Most warts can be diagnosed with the naked eye, but some cannot be diagnosed clinically for a while, and rely on laboratory or other reagents to diagnose, such as with flat warts, you need to do a syphilis serologic test. The following are a few of the commonly used tests.
A. Acetate test
The white acetate test is mainly used to detect subclinical HPV infection in the clinic, especially for the diagnosis of subclinical HPV infection in the female cervix. It is suitable for general clinical and epidemiological studies because of its good sensitivity and simplicity. If the white acetate test is evaluated together with colposcopy and histopathological methods, it will greatly increase the positive predictive value.
A number of data confirm that subclinical HPV infection is far more frequent than clinically evident lesions and can only be detected by special methods. Subclinical HPV infection is initially found in the cervix and later in the female vulva, male penis, and perianal area with similar damage. Subclinical HPV infections can exist alone or as warts visible to the naked eye. In recent years, a number of studies have confirmed the relationship between HPV infection and tumors, so subclinical HPV infection is gaining more and more attention. There are many methods to detect HPV infection in the laboratory, commonly used are immunological methods, deoxyribonucleic acid molecular hybridization techniques and polymorphic enzyme chain reaction. There are no serological tests available and HPV cannot be cultured. Although the above experimental methods have certain sensitivity and specificity, they are difficult to be applied in general clinical and epidemiological practice because they require more complex experimental equipment and techniques. In recent years, some scholars have suggested using the property of acetic acid to turn the HPV-infected area white to detect subclinical HPV infection, believing that this method, when combined with amplification techniques, would be a simpler, applicable and more accurate method. While the method was first used to identify suspicious areas of HPV infection in the female cervix, more recent research interest has shifted to detecting subclinical damage in male sexual partners of women with genital warts damage or intraepithelial growths. In a recent investigation, 72% of 51 male sexual partners of women with histologic manifestations of condyloma acuminata were identified as asymptomatic HPV infection by colposcopy after application of 5% acetic acid solution to normal penile surface skin.
Methods and principles
A cotton swab dipped in 3% to 5% acetic acid solution was applied to the suspected damaged skin and the result of whitening (also known as white acetate – acetonhite) was generally observed after 1 minute. However, in the female vulva, the whitening of acetic acid needs to be observed after 3 to 5 minutes. When a male is examined, the penis and scrotum should be covered with 3% to 5% acetic acid-soaked gauze for 3 to 5 minutes, and then the external skin should be observed with 10 to 16 times magnification to look for hidden warts or acetic acid whitening areas. A longer period of time, typically 15 minutes, may be required to whiten the perianal lesions with confidence. The white acetate test has been improved by the use of magnification techniques and especially by the simultaneous use of colposcopic observation. Magnification with a colposcope makes the acetic white areas more visible, but in most cases, the use of a simple magnifying glass is sufficient.
The mechanism of action of the white acetate test, which makes inconspicuous warts visible to the naked eye, has not been clarified. One theory suggests that the whitening is the result of protein coagulation, and that this protein reflects the abnormal cells and cellular excesses characteristic of HPV-infected epithelium. Another theory postulates that HPV-infected epithelium has different keratin from normal uninfected epithelium and that only the former can be whitened by acetic acid.
Presentation on colposcopy
Following the use of acetic acid, colposcopic features of subclinical HPV infection of the cervix are shiny, snow-white lesions with irregular jagged, angular or feathery margins. There are diffuse, satellite-like lesions beyond the metastatic area, while capillary images may be seen, usually consisting of vessels of the same caliber in a loose, disorganized arrangement often resembling a sieve on a horizontal surface, and dilated capillary collaterals may also extend vertically to the surface, spreading throughout the lesion with the same canal diameter. If the vascular image is not clear enough due to acetic acid-induced constriction of small vessels, a green filter can be added to the magnifying glass to make the image clearer.
Like cervical colposcopy, the acetic acid whitening area of male genital HPV infection is bright, snowy white, and shiny on colposcopy. Notably, 22% of subclinical HPV infections in men occur at the scrotum, which may be an important site of HPV transmission because little attention has been paid to this anatomic area as a possible refuge for HPV.Wikstrom has classified the presentation of white acetate in the male genitalia into three types based on the difference in presentation under magnification.
1. typical: easily distinguishable, slightly elevated marginal damage and punctate central capillaries associated with or unrelated to epithelial depressions are visible.
2, distinct, visible damage with easily distinguishable, slightly elevated margins but lacking distinct punctate capillaries.
3, atypical: damage showing a high and low margin and lacking punctate capillaries.
Clinical application
Wikstrom’s investigation showed that 62% of typical and obvious HPV infections by white acetate test were consistent with histopathological findings. In contrast, only 11% of atypical presentations were consistent. When based on histopathological findings, the sensitivity of the white acetate test is 85% and the specificity is 12%. If based on nucleic acid blotting and in situ hybridization techniques, the sensitivity is 85% and the specificity is 11%. Because of the sensitivity of the white acetate test and the simplicity of the method, which does not require complex instrumentation, it is suitable for general clinical and epidemiological studies.
The white acetate test is mainly used in the clinical setting to detect subclinical HPV infection. Since most cervical HPV infections in women are subclinical, they are only visible with the use of acetic acid. In women, vulvar condyloma is common, but most have a combination of subclinical infections, so the use of the white acetate test in the vulvar area to detect subclinical HPV infection can identify the source of recurrent condyloma. Secondly, it is possible to recognize subclinical HPV infection as a possible cause of previously unexplained vestibular itching, burning sensation, and discomfort during intercourse, which can be very distressing for the patient. The latter condition generally has a long course and can be misdiagnosed as a Candida infection, which is only aggravated by long-term ineffective treatment.
Subclinical HPV infection of the male genitalia can cause small, non-tipped lesions that are not easily detected by the naked eye, especially papules and patchy lesions that are difficult to detect even with very careful examination, and with the use of acetic acid, the lesions can appear as well-defined “acetic acid whitening areas”. Screening for subclinical HPV infection in high-risk men is important for the prevention of cervical cancer in women. It has been suggested that men with clinically insignificant HPV infection can be a source of potentially carcinogenic HPV in women. A London survey of 75 male sexual partners of women with histologically diagnosed cervical intraepithelial neoplasia (CIN grade III) found that 49 (65%) had histologically confirmed penile HPV infection, 16% presented with clinically visible damage, and 49% had subclinical HPV infection confirmed by white acetate test and colposcopy.
Subclinical HPV infection is common, and some cases identified by white acetate testing should in principle be treated, but this is difficult to do in practice. Currently most clinicians only try to get rid of warts that are visible to the naked eye, but do not pay attention to finding subclinical damage. Acetate white test is done only for those patients who have frequent recurrences and for screening the sexual couples of patients with normal clinical appearance.
If there is viral antigen in the lesion, the antigen-antibody combination. In the peroxidase-anti-peroxidase (PAP) method, the nucleus can be stained red. This method is specific and more rapid, which is helpful for diagnosis.
Pathological examination
It is mainly incomplete keratinization, highly hypertrophic spiny layer, papilloma-like hyperplasia, thickened and prolonged epidermal protrusions, and the degree of hyperplasia can resemble pseudoepithelioma-like. The spiny cells and basal cells have a considerable amount of nuclear division and resemble carcinoma. However, the cells were regularly arranged and the boundary between the proliferating epithelium and dermis was clear. It is characterized by the formation of vacuoles in the granular layer and the upper cells of the spiny layer. These vacuolated cells are larger than normal, with light cytoplasmic coloration and large, round, deeply basophilic nuclei in the center. Bushke-loewenstein giant condyloma acuminatum has extreme downward epidermal growth that replaces the underlying tissue and is easily mixed with squamous cells, so multiple biopsies are required. If there is a tendency for slow progression, it is a low-grade malignant process, known as verrucous carcinoma.
V. Genetic diagnosis
To date, HPV is difficult to detect by traditional viral culture and serological techniques, the main experimental diagnostic technique is nucleic acid hybridization. The PCR method developed in recent years has the advantages of specificity, sensitivity, simplicity and rapidity, opening up new ways for HPV detection.
(A) Specimen collection and processing
1. Specimen collection and pre-treatment: Take secretions and cells from the vagina and ectocervix with a scraper or saline moistened cotton swab. While doing cytological examination, put the specimen into 5ml of PBS containing 0.05% thimerosal, wash twice with PBS by centrifugation (3000g, 10min), resuspend the deposited cells in 1ml of PBS, and take 0.5ml of cell suspension for DNA extraction.
2. Extraction of nucleic acids from specimens: 1 volume of cell suspension plus 10 times the volume of cell lysis solution (10 mmol/L Tris-HCl, pH 7.4, 10 mmol/L EDTA, 150 mmol/L NaCl, 0.4% SDS, 1.0 mg/ml proteinase K) was treated overnight at 37°C; and equal volumes of phenol/chloroform (1:1), chloroform/iso (24:1) each twice; add 1/10 volume of 3 mol/L NaAc (pH 5.2) and 2.5 times volume of anhydrous ethanol at -20°C for 2h or overnight to precipitate DNA; add 1 volume of ethanol and wash once; lyse with 60 μl of TE solution (10 mmol/L Tris-HCl, pH 8.0, 1.0 mmol/L EDTA) and incubate for 30 min at 37°C.
(II) PCR amplification
1., Primer design and synthesis: The HPV genome can be divided into early (E) and late (L) regions, each containing a series of open reading frames (ORFs). Sequence analysis showed that there are conserved sequences in the non-coding regions and E1, E6, E7 and L1 regions of each type of HPV. Manos et al. designed synthetic primers MY11 and MY09 by selecting conserved sequences from the L1 region of HPV as shown in Table 1, which have complementary sequences with HPV types 6, 11, 16, 18 and 33 and can also amplify other types of HPV.
2, PCR reaction reagents: Taq DNA polymerase (2U/ml), 10mmol/L dNTP reservoir (10mmol/L each of dATP, dCTP, dGTP, dTTP), 10×PCR buffer (500mmol KCl, 40mmol/L MgCl2, 100mmol/L Tris-HCl, pH 8.5), 100 μmol/L MY11 and MY09 reservoirs, and distilled water prepared in sterilized glass stills.
3. PCR amplification method and procedure: 100 μl PCR reaction solution, sterile 0.5 ml siliconized plastic centrifuge tubes were used as reaction tubes for amplification reactions.
(1) Pre-mixed reaction reagents were prepared and dispensed before the experiment. Pre-mixed reagents include various PCR reagents except specimen DNA.
(2) Add 10 μl of specimen and 90 μl of premix reagent to each reaction tube in turn.
(3) Add 80-100μl of paraffin oil and centrifuge rapidly for a few seconds on a benchtop centrifuge so that each reaction reagent is collected under the oil layer. At present, PCR reagents have been commercialized in 25 μl reaction volume, and only specimen DNA can be added when used.
(4) Place the reaction tube on the PCR amplification instrument, the cycle parameters are 95℃ 30s, 55℃ 40s, 72℃ 50s cycle 35 times, and finally 72℃ extension 5min.
4. Positive and negative controls should be set up for each test. Recombinant plasmid containing HPV (100pg per reaction) or cell line containing HPV (e.g. Caski, HeLa) DNA as positive control, and human cell line DNA without HPV as negative control.
(iii) Detection and analysis of amplification products
1, gel electrophoresis: After the amplification reaction, remove the reaction tube, cool to room temperature, take 10 μl of amplification products with 5%-7% polyacrylamide gel or 1.5% agarose electrophoresis, ethidium bromide staining, UV analyzer to analyze the results, the molecular weight of about 450bp at the appearance of obvious DNA bands.
2, nucleic acid hybridization: If there is no clear DNA by gel electrophoresis or need to determine the specificity of the DNA band, the labeled common mixed probe and (or) type-specific probe can be used for Southern blot hybridization, spot hybridization verification.
The 32P ATP-labeled oligonucleotide probe is made according to standard methods and needs to achieve specific activity of about 107 cpm/pmol. The hybridization solution should contain 2×106-5×106cpm probe/ml. hybridize at 55℃ with slow shaking for 2-3h, followed by rapid rinsing of the hybridized membrane with washing solution (2×SSC, 0.1% SDS) at 30-55℃ to remove the excess probe. Then the membrane was washed under different conditions depending on the probes used: for the public mixed probe, the membrane was washed at 55℃ for 10 min; for MY12, MY13 and MY16 probes, the membrane was washed at 56-57℃ for 10 min and rewashed once with a change of liquid; for MY14 and WD74 probes, the membrane was washed at 58-59℃ for 10 min and also rewashed once with a change of liquid.
The detection of HPV by PCR is superior to nucleic acid hybridization methods. Its high sensitivity, GP-PCR method to gel electrophoresis to directly analyze the results, can detect 200 copies of HPV DNA in the specimen, if the detection of PCR products by nucleic acid hybridization, sensitivity increased, can detect 10 copies of HPV DNA.
In view of the high sensitivity of PCR technology, the use of genital tract exfoliated cells as the test material is sufficient to meet the requirements of the test, avoiding the tedious operation of biopsy sampling and grinding tissue. In general, PCR amplification products are gel electrophoresed and the resulting DNA can be observed to make a direct diagnosis. Therefore, the PCR technique for HPV detection is short, simple and rapid.