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Test ID: SHPV    
Human Papillomavirus (HPV) DNA Detection with Genotyping, High Risk Types by PCR, SurePath

Useful For Suggests clinical disorders or settings where the test may be helpful

Detection of high-risk (HR) genotypes associated with the development of cervical cancer

 

An aid in triaging women with abnormal Pap smear results

 

Individual genotyping of human papillomavirus (HPV)-16 and/or HPV-18, if present

 

Results of HPV-16 and HPV-18 genotyping can be used as an aid in triaging women with positive HR-HPV but negative Pap smear results.

Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test

Persistent infection with human papillomavirus (HPV) is the principal cause of cervical cancer and its precursor cervical intraepithelial neoplasia (CIN).(1-3) The presence of HPV has been implicated in >99% of cervical cancers worldwide. HPV is a small, non-enveloped, double-stranded DNA virus, with a genome of approximately 8,000 nucleotides. There are more than 118 different types of HPV and approximately 40 different HPVs that can infect the human anogenital mucosa. However, data suggest that 14 of these types (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) are considered high-risk (HR) for the development of cervical cancer and its precursor lesions. Furthermore, HPV types 16 and 18 have been regarded as the genotypes most closely associated with progression to cervical cancer. HPV-16 is the most carcinogenic, and is associated with approximately 60% of all cervical cancers, while HPV-18 accounts for approximately 10% to 15% of cervical cancers.(1-3)

 

Although persistent infection with HR HPV is necessary for the development of cervical cancer and its precursor lesions, only a very small percentage of infections progress to these disease states. Sexually transmitted infection with HPV is extremely common, with estimates of up to 75% of all women being exposed to HPV at some point. However, almost all infected women will mount an effective immune response and clear the infection within 2 years without any long term health consequences. An infection with any HPV type can produce CIN although this also usually resolves once the HPV infection has been cleared.

 

In developed countries with cervical cancer screening programs, the Pap smear has been used since the mid-1950s as the primary tool to detect early precursors to cervical cancer. Although it has decreased the death rates due to cervical cancer dramatically in those countries, the Pap smear and subsequent liquid based cytology methods require subjective interpretation by highly trained cytopathologists and misinterpretation can occur. Cytological abnormalities are primarily due to infection with HPV; however, various inflammatory conditions or sampling variations can result in false positive cytology results. Triage of an abnormal cytology result may involve repeat testing, colposcopy and/or biopsy. A histologically confirmed high-grade lesion must be surgically removed or ablated in order to prevent the development of invasive cervical cancer.

 

Nucleic acid (DNA) testing by PCR has become a standard, noninvasive method for determining the presence of a cervical HPV infection. Proper implementation of nucleic acid testing for HPV may 1) increase the sensitivity of cervical cancer screening programs by detecting high-risk lesions earlier in women 30 years and older with normal cytology and 2) reduce the need for unnecessary colposcopy and treatment in patients 21 and older with cytology results showing atypical squamous cells of undetermined significance (ASC-US).

 

Recently, data suggest that individual genotyping for HPV types 16 and 18 can assist in determining appropriate follow-up testing and triaging women at risk for progression to cervical cancer. Studies have shown that the absolute risk of CIN-2 or worse in HPV-16 and/or HPV-18 positive women is 11.4% (95% confidence interval [CI] 8.4%-14.8%) compared with 6.1% (95% CI, 4.9%-7.2%) of women positive for 'other' HR-HPV genotypes and 0.8% (95% CI, 0.3%-1.5%) in HR-HPV negative women.(4) Based in part on these data, the American Society for Colposcopy and Cervical Pathology (ASCCP) now recommends that HPV 16/18 genotyping be performed on women that are positive for HR-HPV but negative by routine cytology. Women that are found to be positive for HPV-16 and/or -18 may be referred to colposcopy, while women that are negative for genotypes 16 and/or 18 may have repeat cytology and HR HPV testing in 12 months.(1)

Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.

Negative for HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68

Interpretation Provides information to assist in interpretation of the test results

A positive result indicates the presence of human papillomavirus (HPV) DNA due to 1 or more of the following genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.

 

A negative result indicates the absence of HPV DNA of the targeted genotypes.

 

For patients with atypical squamous cells of undetermined significance (ASC-US) Pap smear result and who are positive for high-risk (HR) HPV, consider referral for colposcopy, if clinically indicated.

 

For women aged 30 years and older with a negative Pap smear result but who are positive for HPV-16 and/or HPV-18, consider referral for colposcopy, if clinically indicated.

 

For women aged 30 years and older with a negative Pap smear, positive HR HPV test result, but who are negative for HPV-16 and HPV-18, consider repeat testing by both cytology and a HR HPV test in 12 months.

Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances

The cobas human papillomavirus (HPV) test is FDA-approved for cervical/endocervical samples collected in PreservCyt (ThinPrep) media.  Other sample types (eg, vaginal) collected in media, such as SurePath, are not considered FDA-approved sources; however, verification studies have been completed in compliance with CLIA-regulations by Mayo Medical Laboratories.

 

Prolonged storage (>14 days) of clinical samples in SurePath media may impact the detection of high-risk (HR) HPV, especially if the amount of nucleic acid present in the sample is initially at a low concentration.  Therefore, samples should be submitted for testing as soon as possible following collection.

 

The cobas HPV test detects DNA of the high-risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. This test does not detect DNA of HPV low-risk types (eg, 6, 11, 42, 43, 44) since these are not associated with cervical cancer and its precursor lesions.

 

The cobas HPV test is not recommended for evaluation of suspected sexual abuse.

 

Prevalence of HPV infection in a population may affect performance. Positive predictive values decrease when testing populations with low prevalence or individuals with no risk of infection.

 

Infection with HPV is not an indicator of cytologic high grade intraepithelial lesion (HSIL) or underlying high-grade cervical intraepithelial neoplasia (CIN), nor does it imply that CIN2-3 or cancer will develop. Most women infected with 1 or more HR HPV types do not develop CIN2-3 or cancer.

 

A negative HR HPV result does not exclude the possibility of future cytologic HSIL or underlying CIN2-3 or cancer.

Supportive Data

To assess the accuracy of the Roche cobas human papillomavirus (HPV) test using cervical/endocervical and vaginal samples collected in SurePath media, a combination of spiking and comparison testing was performed. For spiking studies, 30 analyte-negative clinical samples (cervical/endocervical or vaginal matrix in SurePath media) were spiked with AcroMetrix HPV positive genotype controls (type 68 [n=10], type 16 [n=10], type 18 [n=10]) at 1 dilution above the limit of detection (LoD). The results are summarized in Table 1 below:

 

Table 1. Verification of accuracy for the Roche cobas HPV test using spiked cervical/endocervical samples in SurePath media.

Source

Positives

Negatives

Agreement

HPV Genotype 68

10/10

0

100%

HPV Genotype 16

10/10

0

100%

HPV Genotype 18

10/10

0

100%

 

 

In addition to the spiking studies described above, clinical samples (n=26) collected in SurePath media and initially tested by the Roche cobas HPV assay at an outside laboratory were tested at Mayo Medical Laboratories in a blinded fashion. The results are summarized in Table 2 below:

 

Table 2. Comparison of SurePath samples tested by Roche cobas HPV at an outside laboratory and the Virology Laboratory at Mayo Medical Laboratories (MML).

 

Roche cobas 4800 –

Outside Laboratory

 

 

Positive

Negative

Total

 

 

 

 

 

Roche cobas 4800 - MML

Positive

14

0

14

Negative

2

10

12

Total

16

10

26

Agreement: 92.3% (74.7 – 99.0%)

 

 

Reference Range:

Cervical/endocervical samples (n=27) and vaginal samples (n=22) collected in SurePath media for routine Pap smear screening were tested by the Roche cobas HPV assay.

 

49 out of 49 (100%) of these samples had negative Pap results and negative Roche cobas HPV 4800 results.

 

The reference range for the Roche cobas HPV test is negative.

 

Limit of Detection (Analytical Sensitivity):

To assess the analytical sensitivity of the Roche cobas HPV test, pools of cervical/endocervical specimens in SurePath media, vaginal specimens in SurePath media were created. Pools were spiked at a high starting concentration using each of the three AcroMetrix HPV Genotype controls (genotypes 16, 18 and 68). Serial dilutions were made into analyte-negative sample containing cells to achieve dilution of the analyte to the point of extinction. At least 6 replicates of each dilution were tested, including the panel member that was 1 dilution below the predicted LoD. The LoD was established as the highest dilution in which 6 of 6 replicates were positive.

 

The LoD of the Roche cobas HPV genotype 16, 18, and 'Other' tests for cervical/endocervical/vaginal cells in SurePath media was determined to be 50 cells/mL, 1250 cells/mL and 250 cells/mL, respectively.

 

Analytical Specificity:

A full specificity panel has been tested by the manufacturer, which included bacteria, fungi and viruses, including those commonly found in the female urogenital tract, as well as several HPV types classified as low or undetermined risk were tested with the cobas HPV test to assess analytical specificity. Results indicated that none of these organisms interfered with detection of HPV 31, HPV16 and HPV18 or produced false positive results in the HPV negative specimen.

 

Specimen Stability:

The stability of SurePath samples (endocervical/cervical and vaginal) at ambient (18-24 degrees C) was assessed using spiking studies and clinical samples. These results are summarized in Tables 3 and 4 below:

 

Table 3. Negative SurePath samples spiked with AcroMetrix positive genotype controls (68, 16 or 18). Samples were held at ambient temperature for 14 days.

 

 

Type 68–

5000 cells/ml

Type 16–

5000 cells/ml

Type 18–

5000 cells/ml

Day

Crossing Point

Crossing Point

Crossing Point

31.6

31.8

36.7

7

32.5

31.4

36.6

14

34.2

31.9

39.2

AVERAGE

32.3

31.7

37.5

% CV

4.03

0.83

3.93

 

In addition to the spiking studies described above, clinical samples collected in SurePath media at an outside laboratory were held at ambient temperature over a period of 14 days and tested by the Roche cobas HPV assay.

 

Table 4. Positive SurePath pooled patient material collected at an outside laboratory were held at ambient temperature and tested over 14 days.

 

 

Patient Genotype

'Other HR HPV'

Patient Genotype

HPV-16

Patient Genotype

HPV-18

Day

Crossing Point

Crossing Point

Crossing Point

37.2

27.5

29.6

7

36.4

28.7

30.4

14

37.4

28.9

29.8

AVERAGE

37.0

28.4

29.9

% CV

1.43

2.66

1.39

Clinical Reference Provides recommendations for further in-depth reading of a clinical nature

1. Saslow D, Solomon D, Lawson HW, et al: American Cancer Society, American Society for Colposcopy and Cervical Pathology, and American Society for Clinical Pathology Screening Guidelines for the Prevention and Early Detection of Cervical Cancer. J Low Genit Tract Dis 2012;16(3):175-204

2. Walboomers JM, Jacobs MV, Manos MM, et al: Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:12-19

3. de Sanjose S, Quint WG, Alemany L, et al: Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol 2010;11:1048-1056

4. Wright TC Jr, Stoler MH, Sharma A, et al: Evaluation of HPV-16 and HPV-18 genotyping for the triage of women with high-risk HPV positive, cytology-negative results. Am J Clin Pathol 2011 Oct;136(4):578-586

5. Procedure manual and package insert: cobas HPV test. Roche Diagnostics. Indianapolis, IN, version 05641268001-01EN

6. Gilbert L, Oates E, Ratnam S. Stability of cervical specimens in SurePath medium for HPV testing with the Roche cobas 4800. J Clin Microbiol 2013 Oct;51(10):3412-3414