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Type-specific detection of human papillomaviruses in a followed-up French cohort

Guillaume Besson 1 , Cyrille Coissard 1 , Jean-Paul Bory 2 , Joel Cucherousset 1 , Stéphanie Caudroy 1 , Jean-Marie Nou 1 , Christian Quereux 2 , Philippe Birembaut 1 , Christine Clavel 1

1 Laboratoire Pol Bouin, C.H.U. de Reims, 45 rue Cognacq-Jay, 51100 Reims, France; 2 Department of Obstetrics and Gynecology, C.H.U. de Reims, 51100 Reims, France

Introduction

It is now clear that high-risk (HR) types of human papillomavirus (HPV) are the cause of cervical carcinomas

The persistence of HR HPV infection is significantly associated with progressive disease 1

Women with normal smears and HR HPV genotypes have a 116-fold greater risk of developing high-grade squamous intraepithelial lesions than women without HR HPV 2

The prevalence and significance of a broad spectrum of HR HPV types needs to be fully investigated in cytologically normal and abnormal cervical specimens, in order for HPV testing to be best integrated into cervical screening

One question to be answered is whether co-infection with multiple HPV types is a high risk factor for disease progression

PCR-based genotyping using the Roche prototype line blot assay 3,4 provides a valuable tool to identify HR HPV types and to assess the implications of HPV status. This test provides:

Highly sensitive detection in diverse clinical materials

Accurate discrimination of 37 genotypes

Reliable and sensitive assessment of co-infections

Objective

To use Roche line blot assay HPV genotyping to assess differences in the persistence of HPV infection and risk profiles in a French population

Materials and methods

Primary screening consisted of ThinPrep® liquid-based cytology and HPV testing (for the presence of HR HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68), using the Digene Hybrid Capture® 2 (hc2) test

During the follow-up of 59 women, the Roche line blot assay was performed on samples stored in liquid medium (Figure 1)

Women underwent at least two smears/scrapes, at a minimum interval of 6 months

Follow-up included cytology, biopsy (if necessary), HPV testing using hc2 and, after DNA extraction, HPV genotyping with the Roche line blot assay

HPV genome β-globin Biotin-labeled Biotin-labeled PCR PGMY09/11 β-globin primer pools primer pair Add
HPV genome
β-globin
Biotin-labeled
Biotin-labeled
PCR
PGMY09/11
β-globin
primer pools
primer pair
Add avidin-HRP
conjugate
HPV-positive sample
Wash
result
Develop color
HPV 16
HPV 31
High β-globin
Low β-globin
HPV 11
Denature and hybridize Reference line
Denature and
hybridize
Reference line

Denatured amplicon added to membrane-bound oligonucleotide probe

HPV: Human papillomavirus; PCR: Polymerase chain reaction; HRP: Horseradish peroxidase

Figure 1. Roche line blot assay

Results

Regression was observed in 14 women:

Four women had persistence of the same HPV genotypes:

Type 6 (n=2); types 16 and 52 (n=1); and types 16 and 58

(n=1)

Eight women had persistence of at least one common HPV genotype (Table 1)

Two women had infection with different HPV genotypes:

One case of type16, followed by type 31

One case of type 59, followed by type IS39

Table 1. Patients with clinical regression and at least one common HPV type detected in each sample

Patient

HPV types detected

 
 

Sample 1

Sample 2

1

16

 

16

 

33

2

16

16

 

66

3

16

16

 

52

4

16

16

52

5

16

 

52

52

6

16

 

16

53

7

16

 

16

68

52

8

16

 

16

68

Persistent HPV infection without clinical progression was observed in 26 women:

12 women had persistence of the same HPV genotypes:

Type 16 (n=5); type 51 (n=1); type 58 (n=1); types 16 and 31 (n=2); and types 16 and 52 (n=3)

12 women had persistence of at least one common HPV genotype (Table 2)

Two women had infection with different HPV genotypes:

One type 18, followed by types 16, 31 and 33

One type 18, followed by types 56 and 58

Table 2. Patients with persistent HPV infection, without clinical progression, and at least one common HPV type detected in each sample

Patient

HPV types detected

Sample 1

Sample 2

Sample 3

1 16

16

51

2 16

31

51

52

52

3 16

16

53

4 16

16

16

53

5 16

16

52

6 16

16

31

31

52

67

7 51

16

16

66

52

66

 

66

8 16

16

51

9 16

16

81

10 16

16

33

45

62

11 16

16

58

12 16

16

52

18

 

31

Persistent HPV infection with cytological progression was observed in 19 women:

10 high-grade lesions were detected and confirmed by histology:

Six women had persistence of the same HPV genotypes:

Type 16 (n=2); types 16 and 31 (n=1); types 16 and 51 (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 genotype (Table 3)

Nine low-grade lesions were observed by cytology; two cases were confirmed by histology:

Four women had persistence of the same HPV genotypes:

Type 16 (n=2); type 45 (n=1); and types 6, 52 and 53

(n=1)

Four women had persistence of at least one common HPV

genotype (Table 3)

One woman had infection with different HPV genotypes:

Ty pe 16, followed by types 52 and 53

Table 3. Patients with low- and high-grade lesions and at least one common genotype detected in each sample

Patient

HPV types detected High-grade

Sample 2

Sample 1

HPV types detected Low-grade

Sample 2

Sample 1

1 16

16

16

16

31

52

2 16

16

56

16

52

52

 

56

3 16

16

16

16

33

31

 

52

4 16

16

16

16

31

31

53

CP6108

52

53

Follow-up began in 1997 and is ongoing

The most common HPV type associated with progression was type 16

Double or multiple infections were common, but were not strongly associated with the development of high-grade lesions

Conclusions

These preliminary results confirm the role of

persistent infection with the same HPV type,

particularly type 16, in the development of

high-grade lesions

The detection of co-infections cannot currently

be considered a high-risk prognostic factor

Further studies using the Roche line blot assay

have the potential to expand our knowledge of

disease epidemiology

References

1. Remmink AJ, Walboomers JM, Helmerhorst TJ et al. Int J Cancer 1995;61:

306–11

2. Rozendaal L, Walboomers JM, van der Linden JC et al. Int J Cancer

1996;68:766–9

3. Gravitt PE, Peyton CL, Apple RJ et al. J Clin Microbiol 1998;36:3020–7

4. Gravitt PE, Peyton CL, Alessi TQ et al. J Clin Microbiol 2000;38:357–61