Use of Multiplex Polymerase Chain Reaction for Detection of High-Risk Human Papillomavirus Genotypes in Women Attending Routine Cervical Cancer Screening in Harare.
In Zimbabwe, cervical cancer is screened through cytology and visual inspection with acetic acid and cervicography (VIAC). The effectiveness of these methods can be increased if complemented by a human papillomavirus DNA detection tool since most cervical cancer cases are caused by persistent infection with high-risk human papillomavirus (HR-HPV) genotypes. Moreover, the possibility of multiple-genotype HR-HPV infections warrants the need for HPV detection tools with the capacity to detect both single and multiple infections. The aim of this study was to detect HR-HPV genotypes (HPV 16, 18, 31, 33, 35, 45, 51, 52, 56, and 58), using multiplex polymerase chain reaction (PCR), in stored cervicovaginal swabs from both HIV-positive and HIV-negative women reporting for routine cervical cancer screening.
Stored cervicovaginal swabs from sexually active women who underwent VIAC at the Parirenyatwa Referral Hospital in Harare, Zimbabwe, between February and April 2015 and had received HIV counselling and testing were genotyped for the selected 10 HR-HPV genotypes using in-house multiplex PCR. The results from the multiplex PCR were compared to those previously obtained when the same samples were HPV genotyped with next-generation sequencing (NGS) on an MiSeq platform (Illumina; USA).
A total of 136 women were recruited and all 10 HR-HPV genotypes were detected. Quality control failed in 3 of the 136 swabs during the multiplex PCR reactions. The prevalence of HR-HPV genotypes in the study subjects was 53% (70/133). HIV-infected women were 1.67 times more likely to be infected with HR-HPV than were HIV-negative women (OR 1.67; p = 0.17). Of the 70 HR-HPV-positive cases, 37% (26/70) had multiple HR-HPV infections, and the majority of them were HIV infected. HIV-infected women were 1.86 times more likely to have multiple HR-HPV infections than HIV-negative women (OR 1.86; p = 0.20). Multiplex PCR and NGS had an almost perfect concordance rate in -HR-HPV detection (κ = 0.960), with only 3 discordant cases (negative with NGS and positive for HPV16 with multiplex PCR).
Multiplex PCR can detect HR-HPV genotypes that are common in Zimbabwe and could be used to detect HR-HPV genotypes from women attending cervical cancer screening programs at the Parirenyatwa VIAC clinic in Harare.
Marembo T
，Dube Mandishora R
，Borok M
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Detection and genotyping of HPV-DNA through different types of diagnostic platforms in liquid-based cervical-cytology samples.
At present cervical cancer represents the second most common cancer in women worldwide and it reaches a global mortality rate of 52%. Only the early detection and the adequate treatment of pre-neoplastic lesions and early-stage cervical cancer decrease the mortality rate for this type of cancer. Cervical carcinoma screening, as a method of second prevention, is currently feasible through molecular research of high-risk HPV genotypes and in lots of organized screening programs the Pap-test is performed only in women with positive HPV-test. Currently, there are various diagnostic platforms detecting and molecular genotyping HPV, which are based on different procedures, determining uneven viral genotypes panels and using diverse type of vials to collect and store the samples. Previous studies have pointed out that DNA-HPV test can be negative in pre-neoplastic lesions, even of high grade, or in presence of cervical cancer. Therefore, it's important to assess the risk of false negative diagnoses using DNA-HPV molecular test, because in this circumstance women do not undergo immediately Pap-test, but they are submitted to second round screening with DNA-HPV test after 5 years: this protocol could increase the incidence of "interval cancers". The present study aims at comparing the results of HPV detection and genotyping on liquid based cervical cytology, using some of the most relevant diagnostic platforms in commerce.
The study is based on a group of patients which went to their private gynecologist in a contest of opportunistic screening. The vial used in the examined population has been EASYPREP® preservative solution (YD Diagnostics CORP-Republic of Korea); liquid-based cervical cytology sampling has been done using a single device (plastic brush), allowing to collect simultaneously cytological material from exocervix and endocervix (Rovers® Cervex-Brush®). The diagnostic platforms employed have been the following: A) Digene HC2 HPV DNA Test, on RCS System (QIAGEN); B) BD Onclarity™ HPV test, on automate platform BD Viper™ LT (Becton Dickinson); C) Xpert® HPV, on GeneXpert® Infinity Systems platform (Cepheid). Every platform researched high-risk HPV genotypes panels (hr-HPV). Part of the clinical records has also been analyzed through PCR and genes L1 and E6/E7 complete sequencing, in order to further typing the viral population.
We have examined 1284 samples of women aged 16 to 73 years: 1125 have been tested using HC2 procedure, 272 samples with Onclarity method, 159 with Xpert® method and 55 samples have been analyzed using PCR and sequencing of gene L1 and gene E6/E7. HPV-DNA was detected with Onclarity method in 15,07%, with Xpert® method in 13,83% and using HC2 procedure in 12,27% of samples. The comparison between the three molecular methods revealed diagnostic discrepancies in 3,14% of our records between Onclarity test and Xpert® method and in 2,20% (6/272) between HC2 test and Onclarity test. Globally, in 431 tests, compared using different diagnostic platforms, discrepant diagnoses, referring to hr-HPV presence or to detected genotype, have been observed 11 times (2,55%). Genotype 16 appeared the most expressed in the positive samples (20,99%), whereas genotype 18 resulted the less expressed in the examined population (4,94%).
The present study highlights the following: 1) Positive results' percentage for high-risk HPV-DNA genotypes, deriving from the three diagnostic platforms used and with the same vial to collect and store samples, does not significantly vary on the basis of the type of equipment and it is congruent with the Italian percentage already detected during organized screening programs. 2) Even the molecular diagnostic approach could give false negative results, preventing the detection in the screened population of cervical HPV-related lesions and theoretically endangering women to develop "interval cancer". 3) In the population examined, genotype 16 has been the most expressed, whereas genotype 18 was among the less frequently detected. Other genotypes often noticed have been: 56-59-66 (Onclarity P3 group), 31, 51 and 35-39-68 (Onclarity P2 group). This remark emphasizes the importance of HPV infection and genotypes distribution's continuous monitoring, considering that HPV-vaccines planned in Italy in the "National vaccination prevention program 2017-2019" are not specific for the majority of these genotypes. 4) The necessity to improve the screening program to identify cervical carcinomas and pre-neoplastic cervical lesions is remarked by the detection during HPV-test of possible coinfection (present at least in 8,76% of our records). In fact, the risk of development of cervical cancer might be associated with type-specific interactions between genotypes in multiple infections and, in addition, other genotypes, not targeted by quadrivalent HPV-vaccine, can increase the risk of cervical carcinoma. 5) As there's a different combination of HPV-genotypes in diagnostic categories used by the HPV screening platforms, it's important that anyone who is in charge of this diagnostic analysis promotes among clinicians the adequate rendition of the laboratory's data in the patient records, reporting both the diagnostic result and the method through which it has been obtained.
Cassani B
，Soldano G
，Finocchiaro D
，Conti S
，Bulfamante A
，Lemorini G
，Bulfamante G
... -
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Genotypic diversity of anogenital human papillomavirus in women attending cervical cancer screening in Harare, Zimbabwe.
Although anogenital cancers have been on a gradual rise in developing countries in the past few decades, they have been understudied. The objective was to investigate genotypic diversity of anogenital HPV amongst women reporting for routine cervical cancer screening in Harare in Zimbabwe. A cross-sectional study that enrolled 144 women ≥18 years from a cervical cancer-screening clinic was performed. Each woman provided a self-collected cervico-vaginal swab (VS) and a clinician-collected anal swab (CCAS). HIV testing was offered and cervical cytology was performed. Both VS and CCAS samples were HPV genotyped, using amplicon sequencing of the L1 gene region with Illumina technology. Mean age of the women was 39.9 (range 18-83 years, SD ± 11.0). HPV prevalence was 72% (104/144) in VS and 48% (69/144) in CCAS. The most common genotypes detected in both VS and CCAS were HPV18, HPV52, and HPV16. Sixty two percent of the subjects had multiple genotypic HPV infections. The odds of being HPV-positive among HIV-infected women were higher than in HIV-negative women in both the vagina and the anus (CCAS OR = 4.8; CI 2.4-9.8, P < 0.001) and (VS OR = 2.9; CI 1.3-6.4, P = 0.005). High HPV prevalence and diverse genotypes were detected in both the vagina and anus. Anal oncogenic HPV infection was common. HPV 52 was one of the most common oncogenic genotypes in both the vagina and anus. HIV co-infection played a significant role in the prevalence of HPV. These data have implications for design of primary and secondary programs for prevention of anogenital cancer in Zimbabwe.
Dube Mandishora RS
，Christiansen IK
，Chin'ombe N
，Duri K
，Ngara B
，Rounge TB
，Meisal R
，Ambur OH
，Palefsky JM
，Stray-Pedersen B
，Chirenje ZM
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