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Resolving repeatedly borderline results of Hybrid Capture 2 HPV DNA Test using polymerase chain reaction and genotyping
Journal of Virological Methods 134 (2006) 252–256
Short communication
Resolving repeatedly borderline results of Hybrid Capture 2 HPV DNA Test using polymerase chain reaction and genotyping Katja Seme, Kristina Fujs, Boˇstjan J. Kocjan, Mario Poljak ∗ Institute of Microbiology and Immunology, Medical Faculty of Ljubljana, Zaloˇska 4, 1000 Ljubljana, Slovenia Received 30 August 2005; received in revised form 6 December 2005; accepted 8 December 2005 Available online 18 January 2006
Abstract The Hybrid Capture 2 HPV DNA Test (hc2) (Digene Corporation, Gaithersburg, MD) is at present the only FDA approved assay for routine detection of human papillomavirus (HPV) infections. A significant analytical inaccuracy of the hc2 near to cut-off was reported recently. To address this problem, 240 samples with repeatedly borderline/equivocal/indeterminate hc2 results (samples with repeated RLU/CO values between 0.4 and 4.0) were tested using the PGMY09/PGMY11 consensus PCR and genotyping in order to resolve their high-risk HPV status. All PGMY09/PGMY11 PCR negative samples were tested in addition using CPI/IIg consensus PCR. A false negative rate of 11.3% and false positive rate of 19.1% were recorded in the samples with repeatedly borderline hc2 results. The corresponding hc2 false reactivity rates in 95 samples selected at random which were clearly hc2 negative (samples with RLU/CO values less than 0.4) and 124 samples selected at random which were clearly hc2 positive (samples with RLU/CO values more than 4.0) were 4.2% and 5.6%, respectively. The proportion of hc2 false reactivity increased with proximity to the hc2 cut-off value. According to the results of the present study, the introduction of an hc2 grey-zone and retesting of samples with repeatedly borderline hc2 results by an alternate HPV detection method, such as the PGMY09/PGMY11 consensus PCR and genotyping, is recommended. © 2005 Elsevier B.V. All rights reserved. Keywords: HPV; hc2; Hybrid capture; Borderline results
Persistent infection with a subgroup of at least 15 highrisk human papillomavirus (HPV) genotypes is a necessary but not sufficient etiological factor for the development of cervical carcinoma—neoplasm, which has a global incidence rate of 400,000 cases annually (de Villiers et al., 2004; Munoz et al., 2003; Walboomers et al., 1999). HPV testing has consequently become an important part of cervical carcinoma screening and detection algorithms during the last decade (Cuschieri and Cubie, 2005; Iftner and Villa, 2003). The Food and Drug Administration of the USA (FDA) has approved recently concurrent HPV and Pap smear screening of women aged 30 years and over. Additionally, several consensus guidelines recommend HPV testing when evaluating patients with a cytological diagnosis of atypical squamous cells of undetermined significance (ASC-US) (Smith et al., 2003; Wright et al., 2002). The Hybrid Capture 2 HPV DNA Test (hc2) (Digene Corporation, Gaithersburg, MD) is at present the only FDA approved
∗
Corresponding author. Tel.: +386 1 543 7453; fax: +386 1 543 7418. E-mail address: [email protected] (M. Poljak).
0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.12.004
assay for routine detection of HPV infections and the only commercially available HPV DNA assay with sufficient scientific data to support its performance in a clinical setting (Iftner and Villa, 2003; Davies et al., 2001; van Doorn et al., 2001). hc2 is a signal amplified hybridization microplate-based assay designed to detect 18 HPV genotypes using two probe cocktails for highrisk HPV genotypes 16/18/31/33/35/39/45/51/52/56/58/59/68, and low-risk HPV genotypes 6/11/42/43/44 (L¨orincz, 1996; L¨orincz and Anthony, 2001; Poljak et al., 1999). According to the manufacturer’s criteria, specimens with a relative light units/cut-off (RLU/CO) ratio of more than 1.0 are considered HPV positive and, surprisingly, no grey-zone is recommended (L¨orincz, 1996; L¨orincz and Anthony, 2001). The hc2 threshold of 1.0 RLU/CO is assigned arbitrarily using a series of controls and this threshold is supported by clinical studies that indicate a very high sensitivity of hc2 for cervical intraepithelial neoplasia grade 3 or high-grade squamous intraepithelial lesions at this hc2 cut-off value (Clavel et al., 2001; Cuzick et al., 1999; Petry et al., 2003; Soderlund-Strand et al., 2005; Vrtacnik-Bokal et al., 2005). However, 100% sensitivity of hc2 for the detection of cervical intraepithelial neoplasia grade 3 or high-grade squamous
K. Seme et al. / Journal of Virological Methods 134 (2006) 252–256
intraepithelial lesions has only been achieved in a few studies (Clavel et al., 2001; Cuzick et al., 1999). Although hc2 is the HPV test used most widely, and many studies have demonstrated that the assay is sensitive and reliable for routine high-risk HPV screening (Castle et al., 2004; Gravitt et al., 2003; Kulmala et al., 2004; L¨orincz and Anthony, 2001; Peyton et al., 1998; Poljak et al., 1999), several recent studies have shown significant analytical inaccuracy and reproducibility problems of the hc2 near to cut-off, due mainly to the cross-reactivity of its high-risk probe cocktail (de Cremoux et al., 2003; Federschneider et al., 2004; Poljak et al., 2002; Schneede et al., 2001; Solomon et al., 2000; Terry et al., 2001; Yamazaki et al., 2001). In response to these shortcomings, the manufacturer modified recently the test procedure to enhance the reproducibility of hc2 by simple reversion of the order of probe and specimen addition and the introduction of short additional incubation. Although the introduction of these modifications has reduced the occurrence of equivocal/indeterminate results and
253
since the HPV status of some borderline samples can be resolved by repeated hc2 testing (Castle et al., 2003; Federschneider and Crum, 2003), a substantial proportion of hc2 borderline samples still needs to be tested by an alternative HPV detection method (de Cremoux et al., 2003; Federschneider and Crum, 2003; Federschneider et al., 2004). Additionally, in the hc2 package insert, the manufacturer recommends “to consider alternate testing methods and/or a repeat specimen if the RLU/CO of a specimen is close to but less than 1.0 and high-risk HPV infection is suspected”, but without specifying exactly which RLU/CO values are “close to but less than 1.0”. Since the proportion of equivocal/indeterminate/borderline hc2 results in routine virological laboratory can be as high as 8% and the procedure for resolving the HPV status of such specimens is at present unclear, it was decided to study this problem in detail in the form of a prospective study in the setting of a routine diagnostic laboratory. In January 2003, therefore, our laboratory decided to report without additional testing as HPV
Fig. 1. Resolving HPV status of 240 samples with repeatedly borderline hc2 results (defined as samples with repeated hc2 RLU/CO values ranging between 0.4 and 4.0) using PGMY09/PGMY11 in-house PCR and HPV genotyping.
254
K. Seme et al. / Journal of Virological Methods 134 (2006) 252–256
high-risk negative only those samples with hc2 RLU/CO less than 0.4 (referred to as clearly hc2 HPV negative samples), and as HPV high-risk positive only those samples with hc2 RLU/CO more than 4.0 (referred to as clearly hc2 HPV positive samples). All samples in the RLU/CO range between 0.4 and 4.0 were retested once with hc2 and, if they again scored 0.4–4.0, they were tested in addition using PGMY09/PGMY11 in-house HPV consensus PCR targeting approx. 450 bp of L1 gene, as described previously (Gravitt et al., 2000; Poljak et al., 2002). To exclude definite HPV infection, all PGMY09/PGMY11 PCR negative samples were additionally tested using CPI/IIg in-house HPV consensus PCR targeting 188 bp of E1 gene, as described previously (Kocjan et al., 2005; Tieben et al., 1993). The quality of each DNA sample was verified by the amplification of a 536 bp fragment of the ubiquitous human beta-globin gene, as described previously (Greer et al., 1991; Poljak et al., 1998a,b). In all HPV PCR positive samples, HPV genotype(s) were determined by restriction fragment analysis of PGMY09/PGMY11 PCR products using seven restriction endonucleases (Bernard et al., 1994) or, when necessary, by sequencing either PGMY09/PGMY11 or CPI/IIg PCR products and using INNO-LiPA HPV Genotyping Assay (Innogenetics, Gent, Belgium). A total of 240 samples with repeatedly borderline hc2 results (defined as specimens with repeated hc2 RLU/CO values ranging between 0.4 and 4.0) were identified during routine hc2 testing between January 2003 and October 2004. One hundred and forty-one samples were in the RLU/CO range 0.4–0.99 and 99 samples in the RLU/CO range 1.0–4.0. After additional HPV PCR testing targeting two different HPV genes and HPV genotyping, as described above, repeatedly borderline hc2 samples were placed into four groups, as summarized in Fig. 1. Thus, 114 samples were HPV negative using hc2 and either negative using both “in-house” PCRs or PCR positive but contained none of the 13 high-risk HPV genotypes included in the hc2 high-risk probe
cocktail represented (considered as hc2 true-negative samples). Fifty-three samples were hc2 and PCR positive and contained at least one of the 13 high-risk HPV genotypes included in the hc2 high-risk probe cocktail (considered as hc2 true-positive samples). Twenty-seven samples were hc2 negative but PCR positive and contained at least one of the 13 high-risk HPV genotypes included in the hc2 high-risk probe cocktail (considered as hc2 false negative samples). Finally, 46 samples were hc2 positive but either negative using both in-house PCRs or PCR positive but contained none of the 13 high-risk HPV genotypes included in the hc2 high-risk probe cocktail (considered as hc2 false positive samples). It should be pointed out that the absence of any of the 13 high-risk HPV genotypes included in the hc2 high-risk probe cocktail in all 17 PCR positive samples considered as hc2 false positive samples (Fig. 1) was confirmed by INNO-LiPA HPV Genotyping Assay. In addition to 240 hc2 borderline specimens, 124 samples selected at random which were clearly hc2 positive (samples with hc2 RLU/CO values more than 4.0) and 95 samples selected at random which were clearly hc2 negative (samples with hc2 RLU/CO values less than 0.4) were additionally tested using the same diagnostic strategy as for hc2 borderline specimens to determine the level of “background” false-reactivity of hc2. Thus, among 124 clearly hc2 positive samples, 117 samples tested PCR positive and contained at least one of the 13 highrisk HPV genotypes included in the hc2 high-risk probe cocktail, while 7 (5.6%) samples tested HPV positive but contained none of the 13 high-risk HPV genotypes included in the hc2 high-risk probe cocktail (considered as hc2 false positive samples). Of 95 clearly hc2 negative samples, 91 samples tested HPV negative with both in-house PCRs, while 4 (4.2%) samples tested PCR positive and contained at least one of the 13 high-risk HPV genotypes included in the hc2 high-risk probe cocktail (considered as hc2 false negative samples).
Fig. 2. The relation between the proportion of hc2 false-reactivity and hc2 RLU/CO values. The white part of each bar represents percentage of hc2 false results and grey part percentage of hc2 true results.
K. Seme et al. / Journal of Virological Methods 134 (2006) 252–256
The relation between the proportion of hc2 false-reactivity and hc2 RLU/CO values is shown in Fig. 2. As expected, the proportion of both hc2 false negativity and false positivity increases considerably with proximity to cut-off value (1.0 RLU/CO), indicating the necessity of introducing a grey-zone. In conclusion, the results of this prospective study showed a significant low-end analytical inaccuracy of the current hc2 HPV high-risk cocktail. It is therefore considered that the introduction of an “in-house” hc2 grey-zone and retesting of samples with hc2 high-risk repeatedly borderline/equivocal/indeterminate results by an alternative, preferably amplification-based HPV detection method, is highly advisable. Since HPV analytical standards or proficiency testing samples are still not established or available commercially, the resolution of the HPV status of any sample with discordant results of two HPV diagnostic tests is problematic. In such cases, therefore, one method (or combination of methods) should be used as the reference standard. In our study, as with early evaluations of molecular methods for Chlamydia trachomatis, two highly sensitive and specific consensus PCRs targeting two different HPV genes associated with reliable genotyping methods provided the reference standard and proved an effective strategy. Other “in-house” PCR protocols (Federschneider et al., 2004) or commercial amplification methods such as SPF10 -LiPA System (Perrons et al., 2005) and Amplicor HPV Test (Poljak et al., 2005) would also be appropriate for resolving the HPV status of repeatedly borderline hc2 samples. References Bernard, H.U., Chan, S.Y., Manos, M.M., Ong, C.K., Villa, L.L., Delius, H., Peyton, C.L., Bauer, H.M., Wheeler, C.M., 1994. Identification and assessment of known and novel human papillomaviruses by polymerase chain reaction amplification, restriction fragment length polymorphisms, nucleotide sequence, and phylogenetic algorithms. J. Infect. Dis. 170, 1077–1085. Castle, P.E., Lorincz, A.T., Scott, D.R., Sherman, M.E., Glass, A.G., Rush, B.B., Wacholder, S., Burk, R.D., Manos, M.M., Schussler, J.E., Macomber, P., Schiffman, M., 2003. Comparison between prototype hybrid capture 3 and hybrid capture 2 human papillomavirus DNA assays for detection of high-grade cervical intraepithelial neoplasia and cancer. J. Clin. Microbiol. 41, 4022–4030. Castle, P.E., Wheeler, C.M., Solomon, D., Schiffman, M., Peyton, C.L., ALTS Group, 2004. Interlaboratory reliability of Hybrid Capture 2. Am. J. Clin. Pathol. 122, 238–245. Clavel, C., Masure, M., Bory, J.P., Putaud, I., Mangeonjean, C., Lorenzato, M., Nazeyrollas, P., Gabriel, R., Quereux, C., Birembaut, P., 2001. Human papillomavirus testing in primary screening for the detection of highgrade cervical lesions: a study of 7932 women. Br. J. Cancer 84, 1616– 1623. Cuschieri, K.S., Cubie, H.A., 2005. The role of human papillomavirus testing in cervical screening. J. Clin. Virol. 32 (Suppl. 1), S34–S42. Cuzick, J., Beverley, E., Ho, L., Terry, G., Sapper, H., Mielzynska, I., Lorincz, A., Chan, W.K., Krausz, T., Soutter, P., 1999. HPV testing in primary screening of older women. Br. J. Cancer 81, 554–558. Davies, P., Kornegay, J., Iftner, T., 2001. Current methods of testing for human papillomavirus. Best Pract. Res. Clin. Obstet. Gynaecol. 15, 677–700. de Cremoux, P., Coste, J., Sastre-Garau, X., Thioux, M., Bouillac, C., Labbe, S., Cartier, I., Ziol, M., Dosda, A., Le Gales, C., Molinie, V., VacherLavenu, M.C., Cochand-Priollet, B., Vielh, P., Magdelenat, H., French Society of Clinical Cytology Study Group, 2003. Efficiency of the Hybrid
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Short communication
Resolving repeatedly borderline results of Hybrid Capture 2 HPV DNA Test using polymerase chain reaction and genotyping Katja Seme, Kristina Fujs, Boˇstjan J. Kocjan, Mario Poljak ∗ Institute of Microbiology and Immunology, Medical Faculty of Ljubljana, Zaloˇska 4, 1000 Ljubljana, Slovenia Received 30 August 2005; received in revised form 6 December 2005; accepted 8 December 2005 Available online 18 January 2006
Abstract The Hybrid Capture 2 HPV DNA Test (hc2) (Digene Corporation, Gaithersburg, MD) is at present the only FDA approved assay for routine detection of human papillomavirus (HPV) infections. A significant analytical inaccuracy of the hc2 near to cut-off was reported recently. To address this problem, 240 samples with repeatedly borderline/equivocal/indeterminate hc2 results (samples with repeated RLU/CO values between 0.4 and 4.0) were tested using the PGMY09/PGMY11 consensus PCR and genotyping in order to resolve their high-risk HPV status. All PGMY09/PGMY11 PCR negative samples were tested in addition using CPI/IIg consensus PCR. A false negative rate of 11.3% and false positive rate of 19.1% were recorded in the samples with repeatedly borderline hc2 results. The corresponding hc2 false reactivity rates in 95 samples selected at random which were clearly hc2 negative (samples with RLU/CO values less than 0.4) and 124 samples selected at random which were clearly hc2 positive (samples with RLU/CO values more than 4.0) were 4.2% and 5.6%, respectively. The proportion of hc2 false reactivity increased with proximity to the hc2 cut-off value. According to the results of the present study, the introduction of an hc2 grey-zone and retesting of samples with repeatedly borderline hc2 results by an alternate HPV detection method, such as the PGMY09/PGMY11 consensus PCR and genotyping, is recommended. © 2005 Elsevier B.V. All rights reserved. Keywords: HPV; hc2; Hybrid capture; Borderline results
Persistent infection with a subgroup of at least 15 highrisk human papillomavirus (HPV) genotypes is a necessary but not sufficient etiological factor for the development of cervical carcinoma—neoplasm, which has a global incidence rate of 400,000 cases annually (de Villiers et al., 2004; Munoz et al., 2003; Walboomers et al., 1999). HPV testing has consequently become an important part of cervical carcinoma screening and detection algorithms during the last decade (Cuschieri and Cubie, 2005; Iftner and Villa, 2003). The Food and Drug Administration of the USA (FDA) has approved recently concurrent HPV and Pap smear screening of women aged 30 years and over. Additionally, several consensus guidelines recommend HPV testing when evaluating patients with a cytological diagnosis of atypical squamous cells of undetermined significance (ASC-US) (Smith et al., 2003; Wright et al., 2002). The Hybrid Capture 2 HPV DNA Test (hc2) (Digene Corporation, Gaithersburg, MD) is at present the only FDA approved
∗
Corresponding author. Tel.: +386 1 543 7453; fax: +386 1 543 7418. E-mail address: [email protected] (M. Poljak).
0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.12.004
assay for routine detection of HPV infections and the only commercially available HPV DNA assay with sufficient scientific data to support its performance in a clinical setting (Iftner and Villa, 2003; Davies et al., 2001; van Doorn et al., 2001). hc2 is a signal amplified hybridization microplate-based assay designed to detect 18 HPV genotypes using two probe cocktails for highrisk HPV genotypes 16/18/31/33/35/39/45/51/52/56/58/59/68, and low-risk HPV genotypes 6/11/42/43/44 (L¨orincz, 1996; L¨orincz and Anthony, 2001; Poljak et al., 1999). According to the manufacturer’s criteria, specimens with a relative light units/cut-off (RLU/CO) ratio of more than 1.0 are considered HPV positive and, surprisingly, no grey-zone is recommended (L¨orincz, 1996; L¨orincz and Anthony, 2001). The hc2 threshold of 1.0 RLU/CO is assigned arbitrarily using a series of controls and this threshold is supported by clinical studies that indicate a very high sensitivity of hc2 for cervical intraepithelial neoplasia grade 3 or high-grade squamous intraepithelial lesions at this hc2 cut-off value (Clavel et al., 2001; Cuzick et al., 1999; Petry et al., 2003; Soderlund-Strand et al., 2005; Vrtacnik-Bokal et al., 2005). However, 100% sensitivity of hc2 for the detection of cervical intraepithelial neoplasia grade 3 or high-grade squamous
K. Seme et al. / Journal of Virological Methods 134 (2006) 252–256
intraepithelial lesions has only been achieved in a few studies (Clavel et al., 2001; Cuzick et al., 1999). Although hc2 is the HPV test used most widely, and many studies have demonstrated that the assay is sensitive and reliable for routine high-risk HPV screening (Castle et al., 2004; Gravitt et al., 2003; Kulmala et al., 2004; L¨orincz and Anthony, 2001; Peyton et al., 1998; Poljak et al., 1999), several recent studies have shown significant analytical inaccuracy and reproducibility problems of the hc2 near to cut-off, due mainly to the cross-reactivity of its high-risk probe cocktail (de Cremoux et al., 2003; Federschneider et al., 2004; Poljak et al., 2002; Schneede et al., 2001; Solomon et al., 2000; Terry et al., 2001; Yamazaki et al., 2001). In response to these shortcomings, the manufacturer modified recently the test procedure to enhance the reproducibility of hc2 by simple reversion of the order of probe and specimen addition and the introduction of short additional incubation. Although the introduction of these modifications has reduced the occurrence of equivocal/indeterminate results and
253
since the HPV status of some borderline samples can be resolved by repeated hc2 testing (Castle et al., 2003; Federschneider and Crum, 2003), a substantial proportion of hc2 borderline samples still needs to be tested by an alternative HPV detection method (de Cremoux et al., 2003; Federschneider and Crum, 2003; Federschneider et al., 2004). Additionally, in the hc2 package insert, the manufacturer recommends “to consider alternate testing methods and/or a repeat specimen if the RLU/CO of a specimen is close to but less than 1.0 and high-risk HPV infection is suspected”, but without specifying exactly which RLU/CO values are “close to but less than 1.0”. Since the proportion of equivocal/indeterminate/borderline hc2 results in routine virological laboratory can be as high as 8% and the procedure for resolving the HPV status of such specimens is at present unclear, it was decided to study this problem in detail in the form of a prospective study in the setting of a routine diagnostic laboratory. In January 2003, therefore, our laboratory decided to report without additional testing as HPV
Fig. 1. Resolving HPV status of 240 samples with repeatedly borderline hc2 results (defined as samples with repeated hc2 RLU/CO values ranging between 0.4 and 4.0) using PGMY09/PGMY11 in-house PCR and HPV genotyping.
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high-risk negative only those samples with hc2 RLU/CO less than 0.4 (referred to as clearly hc2 HPV negative samples), and as HPV high-risk positive only those samples with hc2 RLU/CO more than 4.0 (referred to as clearly hc2 HPV positive samples). All samples in the RLU/CO range between 0.4 and 4.0 were retested once with hc2 and, if they again scored 0.4–4.0, they were tested in addition using PGMY09/PGMY11 in-house HPV consensus PCR targeting approx. 450 bp of L1 gene, as described previously (Gravitt et al., 2000; Poljak et al., 2002). To exclude definite HPV infection, all PGMY09/PGMY11 PCR negative samples were additionally tested using CPI/IIg in-house HPV consensus PCR targeting 188 bp of E1 gene, as described previously (Kocjan et al., 2005; Tieben et al., 1993). The quality of each DNA sample was verified by the amplification of a 536 bp fragment of the ubiquitous human beta-globin gene, as described previously (Greer et al., 1991; Poljak et al., 1998a,b). In all HPV PCR positive samples, HPV genotype(s) were determined by restriction fragment analysis of PGMY09/PGMY11 PCR products using seven restriction endonucleases (Bernard et al., 1994) or, when necessary, by sequencing either PGMY09/PGMY11 or CPI/IIg PCR products and using INNO-LiPA HPV Genotyping Assay (Innogenetics, Gent, Belgium). A total of 240 samples with repeatedly borderline hc2 results (defined as specimens with repeated hc2 RLU/CO values ranging between 0.4 and 4.0) were identified during routine hc2 testing between January 2003 and October 2004. One hundred and forty-one samples were in the RLU/CO range 0.4–0.99 and 99 samples in the RLU/CO range 1.0–4.0. After additional HPV PCR testing targeting two different HPV genes and HPV genotyping, as described above, repeatedly borderline hc2 samples were placed into four groups, as summarized in Fig. 1. Thus, 114 samples were HPV negative using hc2 and either negative using both “in-house” PCRs or PCR positive but contained none of the 13 high-risk HPV genotypes included in the hc2 high-risk probe
cocktail represented (considered as hc2 true-negative samples). Fifty-three samples were hc2 and PCR positive and contained at least one of the 13 high-risk HPV genotypes included in the hc2 high-risk probe cocktail (considered as hc2 true-positive samples). Twenty-seven samples were hc2 negative but PCR positive and contained at least one of the 13 high-risk HPV genotypes included in the hc2 high-risk probe cocktail (considered as hc2 false negative samples). Finally, 46 samples were hc2 positive but either negative using both in-house PCRs or PCR positive but contained none of the 13 high-risk HPV genotypes included in the hc2 high-risk probe cocktail (considered as hc2 false positive samples). It should be pointed out that the absence of any of the 13 high-risk HPV genotypes included in the hc2 high-risk probe cocktail in all 17 PCR positive samples considered as hc2 false positive samples (Fig. 1) was confirmed by INNO-LiPA HPV Genotyping Assay. In addition to 240 hc2 borderline specimens, 124 samples selected at random which were clearly hc2 positive (samples with hc2 RLU/CO values more than 4.0) and 95 samples selected at random which were clearly hc2 negative (samples with hc2 RLU/CO values less than 0.4) were additionally tested using the same diagnostic strategy as for hc2 borderline specimens to determine the level of “background” false-reactivity of hc2. Thus, among 124 clearly hc2 positive samples, 117 samples tested PCR positive and contained at least one of the 13 highrisk HPV genotypes included in the hc2 high-risk probe cocktail, while 7 (5.6%) samples tested HPV positive but contained none of the 13 high-risk HPV genotypes included in the hc2 high-risk probe cocktail (considered as hc2 false positive samples). Of 95 clearly hc2 negative samples, 91 samples tested HPV negative with both in-house PCRs, while 4 (4.2%) samples tested PCR positive and contained at least one of the 13 high-risk HPV genotypes included in the hc2 high-risk probe cocktail (considered as hc2 false negative samples).
Fig. 2. The relation between the proportion of hc2 false-reactivity and hc2 RLU/CO values. The white part of each bar represents percentage of hc2 false results and grey part percentage of hc2 true results.
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The relation between the proportion of hc2 false-reactivity and hc2 RLU/CO values is shown in Fig. 2. As expected, the proportion of both hc2 false negativity and false positivity increases considerably with proximity to cut-off value (1.0 RLU/CO), indicating the necessity of introducing a grey-zone. In conclusion, the results of this prospective study showed a significant low-end analytical inaccuracy of the current hc2 HPV high-risk cocktail. It is therefore considered that the introduction of an “in-house” hc2 grey-zone and retesting of samples with hc2 high-risk repeatedly borderline/equivocal/indeterminate results by an alternative, preferably amplification-based HPV detection method, is highly advisable. Since HPV analytical standards or proficiency testing samples are still not established or available commercially, the resolution of the HPV status of any sample with discordant results of two HPV diagnostic tests is problematic. In such cases, therefore, one method (or combination of methods) should be used as the reference standard. In our study, as with early evaluations of molecular methods for Chlamydia trachomatis, two highly sensitive and specific consensus PCRs targeting two different HPV genes associated with reliable genotyping methods provided the reference standard and proved an effective strategy. Other “in-house” PCR protocols (Federschneider et al., 2004) or commercial amplification methods such as SPF10 -LiPA System (Perrons et al., 2005) and Amplicor HPV Test (Poljak et al., 2005) would also be appropriate for resolving the HPV status of repeatedly borderline hc2 samples. References Bernard, H.U., Chan, S.Y., Manos, M.M., Ong, C.K., Villa, L.L., Delius, H., Peyton, C.L., Bauer, H.M., Wheeler, C.M., 1994. Identification and assessment of known and novel human papillomaviruses by polymerase chain reaction amplification, restriction fragment length polymorphisms, nucleotide sequence, and phylogenetic algorithms. J. Infect. Dis. 170, 1077–1085. Castle, P.E., Lorincz, A.T., Scott, D.R., Sherman, M.E., Glass, A.G., Rush, B.B., Wacholder, S., Burk, R.D., Manos, M.M., Schussler, J.E., Macomber, P., Schiffman, M., 2003. Comparison between prototype hybrid capture 3 and hybrid capture 2 human papillomavirus DNA assays for detection of high-grade cervical intraepithelial neoplasia and cancer. J. Clin. Microbiol. 41, 4022–4030. Castle, P.E., Wheeler, C.M., Solomon, D., Schiffman, M., Peyton, C.L., ALTS Group, 2004. Interlaboratory reliability of Hybrid Capture 2. Am. J. Clin. Pathol. 122, 238–245. Clavel, C., Masure, M., Bory, J.P., Putaud, I., Mangeonjean, C., Lorenzato, M., Nazeyrollas, P., Gabriel, R., Quereux, C., Birembaut, P., 2001. Human papillomavirus testing in primary screening for the detection of highgrade cervical lesions: a study of 7932 women. Br. J. Cancer 84, 1616– 1623. Cuschieri, K.S., Cubie, H.A., 2005. The role of human papillomavirus testing in cervical screening. J. Clin. Virol. 32 (Suppl. 1), S34–S42. Cuzick, J., Beverley, E., Ho, L., Terry, G., Sapper, H., Mielzynska, I., Lorincz, A., Chan, W.K., Krausz, T., Soutter, P., 1999. HPV testing in primary screening of older women. Br. J. Cancer 81, 554–558. Davies, P., Kornegay, J., Iftner, T., 2001. Current methods of testing for human papillomavirus. Best Pract. Res. Clin. Obstet. Gynaecol. 15, 677–700. de Cremoux, P., Coste, J., Sastre-Garau, X., Thioux, M., Bouillac, C., Labbe, S., Cartier, I., Ziol, M., Dosda, A., Le Gales, C., Molinie, V., VacherLavenu, M.C., Cochand-Priollet, B., Vielh, P., Magdelenat, H., French Society of Clinical Cytology Study Group, 2003. Efficiency of the Hybrid
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