|Year : 2019 | Volume
| Issue : 4 | Page : 542-548
Evaluating the PGMY-centre hospitalier universitaire vaudois assay as a cost-effective tool for human papillomavirus genotyping in HIV-infected women
Pallavi Ravindra Baliga1, Raghavendran Anantharam2, Vinotha Thomas3, Priscilla Rupali4, Manu Chopra5, Susanne Pulimood5, Jessie Lionel3, Abraham Peedicayil3, Rajesh Kannangai2, Manu Gnanamony2, Priya Abraham2
1 Department of Microbiology, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Clinical Virology, Christian Medical College, Vellore, Tamil Nadu, India
3 Department of Obstetrics and Gynaecology, Christian Medical College, Vellore, Tamil Nadu, India
4 Department of Infectious Diseases, Christian Medical College, Vellore, Tamil Nadu, India
5 Department of Dermatology, Christian Medical College, Vellore, Tamil Nadu, India
|Date of Submission||11-Mar-2020|
|Date of Acceptance||24-Mar-2020|
|Date of Web Publication||18-May-2020|
Prof. Priya Abraham
Department of Clinical Virology, Christian Medical College, Vellore - 632 004, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Aims: Cervical cancer is one of the leading causes of cancer among women, worldwide. HIV-positive women tend to have persistent infection and infection with multiple human papillomavirus (HPV) types. There is a need for affordable HPV DNA tests as viable alternatives to the existing costly commercial assays. The aim of the study was to establish PGMY-CHUV reverse hybridization assay as a cost-effective tool for HPV genotyping. Study Design: This was a prospective study conducted in a tertiary care centre from March 2011 to July 2012. Subjects and Methods: Fifty cervical brush samples from HIV-infected women and 43 WHO reference samples were tested by both the CHUV assay and linear array (LA). Results: The CHUV assay in comparison to the LA showed a sensitivity of 91%, specificity of 52% and a moderate agreement for all samples that were compared. However, most high-risk HPV types were identified amongst the clinical samples, and the entire range of genotypes in the WHO reference panel was detected. Statistical Analysis: The accuracy indices such as sensitivity, specificity, positive predictive value and negative predictive value were calculated. The level of agreement (kappa value) between the two assays was also calculated. Conclusion: The CHUV assay had an acceptable sensitivity, but it lacked specificity for HPV detection. Despite the lower rates of detection of multiple infections from clinical samples, better results were obtained with the WHO reference samples and the ability of the assay to identify the entire range of genotypes suggests that it can be an efficient tool for genotyping.
Keywords: CD4, cervical neoplasia, HIV, human papillomavirus, vaccination
|How to cite this article:|
Baliga PR, Anantharam R, Thomas V, Rupali P, Chopra M, Pulimood S, Lionel J, Peedicayil A, Kannangai R, Gnanamony M, Abraham P. Evaluating the PGMY-centre hospitalier universitaire vaudois assay as a cost-effective tool for human papillomavirus genotyping in HIV-infected women. Indian J Med Microbiol 2019;37:542-8
|How to cite this URL:|
Baliga PR, Anantharam R, Thomas V, Rupali P, Chopra M, Pulimood S, Lionel J, Peedicayil A, Kannangai R, Gnanamony M, Abraham P. Evaluating the PGMY-centre hospitalier universitaire vaudois assay as a cost-effective tool for human papillomavirus genotyping in HIV-infected women. Indian J Med Microbiol [serial online] 2019 [cited 2020 Sep 30];37:542-8. Available from: http://www.ijmm.org/text.asp?2019/37/4/542/284520
| ~ Introduction|| |
Cervical cancer is still an important cause of cancer death among women, worldwide. Persistent infection with human papillomavirus (HPV) is now known to be a necessary although not sufficient cause of cervical cancer. HPVs are classified based on their association with anogenital cancer into high-risk types, probable high-risk types and low-risk types. Worldwide and in India, HPV16 and 18 are the most frequently detected types in cervical cancer. In most sexually active women, HPV infection is transient and is cleared after 1–2 years. However, women who are immunocompromised such as HIV-infected women are thought to be at an increased risk for persistent infection resulting in an increased risk of cervical neoplasia. They also tend to have multiple HPV types and harbour more non-HPV16/18 infections.,
Line probe assays, based on the amplification of conserved regions of HPV followed by hybridisation to type-specific probes on line blot strips, are also widely used. These are however expensive and may prove cost-effective only in high-throughput laboratories.,,,,,
The PGMY-Centre Hospitalier Universitaire Vaudois (CHUV) assay was developed by the Institute of Microbiology, CHUV, University of Lausanne, Switzerland. This assay was designed as an adjunct to cytology to detect HPV DNA based on the PGMY primers and to genotype the HPV-positive samples using a reusable probe array using the principle of reverse hybridisation. This assay can detect 31 HPV genotypes. The assay has been evaluated for genotyping HPV for the past 10 years in the CHUV Institute, Switzerland, and within the WHO HPV Laboratory Network and found to be suitable for HPV genotyping with successful participation in proficiency panels (PPs) established in this network.
As a reference standard, the commercial CE-IVD approved Linear Array (LA) was used to evaluate the proficiency of the PGMY-CHUV assay in our setting. LA also uses PGMY primers for HPV DNA amplification followed by reverse hybridisation onto an array of 37 HPV genotypes.
The aim of this study was to evaluate the performance of a cost-effective reverse hybridisation assay (CHUV assay) in comparison to a commercial CE-marked reverse hybridisation assay (LA, Roche).
| ~ Subjects and Methods|| |
This was a prospective study of diagnostic accuracy conducted in a tertiary care centre from March 2011 to July 2012. Approval was obtained from the Institutional Review Board (IRB Minute No: 7333, 2010).
Fifty clinical samples and 43 WHO reference samples were included in the study.
Consecutive 50 HIV-positive women attending the infectious diseases clinic and obstetrics and gynaecology outpatient departments were recruited based on the following inclusion and exclusion criteria. Women in the age group of 18–45 years who were recently diagnosed to have HIV infection and who were sexually active were included in the study. Pregnant women, women who were menstruating or had a hysterectomy and HIV-negative women were excluded from the study. A pelvic examination was done, as a part of the management protocol for newly diagnosed HIV-positive women. Pap smears were collected in ThinPrep Pap Smear liquid cytology bottles (Cytyc Corp., Boxborough, Massachusetts, USA) and were sent to the department of pathology for the analysis. CD4 counts were also done for the participants, within 2 weeks of collecting the cervical brush sample.
HPV DNA was collected using the DNAPAP cervical sampler (Qiagen, Gaithersburg, USA) in a specimen transport medium. The cervical brush samples were transported to the department of clinical virology, in an ice bucket at 4°C, aliquoted and stored at −20°C until further testing.
WHO reference panel
Forty-three samples containing 100 μl of purified HPV plasmid DNA in TE buffer with 1 mM EDTA and 10 ng/ml of human placenta DNA (Courtesy Dr. Joakim Dillner, WHO HPV LabNet Global Reference Laboratory, Department of Clinical Microbiology, University Hospital, Malmo, Sweden) were also tested in both the assays. The PP was pretested at GRL Sweden using a modified GP5+/6+ polymerase chain reaction (PCR) followed by Luminex-based typing for HPV genotyping. The PP was further pre-tested by one external laboratory before release, namely the German Cancer Research Centre (DKFZ) in Heidelberg. This panel comprised 42 HPV DNA positives and one negative.
DNA extraction from the clinical samples was done using ethanol precipitation method. The WHO reference samples were obtained as purified plasmids and were directly taken for amplification, without prior extraction in both the assays.
PGMY-Centre Hospitalier Universitaire Vaudois reverse hybridisation assay
The CHUV assay was the index test. The procedure was performed as per the WHO HPV Laboratory Manual (2010). Following DNA extraction using ethanol precipitation, the cervical samples were subjected to PCR with biotin labelled PGMY 09/11 primers targeting the L1 gene (450 bp) of HPV. To confirm adequate DNA extraction and amplification, primers for a cellular target gene, i.e., human leukocyte antigen (amplifying a 230 bp product), were also included as an internal control.
Linear array (Roche diagnostics, Branchburg, NJ USA)
LA was the reference test against which the CHUV assay was compared. All cervical brush samples and the 43 WHO reference samples were tested by LA. The same DNA extracts used for the CHUV assay were used in LA, as per the manufacturer's instructions. The samples were amplified using PGMY09/11 primers targeting the L1 region of HPV DNA, with β-globin as the internal control. The amplified products were then hybridised to oligonucleotide probes on strips followed by the colorimetric detection.
The diagnostic test characteristics of the CHUV assay in comparison to LA – sensitivity, specificity, positive predictive value and negative predictive value – were calculated with a 95% confidence interval. The level of agreement (kappa value) between the two assays was also calculated. All data generated in the study were analysed using the SPSS software-version 16.0 (SPSS Inc. Chicago, IL, USA).
| ~ Results|| |
Comparison of the Centre Hospitalier Universitaire Vaudois assay with the linear array for clinical and WHO reference samples
Demographic data of the women
The median age of the women was 32.2 years. All were married women, 45 of them having only one sexual partner and 5 of them having two or more sexual partners. The median age of onset of sexual activity was 19.9 years, 18 women having the age of coitarche <18 years and 32 women >18 years. Most women were newly diagnosed HIV cases, the duration of HIV diagnosis being <5 years for 41 women and >5 years for 9 women. CD4 counts were <350 for 17 women and >350 for 33 women. Pap smear reports for most women were normal (48/50), low-grade squamous intraepithelial lesion and high-grade squamous intraepithelial lesion being reported in 1 woman each. Partner HIV-status was positive for 43 women, negative for 4 women and undiagnosed/unknown for 3 women.
Four of the 50 clinical samples gave a negative β-globin band in the LA and were excluded from the final analysis, leaving 46 clinical samples for comparison between both the genotyping assays.
Human papillomavirus detection
The overall rate of detection of HPV was higher with the PGMY-CHUV assay when compared to the LA results. Of the clinical samples, 29 (63%) were positive for HPV versus 22 (48%) samples which were positive by LA. Two of the samples that were HPV DNA positive in PGMY-CHUV could not be further genotyped using the reverse hybridisation with genotype specific probes. Among the WHO positives, 36/42 (86%) were positive by CHUV compared to 39/43 (93%) positive by LA. Combining the clinical and WHO samples, the HPV detection rate was 73% by CHUV versus 69% by LA [Figure 1].
|Figure 1: Human papillomavirus positivity in the Centre Hospitalier Universitaire Vaudois assay and linear array using clinical samples|
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Single versus multiple human papillomavirus infection
Among the 27 of 29 clinical samples with HPV genotypes detected, the CHUV assay reported 15/27 (56%) as single and 12/27 (44%) as multiple HPV types, whereas LA reported 8/22 (36%) as single and 14/22 (64%) as multiple HPV types [Table 1]. In the WHO reference panel, the CHUV assay detected 29/34 (85%) as single HPV and 7/8 (88%) as multiple infections, in comparison to LA which detected 31/34 (91%) of single infections and 8/8 (100%) multiple infections among the known 34 single and 8 multiple genotype combinations in the WHO reference panel [Table 2].
|Table 1: Comparison of the Centre Hospitalier Universitaire Vaudois assay with linear array using clinical samples (n=50)|
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|Table 2: Comparison of the Centre Hospitalier Universitaire Vaudois assay and linear array results with the results of the global reference laboratory|
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Both the assays picked up an array of 15 genotypes among the clinical samples [Figure 2] Among the samples, LA identified four additional genotypes compared to CHUV: one each HPV61, HPV62, HPV81 and HPV-CP6108 (not shown), whereas types HPV44 (one sample) and HPV55 two2 samples) were detected only by the CHUV assay. The CHUV assay was able to pick up an additional HPV-56 in a clinical sample. The LA detected additional numbers of genotypes 16, 45, 51 and 59, whereas CHUV detected more numbers of genotypes 6, 31, 42 and 56.
|Figure 2: Range of genotypes detected by Centre Hospitalier Universitaire Vaudois assay and linear array using clinical samples and WHO reference samples|
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The range of HPV types identified by both the assays using the WHO reference samples was similar. However, both the assays missed HPV types 68 (CHUV – five samples and LA –four4 samples) and 56 (CHUV – three samples and LA – one sample) in this reference panel. LA detected additional samples in genotypes 6, 16, 18, 31 and 52 [Figure 3].
|Figure 3: The range of genotypes detected by Centre Hospitalier Universitaire Vaudois and linear array using the WHO reference panel|
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High-risk types detected
[Figure 4] summarises the high-risk HPV types detected by both the assays in the clinical samples. LA identified high-risk type HPV16 in 11 clinical samples compared to 9 samples by CHUV. Both the assays detected the same clinical sample as having HPV18 [Figure 4].
|Figure 4: High-risk human papillomavirus types detected by both the assays using clinical samples|
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Among the other high-risk types detected, HPV31 and HPV56 were detected more frequently by CHUV (five in CHUV vs. two in LA and two in CHUV vs.one in LA, respectively). LA detected HPV 45, 51 and HPV59 (five in LA vs. four in CHUV each) marginally better [Figure 3].
Diagnostic test characteristics
With the LA as the reference standard, the sensitivity of the CHUV assay was 77% and 92%, respectively, for the clinical and WHO samples. The specificity was much lower (50%) for the clinical samples, however, it was nearly 100% for the WHO samples. Overall, the sensitivity improved to 91% when both clinical and the WHO panel were combined; specificity still remained at 52%.
Level of agreement
The level of agreement between the two assays ranged from 0.26 (poor agreement) for the clinical samples to 1.000 (perfect agreement) for the WHO samples. Combining samples from both the panels, there was a moderate agreement of 0.47 between the CHUV and LA assays.
Taking into account the cost of DNA extraction, PCR and genotyping with reverse blotting hybridisation (RBH), the cost of the CHUV assay for one sample was estimated to be Rs. 1030. The cost of testing with LA per sample is Rs. 4375
Comparison of the Centre Hospitalier Universitaire Vaudois assay with the global reference laboratory results for the WHO reference panel
In the WHO reference panel, the CHUV assay was able to detect the entire range of genotypes. HPV genotypes of low DNA concentration in this panel were also detected, including high-risk HPV types such as HPV-16. Three samples with low concentrations of 5 IU, 50 IU and 50 IU (HPV types 6, 16 and 56, respectively) were missed by the CHUV assay.
When the results of CHUV assay were compared to the WHO reference panel results, the sensitivity was around 86% and specificity was 100%. The level of agreement was 1.00 (perfect agreement). [Figure 3] summarises the range of genotypes detected by both the assays in comparison with the global reference laboratory results. [Figure 5] depicts the rate of HPV positivity in the WHO samples. [Figure 6] depicts the high-risk types detected by both the assays in the WHO samples.
|Figure 5: Human papillomavirus positivity by Centre Hospitalier Universitaire Vaudois assay and linear array using the WHO reference panel|
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|Figure 6: High-risk types detected by both the assays using the WHO reference panel|
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The results of the CHUV assay in comparison to the results of LA are summarised in [Table 1].
The results of both the assays for the 43 WHO reference samples were also compared with the results of the WHO HPV global reference laboratory which tested this PP [Table 2].
| ~ Discussion|| |
We evaluated an in-house reverse hybridisation assay(CHUV assay), based on PGMY primers and a reusable probe array, in comparison to the licensed (CE-IVD) LA (Roche Diagnostics) for HPV detection and genotyping. Current guidelines do not validate the PGMY-CHUV assay for the primary screening of cervical cancer; therefore, it was designed as an adjunct to cytology to detect HPV DNA, especially to monitor patients who have had treatment.
LA also uses PGMY primers for HPV DNA amplification and a line probe assay for the detection of 37 HPV genotypes from cervical samples. Several studies have evaluated the proficiency of LA for HPV detection.,,, LA has been found to have an improved rate of detection of multiple genotype combinations and newer HPV genotypes. LA is also found to be superior to the Digene Hybrid Capture 2 HPV kit in the detection of individual carcinogenic HPV types. LA is therefore a thoroughly evaluated assay which is highly sensitive for the detection and genotyping of HPV, particularly multiple genotype HPV infections. The main disadvantage of this assay is its cost (approximately Rs. 4375/sample) which limits its use in low-resource settings.
These two assays were compared because both utilise the principal of reverse hybridisation, employing the same set of L1 region-based primers. Of the 31 and 37 genotypes that the CHUV assay and LA detect, only 28 genotypes are in common. On comparison of both the assays in the detection of these 28 common genotypes, the diagnostic test characteristics remain the same, as the additional HPV types detected by both assays in the clinical samples were part of mixed infections in each sample.
Our study samples included cervical brush samples from 50 asymptomatic HIV-infected women and 43 coded WHO reference HPV plasmids. The samples were included from HIV-infected women as the HPV prevalence is expected to be higher in this group; in addition, as this category of patients is known to harbour multiple HPV genotypes. In addition, the WHO panel of coded samples was included to further evaluate both the assays.
This study found the CHUV assay to have an overall moderate agreement with LA for HPV detection and genotyping. Combining clinical samples and the WHO reference panel, the assay had good sensitivity (91%) but low specificity (52%). The sensitivity of CHUV was lower (77%) for clinical samples when compared to its performance with the WHO panel (92%). Specificity was however higher with the WHO panel.
Both LA and CHUV were able to identify high-risk types HPV16 and HPV18. LA was more efficient in identifying type 16 (11 clinical samples), type 51 and type 59 (five clinical samples each). CHUV identified HPV16 in 9 of 11 samples but was able to detect more numbers of HPV31 (five samples) when compared to LA.
Other factors that may have influenced the discordance between the two assays in the clinical samples are sampling errors – as the cervical brush sample collected is only representative of the cervical epithelium and may contain only limited numbers of HPV-DNA copies, thus producing inconsistencies in an assay with good sensitivity. A study conducted by Quint et al. on HPV genotyping in cervical scrapes and biopsies demonstrated comparable but variable genotyping results. The sample collected is also influenced by the phase of menstrual cycle which can affect the outcome of results in terms of presence or absence of HPV DNA, the accuracy of HPV detection and the detection of multiple genotypes present at different concentrations., The differences in detection of multiple types by the assays, in the clinical samples, could also have been influenced by the relative proportions or distribution of the different HPV types in the cervical sample leading to better detection of some types and inability to detect other types. A recent study by Klug et al. compared various genotyping assays on the same set of cervical samples and clearly showed that the results are not fully congruent. This affects the comparison of newer assays with older licensed genotyping assays with regard to type-specific detection. This may have influenced the comparison of the results of CHUV and LA for the clinical samples. The higher sensitivity of the CHUV assay in clinical samples may also partly be due to the method of post-amplification genotype detection by chemiluminescence, which is a more sensitive method of detection, when compared to the colorimetric detection employed in LA, and therefore, a higher rate of positivity.
An advantage CHUV has over LA is the unambiguous detection of HPV type 52. This is important as HPV52 is among the common genotypes prevalent in many regions in Asia, including India., In our study, CHUV identified type 52 in all single and multiple combinations in the WHO samples, except one sample with types 11, 18, 31 and 52 where it detected only type 11. HPV52 was not detected by CHUV or LA in any of the clinical samples.
Two of our study samples reported negative by LA and were PCR positive by the CHUV assay, although no genotype was detected on RBH. This may be due to the presence of a genotype that is not a part of the CHUV genotype array. The CHUV assay, therefore, has an advantage over LA as the PCR amplicon is available for subsequent nucleotide sequencing to determine the genotype in these indeterminate samples.
The cost of the CHUV assay is estimated to be around Rs. 1030 per sample. The main advantage of the CHUV assay is its reusable membrane and the projected reduction in cost of testing which sums up to approximately Rs. 1030 per sample. This cost is much lower than the cost of doing LA which is Rs. 4375 per sample (cost price).
The results of our study show that the CHUV assay can detect the full range of HPV types and the better results observed with the WHO reference samples indicate that it has the potential for efficient genotyping. Future studies with larger sample size and further fine-tuning by laboratories that have just begun to use this assay will help to assess the CHUV assay's performance characteristics better.
| ~ Conclusion|| |
The CHUV assay had an acceptable sensitivity but it lacked specificity for HPV detection. Despite the lower rates of detection of multiple infection from clinical samples, better results were obtained with the WHO reference samples and the ability of the assay to identify the entire range of genotypes suggests that it can be an efficient tool for genotyping. Future studies with larger sample size and further fine-tuning by laboratories that have just begun to use this assay will help assess the CHUV assay's performance characteristics better.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]