Type-specific detection of human papillomaviruses in a

followed-up French cohort

Guillaume Besson1, Cyrille Coissard1, Jean-Paul Bory2, Joel Cucherousset1,
Stéphanie Caudroy1, Jean-Marie Nou1, Christian Quereux2, Philippe Birembaut1, Christine Clavel1
1Laboratoire Pol Bouin, C.H.U. de Reims, 45 rue Cognacq-Jay, 51100 Reims, France;
2Department of Obstetrics and Gynecology, C.H.U. de Reims, 51100 Reims, France

Introduction – Two women had infection with different HPV genotypes: ● Persistent HPV infection with cytological progression was
observed in 19 women:
• One case of type16, followed by type 31
It is now clear that high-risk (HR) types of human papillomavirus – 10 high-grade lesions were detected and confirmed by histology:
● • One case of type 59, followed by type IS39
(HPV) are the cause of cervical carcinomas • Six women had persistence of the same HPV genotypes:
● The persistence of HR HPV infection is significantly associated Table 1. Patients with clinical regression and at least one common HPV type – Type 16 (n=2); types 16 and 31 (n=1); types 16 and 51
with progressive disease1 detected in each sample (n=1); types 16 and 52 (n=1); and types 16 and 68 (n=1)
• Four women had persistence of at least one common HPV
● Women with normal smears and HR HPV genotypes have a Patient HPV types detected
genotype (Table 3)
116-fold greater risk of developing high-grade squamous Sample 1 Sample 2
intraepithelial lesions than women without HR HPV2 – Nine low-grade lesions were observed by cytology; two cases
1 ● 16 ● 16 were confirmed by histology:
● The prevalence and significance of a broad spectrum of HR HPV ● 33 • Four women had persistence of the same HPV genotypes:
types needs to be fully investigated in cytologically normal and
2 ● 16 ● 16 – Type 16 (n=2); type 45 (n=1); and types 6, 52 and 53
abnormal cervical specimens, in order for HPV testing to be best
● 66
integrated into cervical screening (n=1)
3 ● 16 ● 16
• Four women had persistence of at least one common HPV
● One question to be answered is whether co-infection with multiple ● 52
genotype (Table 3)
HPV types is a high risk factor for disease progression 4 ● 16 ● 16
• One woman had infection with different HPV genotypes:
● PCR-based genotyping using the Roche prototype line blot assay3,4 ● 52
5 ● 16 ● 52
– Type 16, followed by types 52 and 53
provides a valuable tool to identify HR HPV types and to assess the
implications of HPV status. This test provides: ● 52
Table 3. Patients with low- and high-grade lesions and at least one common
6 ● 16 ● 16
– Highly sensitive detection in diverse clinical materials genotype detected in each sample
● 53
– Accurate discrimination of 37 genotypes 7 ● 16 ● 16 Patient HPV types detected HPV types detected
– Reliable and sensitive assessment of co-infections ● 68 ● 52 High-grade Low-grade
8 ● 16 ● 16 Sample 1 Sample 2 Sample 1 Sample 2
● 68
Objective 1 ● 16 ● 16 ● 16 ● 16
● 31 ● 52
To use Roche line blot assay HPV genotyping to assess differences ● Persistent HPV infection without clinical progression was
● 2 ● 16 ● 16 ● 56 ● 16
in the persistence of HPV infection and risk profiles in a French observed in 26 women:
● 52 ● 52
population – 12 women had persistence of the same HPV genotypes:
● 56
• Type 16 (n=5); type 51 (n=1); type 58 (n=1); types 16 and
3 ● 16 ● 16 ● 16 ● 16
31 (n=2); and types 16 and 52 (n=3)
● 33 ● 31
Materials and methods – 12 women had persistence of at least one common HPV ● 52
genotype (Table 2)
● Primary screening consisted of ThinPrep® liquid-based cytology 4 ● 16 ● 16 ● 16 ● 16
and HPV testing (for the presence of HR HPV types 16, 18, 31, 33, – Two women had infection with different HPV genotypes: ● 31 ● 31 ● 53 ● CP6108
35, 39, 45, 51, 52, 56, 58, 59 and 68), using the Digene Hybrid • One type 18, followed by types 16, 31 and 33 ● 52
Capture® 2 (hc2) test • One type 18, followed by types 56 and 58 ● 53
● During the follow-up of 59 women, the Roche line blot assay was
performed on samples stored in liquid medium (Figure 1) Table 2. Patients with persistent HPV infection, without clinical progression, ● Follow-up began in 1997 and is ongoing
and at least one common HPV type detected in each sample
● Women underwent at least two smears/scrapes, at a minimum ● The most common HPV type associated with progression was
interval of 6 months Patient HPV types detected type 16
● Follow-up included cytology, biopsy (if necessary), HPV testing Sample 1 Sample 2 Sample 3
● Double or multiple infections were common, but were not
using hc2 and, after DNA extraction, HPV genotyping with the 1 ● 16 ● 16 strongly associated with the development of high-grade lesions
Roche line blot assay ● 51
2 ● 16 ● 31
● 51 ● 52
HPV genome β-globin
● 52 Conclusions
3 ● 16 ● 16
● 53 ● These preliminary results confirm the role of
4 ● 16 ● 16 ● 16 persistent infection with the same HPV type,
Biotin-labeled Biotin-labeled PCR
PGMY09/11 β-globin ● 53
primer pools primer pair
particularly type 16, in the development of
5 ● 16 ● 16
● 52 high-grade lesions
High β-globin
Low β-globin

Denature and
hybridize
6 ● 16 ● 16
HPV 31

HPV 11
HPV 16

Add avidin-HRP
conjugate
Reference line ● 31 ● 31 ● The detection of co-infections cannot currently
● 52 ● 67
be considered a high-risk prognostic factor
7 ● 51 ● 16 ● 16
HPV-positive sample Wash Denatured amplicon added
result Develop color to membrane-bound ● 66 ● 52 ● 66
oligonucleotide probe
● Further studies using the Roche line blot assay
HPV: Human papillomavirus; PCR: Polymerase chain reaction; HRP: Horseradish peroxidase
● 66
8 ● 16 ● 16 have the potential to expand our knowledge of
Figure 1. Roche line blot assay
● 51 disease epidemiology
9 ● 16 ● 16
● 81
Results 10 ● 16 ● 16
References
● 33
● Regression was observed in 14 women: ● 45
● 62 1. Remmink AJ, Walboomers JM, Helmerhorst TJ et al. Int J Cancer 1995;61:
– Four women had persistence of the same HPV genotypes: 306–11
11 ● 16 ● 16
• Type 6 (n=2); types 16 and 52 (n=1); and types 16 and 58 ● 58 2. Rozendaal L, Walboomers JM, van der Linden JC et al. Int J Cancer
(n=1) 12 ● 16 ● 16 1996;68:766–9

– Eight women had persistence of at least one common HPV ● 52 ● 18 3. Gravitt PE, Peyton CL, Apple RJ et al. J Clin Microbiol 1998;36:3020–7
● 31
genotype (Table 1) 4. Gravitt PE, Peyton CL, Alessi TQ et al. J Clin Microbiol 2000;38:357–61