very effective result. In addition to that, adenocarcinomas and its precursors are often missed by cytology19.

Consequently, there is need for a better screening test. The insight that infection with high risk human papillomavirus (hrHPV) is the causal agent of cervical cancer and its precursors has led to the development of molecular tests for the detection of high risk human papilloma virus (hrPV)19. Strong evidence now supports the use of hrHPV testing in the prevention of cervical cancer20.

From a clinical point of view, testing for hrHPV is only useful when a positive hrHPV test result is informative about the presence or absence of CIN2+ (clinical sensitivity and specificity). Thus, in order to prevent excessive follow-up procedures for women with transient hrHPV infections or hrHPV-positive women without cervical lesions, candidate hrHPV tests to be used for cervical screening should be clinically validated19.

Pathogenesis

Transmission of HPV occurs primarily by skin-to-skin contact21. Basal cells of stratified squamous epithelium may be infected by HPV. Other cells types appear to be relatively resistant. It is assumed that the HPV replication cycle begins with entry of the virus into the cells of the basal layer of the epithelium19.

It is likely that HPV infection of the basal layer requires mild abrasion or microtrauma of the epidermis. Once inside the host cell, HPV DNA replicates progress to the surface of the epithelium. In the basal layer, viral replication is considered to be non-productive, and the virus establishes itself as a low-copy-number episome by using the host DNA replication machinery to synthesize its DNA on average once per cell cycle . In the differenciated keratinocytes of the suprabasallayer of the epithelium, the virus switches to a rolling-circle mode of DNA replication, amplifies its DNA to high copy number, synthesizes capsid proteins, and causes viral assembly21.

Molecular biology-based techniques

The human papillomavirus (HPV) is the causative agent of cervical cancer, but not all genotypes of HPV are causal factors, some cause genital warts. Out of the 100-200 different HPV genotypes, the human papillomavirus (HPV) genotypes most frequently indicated as the causal factor of cervical cancer are HPV16, 18, 31, 33, 35, 45, 52 and 58 which can also be reffered to as the high risk factor of Human Papilloma virus. The Pap smear unquestionably is a successful screening test for cervical cancer. However, recent advances in technology have raised questions regarding whether the conventional Pap smear is still the standard of care22. HPV cannot be propagated in tissue culture, and therefore, in most cases its accurate identification relies on molecular biology techniques. With a double-stranded DNA genome of about 8000 base pairs (bp) and a well-known physical structure and gene organization, the tests of choice for detecting HPV in clinical specimens are based on nucleic probe technology23. The six main possible clinical applications of HPV DNA testing are:

  1. triage of women with equivocal or low-grade cytological abnormalities;
  2. follow-up of women with abnormal screening results who are negative at colposcopy/biopsy;
  3. prediction of the therapeutic outcome after treatment of cervical intraepithelial neoplasia (CIN);
  4. primary screening for HPV DNA testing, alone or in combination with a Pap smear, to detect cervical-cancer precursors.
  5. gain valuable information on the persistence of certain HPV types
  6. Investigation of regional and country-based prevalence of type-specific HPV, to provide baseline values against which the global impact of HPV vaccination can be assessed in the future.

Extraction of DNA from Cervical Smears or Tissue For Molecular Analysis

To carry out the molecular detection of HPV, the patient sits in a litothomy position, a sterile speculum is used to dilate the cervix and then with the use of a sterile swab or with the use of a cytobrush, cervical samples are collected at the squamo-columnar junction, the smears must be fixed immediately in a cytological fixative and thereafter, DNA extraction can be made and then used to analyse cervical cancer molecularly. DNA extraction can also be obtained from Cone biopsy formalin-fixed paraffin-embedded samples for the molecular analysis of cervical cancer. DNA extraction is carried out using;

QIAamp DNA kits – these kits use vacuum procedures or fast spin-column. The DNA binds to the silica-gel membrane while contaminants pass through. The DNA is purified after two efficient wash steps (that removes PCR inhibitors). The DNA is left to be eluted23.

The presence of HPV can be inferred from morphological, serological and clinical findings. However, HPV diagnosis relies on molecular-biology techniques that allow its accurate detection and typing24. These molecular-biology techniques are;

  1. Nucleic acid-hybridization assays
  2. Signal-amplification assays
  3. Nucleic-acid amplification.

Nucleic-Acid Hybridization Assays This has 3 techniques;

These techniques use radio-labelled nucleic acid hybridization assays to detect HPV infection in cervical samples. Although these techniques generated high-quality information, the disadvantages of these direct-probe approaches include low sensitivity, the need for relatively large amounts of purified DNA, and time-consuming procedures23. The southern blot is the gold standard for HPV genomic analysis; it’s a very good technique in the analysis of the presence of HPV in association with their morphology. But the disadvantage of this southern blot technique is that it is time consuming and with a low sensitivity. Southern blot and hybridization cannot use degared DNA25.

B. Signal-Amplification Assays

This has 2 techniques;

Hybrid Capture 2

The Hybrid Capture 2 system is a non-radioactive signal-amplification method based on the hybridization of the target HPV-DNA to labeled RNA probes in solution17. This test detects 13 HR-HPV types (-16,-18,-31,-33,-35,-39,-45,-51,-52,-56,-58,-59 and -68) or 5 LR-types (-6, -11, -42, -43, and -44)17.

This assay distinguishes between HR and LR groups, but was not designed for genotyping single HPV (Cuzick et al, 2008). This is a significant finding, since with persistent infection the risk of a precancerous lesion is between 10 and 15% with HPV types -16/18, and below 3% for all other HR types combined. Therefore, HPV genotyping is very important to identify single oncogenic HPV types and to provide more information regarding risk-stratification as well as persistence of infection 26.

Cervista HPV

The Cervista HPV detects the presence of 14 HR-HPV types, consisting of -16,-18,-31,-33,-35,-39,-45,-51,-52,-56,-58,-59,-66 and -6826. This assay also utilizes a signal-amplification method for the detection of specific nucleic acids.

In comparison with HC2, the Cervista assay demonstrated 100% sensitivity in the detection of CIN III and 98% sensitivity in the detection of CIN II. In addition, this assay showed a lower false-positive rate, and high sensitivity and specificity to genotyping HPV -16/1826.

Nucleic Acid-Amplification Methods Microarray analysis

This method uses probe amplification, the PCR (Polymerase chain reaction) product is hybridized onto a chip, and after a washing step, hybridized signals are visualized with a DNA chip scanner. The microarray-based automated techniques allow for parallel analysis of multiple DNA samples. At present, the two major applications of DNA microarrays are gene-expression profiling and mutation analysis16.

Some studies have demonstrated that DNA microarray analysis coupled with PCR can be successfully applied to detection and genotyping of the HPV. The HPV DNA chip showed higher sensitivity and specificity than gel electrophoresis, and in some cases they produce better results than direct DNA sequencing20.

PapilloCheck®

This assay detects and genotypes 24 HPV types in a single reaction (HPV -6, -11, -16, -18, -31, -33, -35, -39, -40, -42, – 43, -44, -45, -51, -52, -53, -55, -56, -58, -59, -66, -68, -70, – 73, and -82). The assay uses a multiplex PCR with fluorescent primers to amplify a 350 bp fragment of the E1 gene of HPV, comprising 28 probes, each in 5 replicate spots fixed on a DNA chip. Co-amplification of the human ADAT1 gene is used as internal control. The hybridization is performed on a microarray chip, which is automatically scanned and analyzed using the CheckScanner™ at both 532 and 635 nm, and the Check-Report™ software, respectively27.

The main advantage of the PapilloCheck® assay (Greiner Bio-One GmbH, Frickenhausen, Germany) is HR/LR-HPV identification, and detection of multiple infections, and may be considered a reliable screening test. However, this assay does not amplify HPV -35 and -53, the cost is still relatively high, and it requires specific apparatus27.

Polymerase chain reaction (PCR)

The PCR-based techniques are highly sensitive, specific, and widely used. In a conventional PCR, the thermostable DNA polymerase recognizes and extends a pair of oligonucleotide primers that flank the region of interest. In the final process, the PCR can generate one billion copies from a single double-stranded DNA molecule after 30 cycles of amplification28.

The HPV-PCR protocols use consensus primers such as PGMY09/PGMY1 and GP5+/GP6+, which allow amplification of a large number of HPV genotypes in a single reaction. The primers target conserved regions of the HPV genome, such as the L1 capsid gene. After amplification, the HPV genotypes can be determined separately, using techniques such as restriction-fragment length polymorphism (RFLP), linear probe assays, direct sequencing, or genotype-specific primers. Some researchers have used a type-specific PCR, with primers that amplify the long control region L1 and E6/E729.

These PCR techniques also have some drawbacks, mainly in competition for reagents, leading to false negative results for multiple type infections that are contained in samples at lower copy numbers. Because of this problem, the PCR method may not detect all the HPV genotypes that are present in the sample. Another downside is that multiple infections are not uncommon28. Amplification of samples containing DNA from more than one HPV genotype can lead to a much stronger amplification of one of the sequences present, which would complicate the detection of all genotypes in a sample with multiple infections. Sometimes, additional, labor-intensive procedures, such as sequencing or type-specific PCR, are required28.

PCR-RFLP

Genotyping by PCR-RFLP allows the HPV to be typed, and is easier and less expensive than sequencing. The method is simple and robust, does not require sophisticated equipment, and is particularly suited to settings in which financial resources are limited27. PCR-RFLP shows good discriminatory power by differentiating the virus in HR or LR, and it is possible to identify single or multiple infections. In this technique, the amplified DNA is digested by restriction enzymes, resulting in DNA fragments of various lengths. The commonest restriction enzymes are BamHI, Dd6eI, HaeIII, HinfI, PstI and RsaI. However, Santiago et al.,30 used a single restriction enzyme, HpyCH4V, to detect 21 HR- and 31 LR-HPV genotypes 27.

Real-time PCR

This assay is a reliable, sensitive, and specific diagnostic tool for detection and genotyping of targeted HPV genotypes in tissue specimens31 and cellular samples. The advantages of this method are: (i) ability to detect viral load; (ii) with the use of different fluorochromes that emit fluorescence, as the PCR reaction proceeds, the reactions can be performed in multiples and can amplify different nucleic-acid targets; (iii) nucleic acids can be detected even

in a very small concentrations, using a 7-log dynamic range to extrapolate the viral load/concentration over the standard curve; and finally, (iv) it is extremely reproducible, rapid, and applicable to clinical samples32.

Abbott real-time PCR

The Abbott Real-Time HR-HPV test is a novel assay based on concurrent individual genotyping for HPV-16 /18 and pooled detection of 12 HPV genotypes: 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 6823.

COBAS® 4800 HPV test

This test features automated sample preparation combined with Real-Time PCR technology to detect 14 HR-HPV. The PCR amplification and detection occur in a single tube, (i) HPV -16, (ii) HPV -18, (iii) 12 HR (-31, -33, -35, – 39, -45, -51, -52, -56, -58, -59, -66, and -68) as a pool, and

  1. β-globin as the control for extraction and amplification adequacy14.

The agreement between COBAS® 4800 (Roche Molecular Systems, Pleasanton, CA, USA) and Real-Time PCR was strong in a study that determined the reproducibility, involving a sequence of several consecutive steps, both intra- and inter-laboratory. The assay is easy to use because it is adapted for primary specimens, and the results can be obtained approximately 4 hours after the sample is received. COBAS® 4800 fulfils all requirements as defined in the international guidelines to consider it clinically validated for screening, and is reliable in the detection of HR-HPV. This test has been clinically validated for ASC-US triage32.

HPV genome sequencing

The dideoxy chain-termination technique (Sanger technique) was first described for genome sequencing more than three decades ago. Fluorescently labeled nucleotides were incorporated into Sanger sequencing, and advances have led to increasing expansion and development of high-quality, thorough sequencing20. However, it has not been validated for clinical use. Similar to dideoxy sequencing methods, pyrosequencing is applicable to any source of DNA or RNA that can be amplified by PCR (blood, saliva, cell line, plasma, serum, tissue, formalin-fixed paraffin-embedded samples, and whole genome-amplified DNA). The method is based on the detection of the pyrophosphate released during DNA synthesis, and has many advantages over dideoxy sequencing for a wide range of applications that require short-to medium-sequence stretches. The primary advantage is simplicity: the readout sequence itself is obtained, rather than a fluorescent signal that must be converted to a sequence. Secondly, it is faster and less expensive: savings result from its sequence-by-synthesis process where a DNA sequence is read in real time, and it is synthesized by addition of inexpensive, unlabeled nucleotides; and finally, the method is uniquely quantitative22.

CLART® human papillomavirus 2

The CLART® Human Papillomavirus 2 (Genomica, Madrid, Spain) methodology uses biotinylated primers that amplify a 450 bp fragment within the HPV L1 region. Co-amplification of an 892 bp region of the FTR gene and a 1.202 bp fragment of a transformed plasmid provides a control to ensure DNA extraction adequacy and PCR efficiency. Amplicons are detected by hybridization in a low-density microarray containing triplicate DNA probes specific for 35 HPV (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 68, 70, 71, 72, 73, 81, 82, 83, 84, 85 and 89). Semi-quantitative results can be obtained in an automatic reader with highly comparable outcomes, showing excellent sensitivity, specificity, and reproducibility33.

INNO-LiPA

This assay genotypes all 14 HPV that are covered by Real-Time21. INNO-LiPA (LiPA HBV GT; Innogenetics N.V., Ghent, Belgium) is based on the co-amplification of the 65 bp region of the HPV L1 gene and the 270 bp of the human HLA-DP1 gene using SPF10 biotinylated primers, followed by genotyping. Some carcinogenic genotypes such as HPV 35, 39, 52, 56 and 66 were not covered by this method, and it was found to be the least effective genotyping for HPV 42 and 5916.

Although the majority of nucleic-acid amplification methods can reliably detect HPV in cervical-swab specimens, only a few, including Real-Time PCR, are potentially suitable for archival clinical specimens, since they target a relatively small portion of the HPV genome (less than 160 bp). Therefore, the observed differences in internal control amplification efficacy between Real-Time and INNO-LiPA can be attributed most reasonably to the differences in target amplicon length: 136 bp vs. 270 bp, respectively. This kit can be also used on samples taken with swabs, brushes, tampons, and lavage16.

The Linear array®

The Linear Array® HPV Genotyping (Roche Molecular Diagnostics, Pleasanton, CA, USA) is a PCR-based assay coupled with a reversed line blot hybridization. This assay allows the discrimination of 36 HPV, including 15 HR (-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -68, -73 and -82), 3 probable HR (-26, -53 and -66), 10 LR (-6, -11, – 40, -42, -54, -61, -70,-72, -81 and -CP6108) and 9 genotypes for which the risk is still undetermined (-55, -62, -64, -67, -69, -71, -83, -84 and -IS39)34.

The test uses biotinylated PGMY09/11 primers to amplify a 450 bp fragment within the polymorphic L1 region of the HPV genome. Co-amplification of the 268 bp region of the human β-globin gene provides a control to ensure DNA extraction adequacy and PCR efficiency. The hybridization and detection of the amplified product are performed with the Auto – LIPA™ instrument (Innogenetics, Ghent, Belgium), which can process up to 30 strips simultaneously in a perfectly standardized fashion. Colored signals on the strips are read by the naked eye and interpreted according to the Linear Array® reference guide. Equivocal results can be obtained for HPV 52 when HPV 33, 35 or 58 are also present, because it is detected through a cross-hybridization probe for these 4 HPV types. An additional, specific probe is present on the strip to confirm the detection of HPV 33, 35 and 58, but not of HPV 5234.

Clinical arrays® HPV

This kit (Genomica SAU, Madrid, Spain) allows the detection and genotyping of HPV. The DNA extraction method is a modified procedure using absorption columns. The kit employs biotinylated primers to define a sequence of 451 nucleotides within the polymorphic L1 region of the HPV genome. A human cystic-fibrosis transmembrane conductance regulator (CFTR) gene and control plasmids are used in order to check both the PCR procedure and the integrity of the DNA24. This also allows the detection of the 35 genotypes that are individually associated with HR ( 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 and 85) or LR-HPV ( 6, 11, 40, 42, 43, 44, 54, 61, 62, 71, 72, 81, 83, 84 and 89). It is possible to identify simple infections or co-infections24.

Microplate colorimetric hybridization assay (MCHA)

The MCHA (Boehringer Mannheim, Germany) is a method for identifying six HR-HPV ( 16, 18, 31, 33, 39 and 45) and is based on the amplification by PCR of the 150 bp fragment within the L1 region by consensus primers GP5+/6+, followed by colorimetric hybridization to six type-specific probes on microwell plates (Immobilizer™ Amino Surface, Nunc, Roskilde, Denmark)22.

The MCHA showed very good agreement with PapilloCheck® for HPV 31, 33, 45 and higher sensitivity in identifying HPV 16 and 18, but poor agreement for HPV

To improve MCHA for detection of other genotypes, probes for HPV 35, 52, 56 and 58 should be included22.

Conclusion

Cervical cancer develops over a long period, through precursor lesions that may regress spontaneously without treatment. The challenge of cytological screening is to detect the lesions that have a high risk of progression. Consequently, various biomarkers associated with the risk of progression of this cancer have been investigated, and most are associated with high risk-HPV. Molecular techniques are most commonly used for HPV testing, and are the gold standard for diagnosing this viral infection. In spite of their value, molecular techniques still must become more rapid, automated, and low-cost to be of practical use in low-income populations and countries.

REFERENCES

  1. Ali C.I (2016). Cervical cancer: a health limiting condition. Gynecological Obstetrician, 6:378.
  2. Bosch, F., M. M. Manos, N. Munoz, M. Sherman, A. M. Jansen, J. Peto, M. H. Schiffman, V. Moreno, R. Kurman, K. V. Shah, and International Biological Study on Cervical Cancer (IBSCC) Study Group (1995). Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. J. Natl. Cancer Inst. 87:796-802.
  3. Eileen M. Burd (2003). Human Papillomavirus and Cervical Cancer. Clin. Microbiol Rev. 16(1): 1–17.
  4. Khan, M. J., Castle, P. E. and Lorincz, A. T. (2005). The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J. Natl Cancer Inst. 97: 1072- 1079.
  5. Martini, F. H., Timmons, M. J. and Tallitsch, R. B. (2012). Human Anatomy (7th edition). San Francisco:Pearson Benjamin Cummings. Pp. 200-270.
  6. David, C. W. and Marluce, B. (2008). Comprehensive Cytopathology (Third Edition). Pp. 1021 – 1042.
  7. Nainakshi, K., Nadiya K., Sukhpal K. and Sandhya, G. (2019). Risk Factors of Cervical Cancer: A Case-Control Study. Asia Pac J Oncol Nurs. 6(3): 308–314.
  8. Ronco G, Giorgi-Rossi P, Carozzi F, Confortini M, (2010). Ef cacy of human papillomavirus testing for the detection of invasive cervical cancers and cervical intraepithelial neoplasia: a randomised controlled trial. Lancet. Oncol. 11: 249-257.
  9. Mehta, V., Vasanth, V. and Balachandran, C. (2009). Pap smear. Indian J Dermatol Venereol Leprol. 75:214-216.
  10. Moyer, V. A. (2012). Screening for cervical cancer: U.S. Preventive Services Task Force recommendation statement. Ann Intern Med. 156(12):880-891.
  11. Aboubakr, Elnashar. (2014). Screening for cervical cancer: U.S. Preventive Services Task Force recommendation statement. Annals of Internal Medicine., 156 12:880–891.
  12. Castle, P. E., Fetterman, B., Thomas, C. J., Shaber, R., Poitras, N., Lorey, T. and Kinney, W. (2010). The Age-Specific Relationships of Abnormal Cytology and Human Papillomavirus DNA Results to the Risk of Cervical Precancer and Cancer. Obstetrics & Gynecology. 116(1):76-84.
  13. Randall, K. G. and Mark, G. M. (2011). The Impact of Liquid-Based Cytology in Decreasing the Incidence of Cervical Cancer. Rev Obstet Gynecol. 4(1): 02–11.
  14. Zaravinos, A., Mammas, I.N., Sourvinos, G., Spandidos, D.A. (2009). Molecular detection methods of human papillomavirus (HPV). Int Journal of Biological Markers. 24: 215-222.
  15. Cytomolecular Diagnosis of cancers. Annals o f Oncology. 25: 927–935.Arbyn, M., Castellsague, X., de Sanjose, S., Bruni, L., Saraiya, M., Bray, F. (2011). Worldwide burden of cervical cancer. Annals on Oncology. 12: 2675–2686.
  16. Hoheisel, J.D., Diaz, F. (2012). Microarray technology: beyond transcript profiling and genotype analysis. Nat Rev Genet. 7: 200-210.
  17. Rahman, M., Sasagawa, T., Yamada, R., Kingoro, A., Ichimura, H., Makinoda, S. (2011). High prevalence of intermediate-risk human papillomavirus infection in uterine cervices of kenyan women infected with human immunodeficiency virus. Journal of MedicalVirology. 83:1988-1996
  18. Pannier-Stockman, C., Segard, C., Bennamar, S., Gondry, J., Boulanger, J. C. and Sevestre, H, (2008). Prevalence of HPV genotypes determined by PCR and DNA sequencing in cervical specimens from French women with or without abnormalities. Journal of Clinical Virology. 42: 353-360.
  19. Dijkstra, P. F., Snijders, M., Arbyn, H. T. (2014).
  20. Bruni, L., Albero, G., Aldea, M., Serrano, B., Valencia, S., Brotons, M., Mena, M., Cosano, R., Munoz, J., Bosch, F.X., de Sanjosé, S., Castellsagué, X. (2014). ICO Information Centre on HPV and Cancer (HPV Information Centre). Human Papillomavirus and Related Diseases in the World. Summary report. 14:12–18.
  21. Cuzick, J., Arbyn, M., Sankaranarayanan, R., Tsu, V., Ronco,G., Mayrand,M. H. (2008). Overview of human papillomavirus-based and other novel options for cervical cancer screening in developed and developing countries. Int. Journal of Med. Science. 29-41.
  22. Hwang, S. J. and Shroyer, K. R. (2012). Biomarkers of cervical dysplasia and carcinoma. Journal of Oncology. 50: 72-86.
  23. Villa, L. L. and Denny, L. (2006). Methods for detection of HPV infection and its clinical utility. International Journal of Gynaecology & Obstertrics 71-80.
  24. Shen-Gunther, J. and Yu, X. (2011). HPV molecular assays: defining analytical and clinical performance characteristics for cervical cytology specimens. Gynecol Oncology. 123: 263-271.
  25. Gradíssimo, A. and Burk, R. D. (2917). Molecular tests potentially improving HPV screening and genotyping for cervical cancer prevention. Expert Rev. Mol. Diagn. 17(4):379–391.
  26. El-Khatib Z, Tota JE, Kaufmann AM. Progress on human papillomavirus (HPV) infection and cervical cancer prevention in sub-Saharan Africa: highlights of the 27th International Papillomavirus Conference in Berlin, 17-22 September 2011. J Epidemiol Global Health. 2012;2(2):99–102. doi: 10.1016/j.jegh. 2012.04.001. [PubMed] [Cross Ref]
  27. Didelot, M. N., Boulle, N., Damay , A., Costes, V., Segondy, M. (2011). Comparison of the Papillo Check assay with the digene HC2 HPV DNA assay for the detection of 13 high-risk human papillomaviruses in cervical and anal scrapes. Journal of Medical Virology. 83: 1377-1382.
  28. Santos, G., and Saieg, M. A. (2016). Molecular Techniques and methods applied in cytology. In: Yang B, Rao J, editors. Molecular Cytopathology. Switzerland: Springer. Pp. 17–26.
  29. Van-Ballegooijen, M., van-den, Akker-van, Marle, M. E., Warmerdam, P. G., Meijer, C. J., Walboomers, J. M., Habbema, J. D. (1997). Present evidence on the value of HPV testing for cervical cancer screening: a model-based exploration of the (cost-) effectiveness. Br J Cancer. 76 (5): 651–657.
  30. Santiago, E., Camacho, L., Junquera, M. L. and Vázquez, F. (2006). Full HPV typing by a single restriction enzyme. J Clin Virol. 37: 38-46.
  31. Bozzetti, M., Nonnenmacher, B., Mielzinska, I. I., Villa, L. Lorincz, A. and Breitenbach, V. V. (2000). Comparison between hybrid capture II and polymerase chain reaction results among women at low risk for cervical cancer. Annals on Epidemiology. 10: 466-476.
  32. Barcellos, R.B., Almeida, S.E., Sperhacke, R.D., Verza, M., Rosso, F. and Medeiros, R. M. (2011). Evaluation of a novel microplate colorimetric hybridization genotyping assay for human papillomavirus. Journal of Virology Methods. 77: 38-43.
  33. Kocjan, B.J., Seme, K., Poljak, M. (2011). Comparison of the Abbott Real Time High Risk HPVtest and INNO-LiPA HPV Genotyping Extra test for the detection of human papillomaviruses in formalin-fixed, paraffin – embedded cervical cancer specimens. Journal of Virological Methods. 175: 117-119
  34. Snijders, P. J., Heideman, D. A., Meijer, C. J. (2010). Methods for HPV detection in exfoliated cell and tissue specimens, APMIS. 118: 520-528.

 

first time fuck buds.free sex
hot indian college girl fucked by her bf on building roof. https://www.xxvideos.one
http://www.smellslikeafish.com