**5. HPV-DNA screening methods with concurrent or reflex HPV-16 & HPV-18 genotyping**

Reflex HR HPV DNA testing is now recognized as a cost-effective strategy to refer women with an ASC-US cytology for colposcopy [58, 59]. High-risk HPV-DNA-based screening assays with individual or pooled HPV-16 and HPV-18 genotyping are a group of novel HPV assays in which qualitative detection of 13–14 HPV types is combined with concurrent or reflex HPV-16 and HPV-18 genotyping. The Abbott RealTi*me* High Risk HPV test (RealTi*me*; Abbott Molecular, Des Plaines, IL, USA) is a real-time PCR assay based on concurrent individual genotyping for HPV-16 and HPV-18 and pooled detection of 12 other HPVs: HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66 and HPV-68. Amplification of human β-globin is used as an internal control. The assay was launched on the European market in January 2009. RealTi*me* is performed on the *m*2000rt real-time PCR instrument (Abbott Molecular) using a modified GP5+/GP6+ primer mix consisting of three forward and two reverse primers [60]. Analytical sensitivity of RealTime was comparable to that of HC2 (97.6% vs 95.1%), whereas RealTime demonstrated significantly higher analytical specificity compared with HC2 [61, 62].

The cobas 4800 HPV Test (Roche Molecular Diagnostics, Pleasanton, CA, USA) is a real-time PCR assay based on concurrent individual genotyping for HPV-16 and HPV-18 and pooled detection of 12 other HPVs: HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66 and HPV-68 [63-65]. An excellent agreement (>93%) was obtained between cobas 4800 HPV and HC2 or Linear Array (LA) for the detection of HR HPV DNA in CIN [66]. The cobas 4800 HPV test will refer fewer women to colposcopy than automatic referral of all women with ASC-US. It will also distinguish women infected with HPV16 or HPV18 from those infected with other HR HPV genotypes, although this feature is not yet included in clinical guidelines for the management of women with ASC-US. When the cobas 4800 HPV was utilized by a diagnostic laboratory, its performance was equivalent to that of HC2 and show comparable levels of performance including results for women >30 years old with ASCUS cytology [67]. Gage et al found agreement between Cobas and LA to be very good, better than that between Cobas and HC2 [68].

The Cervista HPV 16/18 Test (Hologic) is a signal amplification qualitative test based on the Invader chemistry specifically designed to detect HPV-16 and HPV-18 [69].
