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HPV test by Hybrid Capture II for the diagnosis of HR-HPV persistent infection
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Original article
HPV test by Hybrid Capture II for the diagnosis of HR-HPV persistent infection Test HPV par Hybrid Capture dans le diagnostic de l’infection HPV-HR persistante Y. Serour a,b,∗ , M. Bendahmane a,b , F. Abbou Baker c , M. Medles a,b , B. Moueddene c , R. Kraiba d b
a Faculté des sciences de la nature et de la vie, université Djillali Liabes, Sidi Bel Abbés, Algeria Laboratoire de recherche en environnement et santé (LRES), CHU de Sidi Bel Abbes, Sidi Bel Abbés, Algeria c Service de planning familial de la maternité, Sidi Bel Abbés, Algeria d Institut Pasteur d’Alger, Centre Pierre et Marie Curie, Alger, Algeria
Received 26 January 2016; received in revised form 5 June 2016; accepted 31 May 2017
Abstract Introduction. – Persistent high-risk HPV (HR-HPV) infection is associated with a greater risk of cervical cancer. Patients and methods. – Statistical data on the prevalence of HR-HPV infections in the Algerian population is lacking. We conducted a prospective study of 300 women aged between 25 and 50 years, screened for cervical cancer from 2012 to 2015 in Sidi Bel Abbès, a western region of Algeria. We aimed to assess the reliability of the repeated use of the HC II test (three longitudinal HPV tests 9 months apart from each other) in diagnosing the persistence of HR-HPV infection. Results. – The prevalence of HR-HPV infection was 7.33% and infected women were aged 37.9 ± 3 years. For 90.9% of HR-HPV-positive patients, the infection persisted for a mean of 18.5 months [95% CI: 16.9–22.1 months]. Among these patients, 55.55% developed CIN1 and 11.11% developed CIN2. The sensitivity of the HC II test was 81.74% [95% CI: 71.3–89.6] and its positive predictive value associated with abnormal cervical biopsy was 27.49% [95% CI: 16.0–33.33]. Conclusion. – Repeating the HC II test is a good predictor for identifying women at high risk of cervical cancer. © 2017 Elsevier Masson SAS. All rights reserved. Keywords: Persistent HR-HPV; HC II test; Cervical biopsy
Résumé Introduction. – L’infection persistante à HPV à haut risque (HPV-HR) est associée à un risque élevé de cancer du col utérin. Patients et méthodes. – Vu le manque considérable de données statistiques sur la prévalence de cette infection dans la population algérienne, nous avons entrepris une étude prospective dans la ville de Sidi Bel Abbès, une région de l’ouest Algérien. Cette étude a été effectuée entre 2012 et 2015 auprès de 300 femmes (25–50 ans), dépistées pour le cancer du col de l’utérus. Le but de cette recherche était d’évaluer la fiabilité de l’utilisation répétée du test HC II (trois tests HPV longitudinaux espacés de 9 mois chacun) pour diagnostiquer la persistance de l’infection HPV-HR. Résultats. – Les résultats obtenus ont montré que la prévalence de l’infection HPV-HR était de 7,33 % et que l’âge moyen des femmes affectées était de 37,90 ± 3 ans. Chez 90,9 % des patientes HPV-HR positives, cette infection a persisté pendant une durée moyenne de 18,5 mois [IC à 95 % : 16,9–22,1 mois], dont 55,55 % ont développé une CIN grade 1 et 11,11 % une CIN2. La sensibilité du test HPV était de 81,74 % [IC à 95 % : 71,3–89,6] et sa valeur prédictive positive associée à une biopsie cervicale anormale était de 27,49 % [IC à 95 % : 11,0–33,33]. Conclusion. – La répétition du test HC II constitue un bon prédicteur pour identifier les femmes présentant un risque de cancer du col de l’utérus. © 2017 Elsevier Masson SAS. Tous droits r´eserv´es. Mots clés : HPV-HR persistant ; Test HC II ; Biopsie cervicale ∗
Corresponding author. E-mail address: [email protected] (Y. Serour).
http://dx.doi.org/10.1016/j.medmal.2017.05.013 0399-077X/© 2017 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Serour Y, et al. HPV test by Hybrid Capture II for the diagnosis of HR-HPV persistent infection. Med Mal Infect (2017), http://dx.doi.org/10.1016/j.medmal.2017.05.013
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1. Introduction High-risk HPV (HR-HPV) infections have been more extensively studied than low-risk HPV infections because of their close association with cervical cancer [1,2]. Women presenting with persistent HR-HPV infection are at higher risk of cervical cancer [3]. There is currently no consensus on the definition of a persistent or transient HPV infection [4]. The most common definition refers to two or three (or more) positive tests for HPV during the follow-up period [5–7]. The risk of cervical intraepithelial neoplasia (CIN) is proportional to the number of positive tests for HR-HPV [8]. The time interval between consecutive HPV tests reported in the literature ranges from less than a year in some studies to 1 or 2 years in others [9]. Most HPV infections are transient and spontaneously disappear within 12 to 18 months [10]. For patients presenting with persistent HPV infection, colposcopy is recommended in the second-line diagnostic strategy to assess lesion severity, followed by a biopsy to confirm the histological diagnosis [11]. However, the early diagnosis of lesions in a patient presenting with persistent cytological abnormalities and a negative test supports the hypothesis of a false negative result [12]. We detected HR-HPV using Hybrid Capture II assay (HCII; Qiagen, Gaithersburg, MD). This test is practical but it does not allow for identifying the various genotypes; a positive result thus indicates the presence of one or several HR-HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) without distinguishing them [13]. We aimed to evaluate the reliability of repeated HC II tests (three consecutive tests 9 months apart from one another) in the diagnosis of persistent HR-HPV infection. 2. Patients and methods We performed the study at the maternity of Sidi Bel Abbès and at the Institut Pasteur of Pierre and Marie Curie Center in Alger between March 1, 2012 and May 31, 2015. We performed a prospective study of 300 female patients aged 25–50 years, and looked for HPV lesions at high risk of cervical cancer. Our investigation focused on three successive campaigns; each one including kits for 100 patients. Sample collection was performed in the most visited healthcare facilities of Sidi Bel Abbès province, the family planning clinic of Sidi Bel Abbès maternity, the polyclinic of Ain el berd, and the medical center of Sidi Lahcen. Specific screening guidelines were not issued for patients at risk of cervical lesions as all sexually active women are at risk of cervical cancer. Our study primarily focused on women who had never had any screening Pap smear performed. Midwives performed residual Pap smear collection in liquid media and sent them to the Pierre and Marie Curie laboratory of Alger to look for viral HR-HPV using the Hybrid Capture II technique. The Hybrid Capture test is a homogeneous hybridization assay using unmarked single strand RNA probes, compatible with the targeted sequences. The association kinetics of nucleic
acid strands is boosted in liquid media; the reaction can thus be more rapidly completed. Cellular DNA is denatured and hybridized with specific RNA probes. The resulting hybrid molecules are either captured in the well of a microplate or a tube by polyclonal antibodies specifically targeting RNA-DNA hybrids. Following washing, RNA-DNA molecules are detected by conjugated monoclonal antibodies with alkaline phosphatase. The addition of dioxetane triggers light emission measured by a luminometer. Tests performed were defined as positive or negative, depending on the strength of relative light units (RLU) as compared with 1 pg/mL HPV16 DNA and with a positive control (PC). Samples were considered positive when the RLU/PC ratio was ≥ 1 [14]. HR-HPV positive patients had three HC II tests performed 9 months apart from one another. Patients presenting with HRHPV infection persisting for more than 18 months (three positive HC II tests) had a colposcopy and a guided biopsy performed. Reliability of the HC II tests versus guided cervical biopsies was fundamental and needed to be discussed in our study. The evaluation was based on the histological detection of Grade 1 or more neoplastic lesions (CIN1 or more). The relevance of cotesting (HPV test and guided biopsy) also needed to be clarified to adjust the long-term follow-up of our patients. ® Data was captured on Microsoft Excel , and statistical data was analyzed (sensitivity and positive predictive value) using the BioStat 5.3.0 software.
3. Results and discussion 3.1. Study of HR-HPV prevalence Our findings revealed that the prevalence of HR-HPV infection among all Pap smears analyzed by Hybrid Capture II test was 7.33% (22/300), while most Pap smears (92.66%) were negative. During the three campaigns undertaken in the reference healthcare facilities of Sidi Bel Abbès province, the prevalence of HR-HPV infection was 14% between 2012 and 2013. A drastic drop in prevalence was then observed more than three times (4%) between 2013 and 2015 (Fig. 1). On the basis of data analysis, the prevalence of HR-HPV infection also seemed to be related with the patient’s age. This important factor was able to change result interpretation. We observed a mean age of infected women of 37.90 ± 3 years. This prevalence was low among women aged less than 30 years and increased in women aged over 30 years, with a peak between 30 and 35 years (Fig. 2). The prevalence of HPV infection in Africa widely varies by country, ranging from 12 to 46% [15]. The prevalence is much higher in Europe (29%), Northern America (25%), South and Central America (25%), and Asia (32.6%) [16]. These results highlight that the infection prevalence in Algeria is low (7.33% on average) as compared with that reported in the literature. Literature data indicates that the prevalence of HR-HPV infection is higher among young women [17]. However, we observed a higher prevalence of the infection among older
Please cite this article in press as: Serour Y, et al. HPV test by Hybrid Capture II for the diagnosis of HR-HPV persistent infection. Med Mal Infect (2017), http://dx.doi.org/10.1016/j.medmal.2017.05.013
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Fig. 3. Persistent HR-HPV infection rate by patient’s age. Taux d’infections à HPV-HR persistant selon l’âge des patientes. Fig. 1. Prevalence of HR-HPV infections during the three campaigns (2012–2015). Prévalence des infections à HPV-HR au cours des trois campagnes (2012–2015).
Fig. 2. Prevalence of HR-HPV infection by age. Prévalence des infections à HPV-HR selon l’âge des patientes.
women (37.90 ± 3 years). We also observed that HR-HPVinfected women aged over 30 years were 116 times more likely to have severe cervical dysplasia than non-infected women [18].
decreased between 36 and 40 years, and newly increased between 41 and 50 years (Fig. 3). Repeating the HC II test aimed to evaluate the likelihood of contracting a persistent HR-HPV infection, and to assess the risk of high-grade cervical lesions. A single positive HPV test is indeed not associated with the presence of an underlying cervical lesion [19]. We did not observe any significant difference between the first and the second HC II test (95.45% of patients presented with a persistent infection), although literature data indicates that an HPV infection persisting for more than six months is likely to become permanent and to increase the risk of disease progression [20–23]. Our study also confirmed that the persistence of HR-HPV infections is clearly linked to the patient’s age. Women who tested positive to their second HC II test were older (38.6 ± 7 years on average). On the basis of literature data, the detection of HPV DNA in women aged over 35 years is often associated with a persistent infection [24]. These women are usually considered the real group of patients at risk of cervical cancer [24]; their relative risk of cervical cancer ranges from 4.5 to 20.7 the first two years [25]. However, a very low rate of clearance (4.54%) was observed in a 29-year-old patient included in our study. Indeed, HPV infections in most patients aged below 30 years are transient and associated with the clearing of cytological abnormalities that the virus may have induced [26]. Theoretically, a negative result is predictive of a very low risk of invasive cancer in the following six years [27].
3.2. Study of HR-HPV persistence 3.3. Study of HR-HPV infection progression We performed a second HC II test in HR-HPV-positive patients for the purpose of their follow-up, but also to reassure them. Among followed-up women, 95.45% presented with a persistent infection for an average duration of 9.3 months [95% CI: 9.5–10.1 months], and the infection resolved in only one woman (4.54%). Mean age of women presenting with a persistent HRHPV infection was 38.06 ± 7 years. The infection prevalence increased among women aged between 30 and 35 years,
Women who tested positive to the second HPV test were asked to come back nine months later for a third test. Results of that third test revealed that all HR-HPV infections persisted for an average duration of 18.5 months [95% CI: 16.9–22.1 months]. Eighteen patients of our study (85.71%) had a colposcopy performed: 16.66% were normal and 83.33% revealed abnormalities (72.22% of G1AT [grade 1 atypical transformations] and 11.11% of G2AT [grade 2 atypical transformations]). Histological analyses of cervical biopsies revealed that 33.33%
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Fig. 4. Histological results of cervical biopsies under colposcopic control. Résultats histologiques des biopsies cervicales réalisées sous contrôle colposcopique.
Fig. 5. HC II test performance versus cervical biopsy. Performance du test HC II par rapport à la biopsie cervicale.
of women did not present with any abnormalities; 55.55% developed CIN1 (grade 1 cervical intraepithelial neoplasia), and 11.11% developed CIN2 (Fig. 4). We observed an average sensitivity of the HC II test of 81.74% [95% CI: 71.3–89.6], and the average positive predictive value of a positive HR-HPV test associated with an abnormal biopsy was 27.49% [95% CI: 17.0–33.33]. Both values (sensitivity and positive predictive value) of the HC II test increased between the age of 30 and 45 years and decreased in women aged over 46 years (Fig. 5). We observed that a median persistence time of 18.5 months of the HR-HPV infection increased the progression risk of cervical lesions. It has indeed been already proven that HR-HPV infection persisting for more than 12 or 18 months is a good predictor of current or future lesions [28]. Pre-cancerous cervical lesions are also indicative of immune evasion in papillomavirus context [28]. Colposcopies performed for the study purposes confirmed the HC II test results in 83.33% of cases. The percentage of histologically confirmed lesions by cervical biopsy also highlighted the correlation with the proportion of positive HC II tests in 66.66% of cases. The reliability of the targeted biopsy was called into question in our study. This histological confirmation was required to avoid useless treatments and their major complications [29]. However, its reproducibility is not perfect, especially for low-grade CIN (CIN1) [30].
CIN2 lesions are now considered a questionable diagnosis of pre-cancerous cervical lesions [31]. In some patients, cervical lesions may persist and progress to CIN3 as they have a very low potential for regression [32]. The sensitivity of the HC II test and its positive predictive value observed in our study were consistent with those reported in Schiffman’s study (2000), as he reported a sensitivity of 88.4% at a threshold of 1 pg/mL and a positive predictive value of 29% versus 50% [33]. A few authors reported significant associations between older women and a decreased clearance [34,35]. The mean age of patients (39.06 ± 8 years) who had a third HC II test performed was an excellent factor for determining the persistence of HRHPV infection and the risk of progression to CIN2, mainly observed in women aged above 40 years. The management of diagnosed cervical lesions was crucial in our study. With regard to CIN1 lesions, offering a follow-up using a new cytology at 12 months and/or a HPV test, having both results negative leads to a cytology performed at 24 months while having one or both results positive requires a control test six months later using cytology and/or HPV [36]. A twice yearly cytological follow-up and a colposcopic follow-up for 6 to 12 months is, however, recommended in women presenting with CIN2 lesions [37]. The cytological control performed at six months in our study was consistent with the HC II test results in 66.66% of cases. Among controlled patients, 33.33% had ASCUS (atypical squamous cells of undetermined significance) Pap smears, 44.44% had LSIL (low-grade squamous intraepithelial lesions), and 22.22% had HSIL (high-grade squamous intraepithelial lesions). As for the treatment strategy for histologically confirmed cervical lesions observed in our study, 95% of patients received different tailored treatments. For CIN1 lesions, conization is only indicated when the HPV test remains positive for at least 18 months. CIN2 lesions may be removed by surgical resection (conization) or other physical methods [38]. On the basis of analyzed data, HPV test may be used as a discriminating factor in the management of patients presenting with intraepithelial neoplasia [37]. On top of their medical use, HPV tests provide economic benefits as fees associated with Pap smears and repeated colposcopy are avoided [39]. 4. Conclusion Detection of high risk HPV infection is currently well established in Algeria. Our study aimed to systematically screened HPV in Algerian women aged between 25 and 50 years to detect any persistent HR-HPV infection, as the infection plays a major role in cervical cancer. The prevalence of the infection in our region is on average 7.33%, including 90.9% of persistent HRHPV at high risk of progression, especially among women aged above 30 years. We confirmed the persistent nature of the infection by colposcopy and cervical biopsy, but the mechanism promoting disease progression remains poorly known. Our study findings highlighted that the repeated use of the Hybrid Capture II Qiagen test is a reliable strategy to identify
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women at high risk of cervical cancer. However, a combined use of the HC II test and of other tests (colposcopy and cervical biopsy) will help better detect specific pre-cancerous cervical lesions. Contribution of authors Serour Yamina supervised the study and the writing of the article. Bendahmane Malika designed the study protocol. Abbou baker Fadl Allah performed the post-HPV test followup. Medles Merieme performed the statistical analysis. Kraiba Radia performed the viral search for HR-HPV using Hybrid Capture II test. Disclosure of interest The authors declare that they have no competing interest. Acknowledgment The authors would like to thank all members of the Public Health Department of Sidi Bel Abbès region, especially Dr Abri Miloud and Dr Abdi Malika for their support, advice, and educational overview. References [1] Richardson H, Kelsall G, Tellier P, et al. The natural history of type-specific human papillomavirus infections in female university students. Cancer Epidemiol Biomarkers Prev 2003;12(6):485–90. [2] Herrero R, Castle PE, Schiffman M, et al. Epidemiologic profile of type-specific human papillomavirus infection and cervical neoplasia in Guanacaste, Costa Rica. J Infect Dis 2005;191:1796–807. [3] IARC. Handbooks of cancer prevention. Cervix cancer screening. Lyon: IARC Press; 2005. [4] Franco EL, Villa LL, Rahal P, Ruiz A. Molecular variant analysis as an epidemiological tool to study persistence of cervical human papillomavirus infection. J Natl Cancer Inst 1994;86(20):1558–9. [5] Liaw KL, Glass AG, Manos MM, et al. Detection of human papillomavirus DNA in cytologically normal women and subsequent cervical squamous intraepithelial lesions. J Natl Cancer Inst 1999;91(11):954–60. [6] Schiffman M, Castle PE. The promise of global cervical cancer prevention. N Engl J Med 2005;17(353):2101–4. [7] Cuschieri KS, Cubie HA, Whitley MW, et al. Multiple high risk HPV infections are common in cervical neoplasia and young women in a cervical screening population. J Clin Pathol 2004;57:68–72. [8] Koutsky LA, Holmes KK, Critchlow CW, Stevens, et al. A cohort study of the risk of cervical intraepithelial neoplasia grade 2 or 3 in relation to papillomavirus infection. N Engl J Med 1992;327(18):1272–8. [9] Fender M, Schott J, Baldauf JJ, et al. EVE, une campagne régionale de dépistage du cancer du col de l’utérus. Organisation, résultats à 7 ans etperspectives. Presse Med 2003;32:1545–51. [10] Spence AR, Goggin P, Franco EL. Process of care failures in invasive cervical cancer: systematic review and meta-analysis. Prev Med 2007;45(2–3):93–106. [11] Denny L, et al. Direct visual inspection for cervical cancer screening: an analysis of factors influencing test performance. Cancer 2002;94(6):1699–707.
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Original article
HPV test by Hybrid Capture II for the diagnosis of HR-HPV persistent infection Test HPV par Hybrid Capture dans le diagnostic de l’infection HPV-HR persistante Y. Serour a,b,∗ , M. Bendahmane a,b , F. Abbou Baker c , M. Medles a,b , B. Moueddene c , R. Kraiba d b
a Faculté des sciences de la nature et de la vie, université Djillali Liabes, Sidi Bel Abbés, Algeria Laboratoire de recherche en environnement et santé (LRES), CHU de Sidi Bel Abbes, Sidi Bel Abbés, Algeria c Service de planning familial de la maternité, Sidi Bel Abbés, Algeria d Institut Pasteur d’Alger, Centre Pierre et Marie Curie, Alger, Algeria
Received 26 January 2016; received in revised form 5 June 2016; accepted 31 May 2017
Abstract Introduction. – Persistent high-risk HPV (HR-HPV) infection is associated with a greater risk of cervical cancer. Patients and methods. – Statistical data on the prevalence of HR-HPV infections in the Algerian population is lacking. We conducted a prospective study of 300 women aged between 25 and 50 years, screened for cervical cancer from 2012 to 2015 in Sidi Bel Abbès, a western region of Algeria. We aimed to assess the reliability of the repeated use of the HC II test (three longitudinal HPV tests 9 months apart from each other) in diagnosing the persistence of HR-HPV infection. Results. – The prevalence of HR-HPV infection was 7.33% and infected women were aged 37.9 ± 3 years. For 90.9% of HR-HPV-positive patients, the infection persisted for a mean of 18.5 months [95% CI: 16.9–22.1 months]. Among these patients, 55.55% developed CIN1 and 11.11% developed CIN2. The sensitivity of the HC II test was 81.74% [95% CI: 71.3–89.6] and its positive predictive value associated with abnormal cervical biopsy was 27.49% [95% CI: 16.0–33.33]. Conclusion. – Repeating the HC II test is a good predictor for identifying women at high risk of cervical cancer. © 2017 Elsevier Masson SAS. All rights reserved. Keywords: Persistent HR-HPV; HC II test; Cervical biopsy
Résumé Introduction. – L’infection persistante à HPV à haut risque (HPV-HR) est associée à un risque élevé de cancer du col utérin. Patients et méthodes. – Vu le manque considérable de données statistiques sur la prévalence de cette infection dans la population algérienne, nous avons entrepris une étude prospective dans la ville de Sidi Bel Abbès, une région de l’ouest Algérien. Cette étude a été effectuée entre 2012 et 2015 auprès de 300 femmes (25–50 ans), dépistées pour le cancer du col de l’utérus. Le but de cette recherche était d’évaluer la fiabilité de l’utilisation répétée du test HC II (trois tests HPV longitudinaux espacés de 9 mois chacun) pour diagnostiquer la persistance de l’infection HPV-HR. Résultats. – Les résultats obtenus ont montré que la prévalence de l’infection HPV-HR était de 7,33 % et que l’âge moyen des femmes affectées était de 37,90 ± 3 ans. Chez 90,9 % des patientes HPV-HR positives, cette infection a persisté pendant une durée moyenne de 18,5 mois [IC à 95 % : 16,9–22,1 mois], dont 55,55 % ont développé une CIN grade 1 et 11,11 % une CIN2. La sensibilité du test HPV était de 81,74 % [IC à 95 % : 71,3–89,6] et sa valeur prédictive positive associée à une biopsie cervicale anormale était de 27,49 % [IC à 95 % : 11,0–33,33]. Conclusion. – La répétition du test HC II constitue un bon prédicteur pour identifier les femmes présentant un risque de cancer du col de l’utérus. © 2017 Elsevier Masson SAS. Tous droits r´eserv´es. Mots clés : HPV-HR persistant ; Test HC II ; Biopsie cervicale ∗
Corresponding author. E-mail address: [email protected] (Y. Serour).
http://dx.doi.org/10.1016/j.medmal.2017.05.013 0399-077X/© 2017 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Serour Y, et al. HPV test by Hybrid Capture II for the diagnosis of HR-HPV persistent infection. Med Mal Infect (2017), http://dx.doi.org/10.1016/j.medmal.2017.05.013
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1. Introduction High-risk HPV (HR-HPV) infections have been more extensively studied than low-risk HPV infections because of their close association with cervical cancer [1,2]. Women presenting with persistent HR-HPV infection are at higher risk of cervical cancer [3]. There is currently no consensus on the definition of a persistent or transient HPV infection [4]. The most common definition refers to two or three (or more) positive tests for HPV during the follow-up period [5–7]. The risk of cervical intraepithelial neoplasia (CIN) is proportional to the number of positive tests for HR-HPV [8]. The time interval between consecutive HPV tests reported in the literature ranges from less than a year in some studies to 1 or 2 years in others [9]. Most HPV infections are transient and spontaneously disappear within 12 to 18 months [10]. For patients presenting with persistent HPV infection, colposcopy is recommended in the second-line diagnostic strategy to assess lesion severity, followed by a biopsy to confirm the histological diagnosis [11]. However, the early diagnosis of lesions in a patient presenting with persistent cytological abnormalities and a negative test supports the hypothesis of a false negative result [12]. We detected HR-HPV using Hybrid Capture II assay (HCII; Qiagen, Gaithersburg, MD). This test is practical but it does not allow for identifying the various genotypes; a positive result thus indicates the presence of one or several HR-HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) without distinguishing them [13]. We aimed to evaluate the reliability of repeated HC II tests (three consecutive tests 9 months apart from one another) in the diagnosis of persistent HR-HPV infection. 2. Patients and methods We performed the study at the maternity of Sidi Bel Abbès and at the Institut Pasteur of Pierre and Marie Curie Center in Alger between March 1, 2012 and May 31, 2015. We performed a prospective study of 300 female patients aged 25–50 years, and looked for HPV lesions at high risk of cervical cancer. Our investigation focused on three successive campaigns; each one including kits for 100 patients. Sample collection was performed in the most visited healthcare facilities of Sidi Bel Abbès province, the family planning clinic of Sidi Bel Abbès maternity, the polyclinic of Ain el berd, and the medical center of Sidi Lahcen. Specific screening guidelines were not issued for patients at risk of cervical lesions as all sexually active women are at risk of cervical cancer. Our study primarily focused on women who had never had any screening Pap smear performed. Midwives performed residual Pap smear collection in liquid media and sent them to the Pierre and Marie Curie laboratory of Alger to look for viral HR-HPV using the Hybrid Capture II technique. The Hybrid Capture test is a homogeneous hybridization assay using unmarked single strand RNA probes, compatible with the targeted sequences. The association kinetics of nucleic
acid strands is boosted in liquid media; the reaction can thus be more rapidly completed. Cellular DNA is denatured and hybridized with specific RNA probes. The resulting hybrid molecules are either captured in the well of a microplate or a tube by polyclonal antibodies specifically targeting RNA-DNA hybrids. Following washing, RNA-DNA molecules are detected by conjugated monoclonal antibodies with alkaline phosphatase. The addition of dioxetane triggers light emission measured by a luminometer. Tests performed were defined as positive or negative, depending on the strength of relative light units (RLU) as compared with 1 pg/mL HPV16 DNA and with a positive control (PC). Samples were considered positive when the RLU/PC ratio was ≥ 1 [14]. HR-HPV positive patients had three HC II tests performed 9 months apart from one another. Patients presenting with HRHPV infection persisting for more than 18 months (three positive HC II tests) had a colposcopy and a guided biopsy performed. Reliability of the HC II tests versus guided cervical biopsies was fundamental and needed to be discussed in our study. The evaluation was based on the histological detection of Grade 1 or more neoplastic lesions (CIN1 or more). The relevance of cotesting (HPV test and guided biopsy) also needed to be clarified to adjust the long-term follow-up of our patients. ® Data was captured on Microsoft Excel , and statistical data was analyzed (sensitivity and positive predictive value) using the BioStat 5.3.0 software.
3. Results and discussion 3.1. Study of HR-HPV prevalence Our findings revealed that the prevalence of HR-HPV infection among all Pap smears analyzed by Hybrid Capture II test was 7.33% (22/300), while most Pap smears (92.66%) were negative. During the three campaigns undertaken in the reference healthcare facilities of Sidi Bel Abbès province, the prevalence of HR-HPV infection was 14% between 2012 and 2013. A drastic drop in prevalence was then observed more than three times (4%) between 2013 and 2015 (Fig. 1). On the basis of data analysis, the prevalence of HR-HPV infection also seemed to be related with the patient’s age. This important factor was able to change result interpretation. We observed a mean age of infected women of 37.90 ± 3 years. This prevalence was low among women aged less than 30 years and increased in women aged over 30 years, with a peak between 30 and 35 years (Fig. 2). The prevalence of HPV infection in Africa widely varies by country, ranging from 12 to 46% [15]. The prevalence is much higher in Europe (29%), Northern America (25%), South and Central America (25%), and Asia (32.6%) [16]. These results highlight that the infection prevalence in Algeria is low (7.33% on average) as compared with that reported in the literature. Literature data indicates that the prevalence of HR-HPV infection is higher among young women [17]. However, we observed a higher prevalence of the infection among older
Please cite this article in press as: Serour Y, et al. HPV test by Hybrid Capture II for the diagnosis of HR-HPV persistent infection. Med Mal Infect (2017), http://dx.doi.org/10.1016/j.medmal.2017.05.013
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Fig. 3. Persistent HR-HPV infection rate by patient’s age. Taux d’infections à HPV-HR persistant selon l’âge des patientes. Fig. 1. Prevalence of HR-HPV infections during the three campaigns (2012–2015). Prévalence des infections à HPV-HR au cours des trois campagnes (2012–2015).
Fig. 2. Prevalence of HR-HPV infection by age. Prévalence des infections à HPV-HR selon l’âge des patientes.
women (37.90 ± 3 years). We also observed that HR-HPVinfected women aged over 30 years were 116 times more likely to have severe cervical dysplasia than non-infected women [18].
decreased between 36 and 40 years, and newly increased between 41 and 50 years (Fig. 3). Repeating the HC II test aimed to evaluate the likelihood of contracting a persistent HR-HPV infection, and to assess the risk of high-grade cervical lesions. A single positive HPV test is indeed not associated with the presence of an underlying cervical lesion [19]. We did not observe any significant difference between the first and the second HC II test (95.45% of patients presented with a persistent infection), although literature data indicates that an HPV infection persisting for more than six months is likely to become permanent and to increase the risk of disease progression [20–23]. Our study also confirmed that the persistence of HR-HPV infections is clearly linked to the patient’s age. Women who tested positive to their second HC II test were older (38.6 ± 7 years on average). On the basis of literature data, the detection of HPV DNA in women aged over 35 years is often associated with a persistent infection [24]. These women are usually considered the real group of patients at risk of cervical cancer [24]; their relative risk of cervical cancer ranges from 4.5 to 20.7 the first two years [25]. However, a very low rate of clearance (4.54%) was observed in a 29-year-old patient included in our study. Indeed, HPV infections in most patients aged below 30 years are transient and associated with the clearing of cytological abnormalities that the virus may have induced [26]. Theoretically, a negative result is predictive of a very low risk of invasive cancer in the following six years [27].
3.2. Study of HR-HPV persistence 3.3. Study of HR-HPV infection progression We performed a second HC II test in HR-HPV-positive patients for the purpose of their follow-up, but also to reassure them. Among followed-up women, 95.45% presented with a persistent infection for an average duration of 9.3 months [95% CI: 9.5–10.1 months], and the infection resolved in only one woman (4.54%). Mean age of women presenting with a persistent HRHPV infection was 38.06 ± 7 years. The infection prevalence increased among women aged between 30 and 35 years,
Women who tested positive to the second HPV test were asked to come back nine months later for a third test. Results of that third test revealed that all HR-HPV infections persisted for an average duration of 18.5 months [95% CI: 16.9–22.1 months]. Eighteen patients of our study (85.71%) had a colposcopy performed: 16.66% were normal and 83.33% revealed abnormalities (72.22% of G1AT [grade 1 atypical transformations] and 11.11% of G2AT [grade 2 atypical transformations]). Histological analyses of cervical biopsies revealed that 33.33%
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Fig. 4. Histological results of cervical biopsies under colposcopic control. Résultats histologiques des biopsies cervicales réalisées sous contrôle colposcopique.
Fig. 5. HC II test performance versus cervical biopsy. Performance du test HC II par rapport à la biopsie cervicale.
of women did not present with any abnormalities; 55.55% developed CIN1 (grade 1 cervical intraepithelial neoplasia), and 11.11% developed CIN2 (Fig. 4). We observed an average sensitivity of the HC II test of 81.74% [95% CI: 71.3–89.6], and the average positive predictive value of a positive HR-HPV test associated with an abnormal biopsy was 27.49% [95% CI: 17.0–33.33]. Both values (sensitivity and positive predictive value) of the HC II test increased between the age of 30 and 45 years and decreased in women aged over 46 years (Fig. 5). We observed that a median persistence time of 18.5 months of the HR-HPV infection increased the progression risk of cervical lesions. It has indeed been already proven that HR-HPV infection persisting for more than 12 or 18 months is a good predictor of current or future lesions [28]. Pre-cancerous cervical lesions are also indicative of immune evasion in papillomavirus context [28]. Colposcopies performed for the study purposes confirmed the HC II test results in 83.33% of cases. The percentage of histologically confirmed lesions by cervical biopsy also highlighted the correlation with the proportion of positive HC II tests in 66.66% of cases. The reliability of the targeted biopsy was called into question in our study. This histological confirmation was required to avoid useless treatments and their major complications [29]. However, its reproducibility is not perfect, especially for low-grade CIN (CIN1) [30].
CIN2 lesions are now considered a questionable diagnosis of pre-cancerous cervical lesions [31]. In some patients, cervical lesions may persist and progress to CIN3 as they have a very low potential for regression [32]. The sensitivity of the HC II test and its positive predictive value observed in our study were consistent with those reported in Schiffman’s study (2000), as he reported a sensitivity of 88.4% at a threshold of 1 pg/mL and a positive predictive value of 29% versus 50% [33]. A few authors reported significant associations between older women and a decreased clearance [34,35]. The mean age of patients (39.06 ± 8 years) who had a third HC II test performed was an excellent factor for determining the persistence of HRHPV infection and the risk of progression to CIN2, mainly observed in women aged above 40 years. The management of diagnosed cervical lesions was crucial in our study. With regard to CIN1 lesions, offering a follow-up using a new cytology at 12 months and/or a HPV test, having both results negative leads to a cytology performed at 24 months while having one or both results positive requires a control test six months later using cytology and/or HPV [36]. A twice yearly cytological follow-up and a colposcopic follow-up for 6 to 12 months is, however, recommended in women presenting with CIN2 lesions [37]. The cytological control performed at six months in our study was consistent with the HC II test results in 66.66% of cases. Among controlled patients, 33.33% had ASCUS (atypical squamous cells of undetermined significance) Pap smears, 44.44% had LSIL (low-grade squamous intraepithelial lesions), and 22.22% had HSIL (high-grade squamous intraepithelial lesions). As for the treatment strategy for histologically confirmed cervical lesions observed in our study, 95% of patients received different tailored treatments. For CIN1 lesions, conization is only indicated when the HPV test remains positive for at least 18 months. CIN2 lesions may be removed by surgical resection (conization) or other physical methods [38]. On the basis of analyzed data, HPV test may be used as a discriminating factor in the management of patients presenting with intraepithelial neoplasia [37]. On top of their medical use, HPV tests provide economic benefits as fees associated with Pap smears and repeated colposcopy are avoided [39]. 4. Conclusion Detection of high risk HPV infection is currently well established in Algeria. Our study aimed to systematically screened HPV in Algerian women aged between 25 and 50 years to detect any persistent HR-HPV infection, as the infection plays a major role in cervical cancer. The prevalence of the infection in our region is on average 7.33%, including 90.9% of persistent HRHPV at high risk of progression, especially among women aged above 30 years. We confirmed the persistent nature of the infection by colposcopy and cervical biopsy, but the mechanism promoting disease progression remains poorly known. Our study findings highlighted that the repeated use of the Hybrid Capture II Qiagen test is a reliable strategy to identify
Please cite this article in press as: Serour Y, et al. HPV test by Hybrid Capture II for the diagnosis of HR-HPV persistent infection. Med Mal Infect (2017), http://dx.doi.org/10.1016/j.medmal.2017.05.013
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women at high risk of cervical cancer. However, a combined use of the HC II test and of other tests (colposcopy and cervical biopsy) will help better detect specific pre-cancerous cervical lesions. Contribution of authors Serour Yamina supervised the study and the writing of the article. Bendahmane Malika designed the study protocol. Abbou baker Fadl Allah performed the post-HPV test followup. Medles Merieme performed the statistical analysis. Kraiba Radia performed the viral search for HR-HPV using Hybrid Capture II test. Disclosure of interest The authors declare that they have no competing interest. Acknowledgment The authors would like to thank all members of the Public Health Department of Sidi Bel Abbès region, especially Dr Abri Miloud and Dr Abdi Malika for their support, advice, and educational overview. References [1] Richardson H, Kelsall G, Tellier P, et al. The natural history of type-specific human papillomavirus infections in female university students. Cancer Epidemiol Biomarkers Prev 2003;12(6):485–90. [2] Herrero R, Castle PE, Schiffman M, et al. Epidemiologic profile of type-specific human papillomavirus infection and cervical neoplasia in Guanacaste, Costa Rica. J Infect Dis 2005;191:1796–807. [3] IARC. Handbooks of cancer prevention. Cervix cancer screening. Lyon: IARC Press; 2005. [4] Franco EL, Villa LL, Rahal P, Ruiz A. Molecular variant analysis as an epidemiological tool to study persistence of cervical human papillomavirus infection. J Natl Cancer Inst 1994;86(20):1558–9. [5] Liaw KL, Glass AG, Manos MM, et al. Detection of human papillomavirus DNA in cytologically normal women and subsequent cervical squamous intraepithelial lesions. J Natl Cancer Inst 1999;91(11):954–60. [6] Schiffman M, Castle PE. The promise of global cervical cancer prevention. N Engl J Med 2005;17(353):2101–4. [7] Cuschieri KS, Cubie HA, Whitley MW, et al. Multiple high risk HPV infections are common in cervical neoplasia and young women in a cervical screening population. J Clin Pathol 2004;57:68–72. [8] Koutsky LA, Holmes KK, Critchlow CW, Stevens, et al. A cohort study of the risk of cervical intraepithelial neoplasia grade 2 or 3 in relation to papillomavirus infection. N Engl J Med 1992;327(18):1272–8. [9] Fender M, Schott J, Baldauf JJ, et al. EVE, une campagne régionale de dépistage du cancer du col de l’utérus. Organisation, résultats à 7 ans etperspectives. Presse Med 2003;32:1545–51. [10] Spence AR, Goggin P, Franco EL. Process of care failures in invasive cervical cancer: systematic review and meta-analysis. Prev Med 2007;45(2–3):93–106. [11] Denny L, et al. Direct visual inspection for cervical cancer screening: an analysis of factors influencing test performance. Cancer 2002;94(6):1699–707.
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Please cite this article in press as: Serour Y, et al. HPV test by Hybrid Capture II for the diagnosis of HR-HPV persistent infection. Med Mal Infect (2017), http://dx.doi.org/10.1016/j.medmal.2017.05.013