HPV typing
Related Terms
Amplicor? HPV test, cervical cancer, cervical dysplasia, chemiluminescence, DNA microarray, DNA probes, dot blot hybridization, electrophoresis, epidemiology, Gardasil?, genotyping, HC II, HIV, HPV microarray systems?, HPV vaccine, HPV viral load, human immunodeficiency virus, human papillomavirus, human papillomavirus typing, hybrid capture, Hybrid Capture IIT test, hybridization, in situ hybridization, LINEAR ARRAY? HPV genotyping test, Pap test, PCR, polymerase chain reaction, signal amplification technique, Southern blot hybridization, SPF10-INNO LiPA? genotyping test, target amplification technique, ThinPrep? Cytyc.
Background
Human papillomavirus (HPV): Papillomaviruses are a diverse group of deoxyribonucleic acid (DNA)-based viruses that infect humans and a wide variety of animals, including birds. HPV is transmitted from one person to another via bodily fluids, including blood, semen, vaginal secretions, and breast milk. It is one of the most common sexually transmitted diseases (STDs).
HPV types and infection: More than 100 different types of HPV have been identified. HPV has been classified based on similarities in genes, which generally correlate with three categories of infections seen clinically, namely, nongenital cutaneous disease, nongenital mucosal disease, and anogenital disease.
Nongenital cutaneous (skin) diseases are caused by certain types of HPV (e.g., types 1, 2, 4, 10, 26, etc.) that usually present as common warts found on the hands and feet. They usually appear as soft, moist, pink, or red swellings that grow quickly.
Nongenital mucosal diseases are caused by certain types of HPV (e.g., types 6, 11, 16, 18, 57, etc.) that infect the mucosal surfaces, most commonly in the mouth. Most often they may present without any symptoms, but may lead to changes in the tissue that may grow into a cancerous growth (precancerous condition). The mucosal surface is the moist membrane lining a body cavity such as the mouth, nose, etc.
Anogenital diseases are caused by certain types of HPV (e.g., 16, 18, 6, 11, 31, 34, 56, etc.) that infect the genital tissues and do not cause any symptoms, but may increase the risk of cancer of the cervix (the lower part of the womb or uterus) in women and other genital cancers in both women and men. These types of HPV have also been associated with the development of precancerous conditions, skin cancer, cancers of the mouth, and anal cancers.
HPV types may also be divided into low-risk and high-risk groups based on whether or not they carry a risk of progressing to cancer. The low-risk types of HPV typically cause genital warts and have a low risk of progressing to cancer. There are about 12 types of low-risk HPV, which are known by the numbers 6, 11, 40, 42, 43, 44, 53, 54, 61, 72, 73, and 81. Types 6 and 11 are the most common forms and cause about 90% of the genital warts. The high-risk types of HPV cause abnormal changes in the cells of the cervix that may progress to cervical cancer if not treated at an early stage.
High-risk types of HPV that cause cervical cancer do not cause any classical symptoms, but develop at later stages. The 13 types of high-risk HPV that are most concerning include: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. Types 16 and 18 are the most common and are associated with about 70% of cervical cancer cases.
HPV DNA typing: An HPV DNA typing test is performed to determine what type of HPV a person is infected with. HPV typing is essential so as to distinguish the low-risk from the high-risk types of HPV. Because cervical cancer is one of the most successfully treated cancers when it is detected early, prompt diagnosis aids in controlling the progression of cervical cancer.
The HPV-based vaccine (e.g., Gardasil?) is given to protect against the development of cervical cancer in women caused by certain types of HPV. A vaccine is an agent or a preparation, which when given, stimulates immune response against an infectious agent, in this case certain HPV infections. Thus, HPV typing is essential before giving the HPV-based vaccine against cervical cancer.
Indications: HPV DNA typing is usually performed in addition to a Pap test if a woman is sexually active, has symptoms of HPV infection (such as genital warts), or is age 30 or older. A Pap test or Pap smear is a simple procedure used to collect a sample of cervical cells. The samples are then analyzed to detect the possibility of cervical cancer.
The American College of Obstetricians and Gynecologists (ACOG) and The U.S. Food and Drug Administration (FDA) recommend that women 30 years or older be offered the HPV DNA test in addition to their Pap test. If the HPV DNA test and Pap test results are negative, ACOG and FDA recommends that the woman wait for three years before having another Pap test and HPV DNA test, if she does not have an underlying health condition such as human immunodeficiency virus (HIV) infection or immunosuppression (very low immunity).
HPV DNA typing is also indicated in women with minimally abnormal cells that may be indicative of cancerous changes and are detected through a Pap test (used to monitor their progression to cervical cancer or as a follow-up procedure in women who have undergone treatment for cervical dysplasia). Thus, HPV typing helps in evaluating the presence of any remaining dysplasia in the cervix.
HPV typing molecular assays: HPV DNA typing test is a method to determine what type of HPV a person is infected with so as to initiate suitable treatment. Several analysis techniques (assays) at the molecular (small particle) level are available for HPV typing. Some of the molecular assays for HPV DNA typing include the Hybrid Capture IIT test (Digene Corporation, Gaithersburg, MD), the Amplicor? HPV test, the LINEAR ARRAY? HPV genotyping test (Roche Molecular Systems, CA,), and the SPF0-INNO LiPA? HPV genotyping test (Innogenetics Group, Ghent, Belgium).
Methods
General: The human papillomavirus (HPV) causes HPV infections, which are sexually transmitted diseases (STDs). More than 50 types of HPV have been identified; some cause genital infections and have been associated with the development of cervical cancer in women. Detection of and distinction between different types of HPV aid in early diagnosis of cervical cancer, which is the most successfully treated cancer when it is detected early.
An HPV DNA typing test is performed to determine what type of HPV a person is infected with, so as to identify individual HPV types and distinguish the low-risk from the high-risk types of HPV. Usually, a sample of cervical cells is collected for HPV typing assays in conjunction with a Pap test.
Pap test: The Pap test is a simple procedure to collect cervical cells, which are analyzed to detect the possibility of cervical cancer. In this procedure, a brush or a spatula is used to scrape the surface of a woman's cervix to collect a sample of cervical cells. In the conventional Pap smear method, the sample is smeared onto a microscope slide, sprayed with a fixative (a fluid that firmly secures and preserves the cells), and sent to the laboratory for further analysis.
The liquid-based Pap test is a recently developed method that requires transfer of the cellular material from the brush or spatula into a preservative vial (bottle) to be processed at a laboratory for further analysis. Examples of liquid-based Pap tests are ThinPrep? (Cytyc, Boxborough, MA) and SurePathTM (BD Diagnostics, TriPath, NJ).
Liquid-based Pap tests are more sensitive and better than conventional Pap smears, as the cervical cells are not clumped together and cell characteristics are easier to observe. Another advantage is that the cervical cell features are not obscured by blood, mucus, or other cells. For HPV typing assays, the sample of cervical cells are collected in another vial with a special liquid preservative, from which the DNA is isolated or extracted for further analysis. HPV tests can also be done on male patients suspected of having an HPV infection, which may cause genital warts, or penile or anal cancer. Genital warts are growths on the penis, testicles, groin, thighs, or anus. An anal swab or brush specimen is used for testing.
HPV molecular typing techniques: The HPV DNA typing techniques are based on direct hybridization techniques with signal amplification, as well as techniques based on nucleic acid amplification (multiplication) such as polymerase chain reaction (PCR). Nucleic acid amplification techniques may be of two types: target amplification techniques, in which the assay amplifies the target nucleic acid sequence (e.g., the AMPLICOR? HPV test or LINEAR ARRAY? HPV genotyping test), and signal amplification techniques, in which the signal generated from each probe is increased by a compound probe or by branched-probe technology (e.g., hybrid capture technology). These have been described below.
Direct nucleic acid probe hybridization: Nucleic acids (e.g., DNA) are formed by long strands of nucleotides. Nucleotides are the building blocks of DNA and are made of nitrogen bases, sugars, and phosphate. Nitrogen bases are of two types in DNA: purines, such as adenine (A) and guanine (G), and pyrimidines, such as cytosine (C) and thymine (T). Probes are single-stranded DNA sequences with base pairs complementary to the target DNA segment, for example, the complementary strand for G-T-A-C is C-A-T-G. These probes are labeled (attached) with radioactive molecules for easy detection by autoradiography. Autoradiography is a technique where the probes labeled (attached) with radioactive molecules can be visualized upon exposure to X-rays. Hybridization is the process of the joining or bonding between the probe and the target DNA sequence, which allows the target molecule to be detected in a biological sample.
Some of the HPV typing by direct nucleic acid probe hybridization techniques include Southern blot hybridization, dot blot hybridization, and in situ hybridization. Generally, these methods are time-consuming, require trained technicians, demand many laboratory reagents (chemical substances in a solution) and equipment, and have low sensitivity. These techniques also require a large amount of DNA in biological samples, and they cannot be carried out when a very minimal quantity of sample is available. Thus, these techniques for HPV DNA typing are not applicable in a large-scale population screening analysis.
Southern blot hybridization is a research technique that is generally used to classify newly identified viral types. In this procedure, the genetic content of HPV (HPV DNA) from the sample is broken into fragments using enzymes (e.g., restriction endonucleases). The fragmented DNA is put on a gel electrophoresis, which uses an electrical current to separate the DNA fragments based on the size of each fragment and employs electric charges as they migrate through a gel matrix to form distinct bands. The separated DNA fragments are transferred to a nitrocellulose membrane, a thin layer formed by a mixture of pulpy or cotton-like polymers of cellulose (a form of glucose), nitric acid, and sulfuric acids, and hybridized (joined) with HPV probes labeled with radioactive molecules. The detection of the labeled DNA hybridization product is performed using autoradiography, indicating the presence of a specific type of HPV in the sample. Commercial kits are not marketed for this HPV typing method.
Dot blot is a simple laboratory technique for DNA detection, analysis, and identification, in which the DNA sample is dotted/spotted directly onto the nitrocellulose membrane and fixed (attached). This is followed by hybridization with probes that are complementary to the target DNA sequence. These probes are labeled with radioactive molecules for easy detection by autoradiography. The dot blot hybridization technique was formerly used in two commercial HPV detection kits, Virapap and Viratype. These kits, previously available through the Digene Corporation, are no longer marketed, as more advanced and efficient kits are available now.
In situ hybridization is a technique used to identify specific HPV DNA sequences while the sequences remain in their original location, i.e., within the cell. This technique is performed on a microscope slide and can be applied to archived cervical smears (old stored samples). The sample is processed to remove the cellular components other than the targeted DNA, and the specimen is heated to separate the DNA strands into single strands (a process called denaturation). Then the probes are introduced; they are hybridized (joined) with the target HPV DNA sequence if it is present in the sample. The hybridized complex is reacted with specific antibodies, which are attached to these probes. Antibodies are proteins produced during an immune response to a stimulus, such as a bacterium, virus, or something that is foreign to the body. This is followed by addition of enzymes, which react with the antibodies that are bound to DNA-probe hybrid complex. This reaction process causes staining of the antibodies, which can be seen under a microscope. Kreatech Biotechnology B.V. (Amsterdam, The Netherlands) has developed a detection kit based on this in situ hybridization technique and is available as a research tool. The kit can detect HPV types 1, 2, 6, 11, 16, 18, 31, and 33; however, it has not been yet approved for commercial use by the U.S. Food and Drug Administration (FDA).
Target amplification: The target amplification technique uses polymerase chain reaction (PCR) to detect and identify HPV types (examples of the technique include the AMPLICOR? HPV test and the LINEAR ARRAY? HPV genotyping test). PCR is an efficient and sensitive laboratory technique to amplify (by replication) a specific sequence of DNA into billions of copies, in this case HPV DNA. The amplification process is performed in the presence of sequence specific oligonucleotide primers (sequences of nucleotides, usually of 20-50 bases long, that are complementary to a target DNA sequence and serve as a starting point for DNA replication) and DNA polymerase (an enzyme that synthesizes new DNA strands using preexisting DNA strands as a template, thereby assisting in DNA replication under controlled conditions (e.g., temperature, time required, etc.)). A fluorescent marker is also incorporated into the PCR product during the amplification process. This assists in seeing the PCR products. The advantage of the PCR -based method is its ability to detect very small amounts of HPV DNA.
The Amplicor? HPV test (Roche Molecular Systems, CA) is a PCR-based test for the detection of 13 high-risk HPV (HR-HPV) types, i.e., types of HPV that cause abnormal changes in the cells of cervix that may progress to cervical cancer if not treated. The 13 HR-HPV types are types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. The Amplicor? HPV test is a highly sensitive method, but it does not have the ability to discriminate between these types. This method detects the PCR products using 13 oligonucleotide probes.
The LINEAR ARRAY? HPV genotyping test (Roche Molecular Systems, CA) is a PCR-based test used for detection and identification of 37 HPV genotypes, including all high-risk and low-risk genotypes. Low-risk types of HPV typically cause genital warts and have a low risk of progressing to cancer. This method detects amplified type-specific HPV DNA with probes that have been fixed (attached) to a nylon membrane. It is a rapid method; individual as well as multiple genotypes are easy to interpret in a single test without requiring expensive and specialized instrumentation.
The SPF10-INNO LiPA? HPV genotyping test (Innogenetics Group, Ghent, Belgium) is a highly sensitive and specific method, capable of detecting and identifying 43 different genotypes and providing type-specific genotype information for 25 different HPV genotypes simultaneously. This reduces the cost and time required, performing multiple tests in a single reaction with the same sample.
Signal amplification technique: The Hybrid Capture IIT (HC II) assay (Digene Corporation, Gaithersburg, MD) uses the signal amplification technique to detect and identify all of the 13 high-risk HPV types (similar to Amplicor?). This technique involves extraction of HPV DNA from the sample of interest (e.g., cervical cells). The HPV DNA is then amplified using PCR, which can make millions of copies of the DNA. PCR is then followed by denaturation, which is a process of breaking down the double-stranded DNA into single-stranded DNA. The single-stranded DNA is hybridized with specific ribonucleic acid (RNA) probes. RNA is a single-stranded nucleic acid that helps in protein synthesis, which is important for growth and maintenance of the body.
The RNA-DNA hybrids are then captured (bound) by one set of antibodies (proteins produced during an immune response to a stimulus, such as a bacterium, virus, or something that is foreign to the body). These antibodies are coated onto the walls of microwell plates (tiny wells on a plate) so as to immobilize the DNA-RNA hybrids.
An additional set of antibodies that have an enzyme, such as alkaline phosphatase, attached to them are then added to the plates that contain the captured antibody-DNA-RNA hybrid complex.
This reaction process causes the enzyme to be separated from the antibody complex, which results in the production of light (chemiluminescence). The emitted light is measured by a device called as luminometer. The intensity of light emitted is proportional to the amount of target HPV DNA in the sample; a brighter light indicates high levels of HPV DNA and a dimmer light indicates a lower amount of HPV DNA in the sample.
DNA microarray: DNA microarrays, or DNA chips, are small pieces of glass or silicon that have many DNA attached on them in a regular pattern for genetic analysis. The tiny, fixed DNA spots may be detected by allowing the microarrays to react with probes tagged (attached) with a fluorescent dye. A probe is a sequence complementary to the target sequence (HPV DNA) on the microarray chip. When the specific target DNA is joined to the DNA probe, the fluorescent marker emits colored light, which is detected by a fluorescent imager. This data is scanned and fed in a computer and then analyzed, which identifies the target sequence. HPV microarray systems? (Biomedlab Company, Seoul, Korea) have the ability to detect up to 22 HPV types. Although this method is rapid and sensitive, it requires expensive equipment and may not be suitable for many developing countries.
Comparison of molecular assays: The Amplicor? test and Hybrid Capture IIT (HC II) assay are both PCR-based HPV DNA typing assays to detect high-risk types of HPV. The Amplicor? test has the advantage of requiring only small amounts of sample DNA and may be done on archival (stored) samples, as well as cervical cells collected along with Pap test. However, the Hybrid Capture IIT (HC II) assay may only be performed on HPV DNA derived from cervical cells collected along with Pap test. There are conflicting results among the studies that have been conducted to compare the tests' sensitivity and specificity. Specificity refers to the distinguishing quality or accuracy of the test; sensitivity indicates the probability of a test to correctly identify the sequence with a very small quantity of the sample. Some researchers have determined that both assays are similar in their ability to detect HPV and in their ability to distinguish between different types, while others indicate that Amplicor? may have better specificity than HC II, as there are chances of cross-reaction of probes with other DNA sequences in hybridization procedures (i.e., the probes may get attached to DNA sequences other than the target DNA sequence). This may lead to false results. However, HC II was found to have good reliability and consistent results when tested in multiple laboratories throughout Europe and North America.
Certain studies have been conducted to compare LINEAR ARRAY? and SPF10-INNO LiPA? HPV genotyping tests, and they have concluded that both the assays are almost equal in terms of achieving results, the time required to run assays, and the sensitivity and specificity of the test. However, LINEAR ARRAY? can detect 37 HPV genotypes, compared to the 43 genotypes that SPF10-INNO LiPA? can; the former include all high-risk HPV types and some low-risk types. In some of the studies, SPF10-INNO LiPA? has been found to be less sensitive in multiple genotypes of HPV infections.
Research
Currently many research studies are being conducted comparing several different types of human papillomavirus (HPV) typing techniques, particularly in a large number of patients and using different types of samples (e.g., cervical brushings or stored cervical smears), so as to establish which HPV typing method is more applicable as a screening test or which method to adopt based on the geographical distribution of HPV types. HPV DNA typing tests are performed to determine what type of HPV a person is infected with. Several HPV typing methods are available, such as the Hybrid Capture IIT test (Digene Corporation, Gaithersburg, MD), the Amplicor? HPV test, the LINEAR ARRAY? HPV genotyping test (Roche Molecular Systems, CA), and the SPF0-INNO LiPA? HPV genotyping test (Innogenetics Group, Ghent, Belgium).
New HPV types are being identified according to recent research reports. Their transformation potential to cervical cancer is determined by several laboratory and population-based studies that utilize these various HPV typing techniques.
Implications
General: The human papillomavirus (HPV) is a DNA-based virus that causes HPV infection, a sexually transmitted disease (STD). HPV has also been associated with the development of cervical cancer.
Detection and confirmation: The detection of HPV and identification of its types, of which there are more than 100, aid in the early diagnosis of cervical cancer, which is the most successfully treated cancer when it is detected early. The early stage of cervical cancer is the cervical dysplasia stage, i.e., when changes in the cervix have an increased chance of progressing to cervical cancer. HPV typing is also useful in monitoring and following up with HPV-positive women so as to prevent the development of cervical cancer. Identification of HPV genotypes provides information that may help physicians choose suitable treatment options, such as the use of anticancer drugs (chemotherapy), surgery, etc. Detection of HPV types 16 and 18 is of prime importance, as they are the most common and cause about 70% of cervical cancers. HPV types 6 and 11 are the most common forms and cause about 90% of genital warts, which usually appear as soft, moist, pink, or red swellings that grow quickly. The high-risk types of HPV cause abnormal changes in the cells of the cervix that may progress to cervical cancer if not treated.
HPV viral load: The Hybrid Capture IIT assay, which is an HPV typing method, determines the amount (viral load) of cancer-causing (oncogenic) HPV virus at the infection site in the cervix, and it detects and identifies high-risk HPV types. According to some studies, a high HPV viral load is suggestive of a viral infection that had persisted for a long time and of disease development, while a low HPV viral load is associated with the clearance of an infection and even a decrease of cervical growth. This determination aids in distinguishing an HPV infection that is more harmful and also in determining the severity of the infection so as to choose suitable anticancer therapies, such as drugs (chemotherapy) or radiation (radiotherapy).
Geographical distribution: It is import to understand the HPV DNA types in different geographical locations and the distribution of various HPV types (low-risk and high-risk) in different populations. Thus, HPV typing methods may be modified so that the HPV types that are present in a particular region or population can be tested for, thereby aiding in early detection, prevention, and treatment of cervical cancer.
HPV vaccination: The HPV-based vaccine is given to protect against the development of cervical cancer in women, when it is caused by certain types of HPV. A vaccine is an agent or a preparation, which when given, stimulates immune response against an infectious agent, in this case certain HPV infections. For example, Gardasil? (Merck) is an HPV vaccine that induces immunity against HPV infection, particularly those resulting from infection with HPV types 16 and 18, which causes about 70% of cervical cancers. In addition, it protects against HPV types 6 and 11, which are the two most common causes of genital warts. Since HPV vaccines are developed to protect against specific HPV types, it is important to know the geographical distribution of different types of HPV infections so as to form the basis for vaccination programs throughout the world. In addition, HPV typing techniques may also be able to assist in the monitoring of the efficacy of HPV-based vaccines and the duration of protection in the population. The duration of protection by the vaccine Gardasil? is not yet established.
Limitations
Human papillomavirus (HPV) DNA typing assays have been found to be more sensitive cervical cancer screening techniques than the Pap test. The Pap test is a simple procedure to collect and analyze a sample of cervical cells to screen for cervical cancer.
However, HPV typing has less specificity than the Pap test in detecting cervical cancers. Specificity refers to the distinguishing quality or accuracy of the test. As a result, this may lead to an increase in the referral of women for unnecessary follow-up tests and potential treatment (and therefore the added costs associated with these medical procedures). This may be overcome by doing a Pap test as a secondary screening test, thus increasing the specificity while maintaining the sensitivity of cancer-causing (oncogenic) HPV DNA testing.
Although HPV typing assay detects and identifies the types of HPV infection in all the women infected, all of them may not progress to cervical cancer, which reduces the specificity of this test.
Future research
Human papillomavirus (HPV) is a virus that causes HPV infection, a sexually transmitted disease (STD). More than 50 types of HPV have been identified, some that cause genital infections and some that have been associated with the development of cervical cancer in women. Co-infection of multiple HPV types or co-infection of HPV and HIV may lead to increased HPV viral multiplication (breeding) and a rapid progression to cancer of the cervix. This may occur as a result of the suppression of the immune system triggered by HIV, resulting in an increased amount of the virus (viral burden), but these findings have to be further studied in a large number of patients so as to establish this finding.
Author information
This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).
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