Rita ; Calado

Rita Almeida Calado

Maria Raquel Rocha

Ricardo Parreira

João Piedade

Aida Venenno

Esteves

Aida Esteves
email: aidaesteves@ihmt.unl.pt

Oligonucleotides used for RT-PCR amplification of HCV genomic sequences Designation Sequence (5'→3') Location in the H77 genome (AF011753) UTR_f UTRi_r UTRo_r CEo_f CEi_f

Keywords: HCV, subtypes, intravenous drug users, Portugal HCV, subtypes, intravenous drug users, Portugal. 16 17

4 samples with detectable HCV RNA were seronegative for HCV *samples with discordant subtyping results (n=1) or with subtype available only for the NS5B region (n=2) were considered indeterminate (ind)

Table III. Average intra-subtype genetic distances (%) of Portuguese HCV strains grouped per coding sequence, and sampling time period (only for subtypes with n>5). 2008-09). Partial coding sequences for E1 and/or NS5B were obtained from 124 9 individuals with HCV viremia, both mono-infected and co-infected with HIV. 10 Phylogenetic analysis showed that, for both time periods, the most prevalent subtypes 11 were 1a and 3a, found, altogether, in 64.9 and 71.6% of the individuals, respectively for 12 the first and the second sampling periods. However, genotype 4 viruses (subtypes 4a 13 and 4d), introduced later, as inferred by comparison of intra-subtype genetic distances, 14 were also relatively frequent even one decade ago (24.6%). This HCV subtype profile 15 for Portuguese intravenous drug users is in agreement with those described for other 16 southern European countries when in association with drug consumption. With the 17 exception of subtype 1b, phylogenetic trees did not show clustering of the Portuguese 18 sequences, but rather phylogenetic mixing of HCV sequences from different geographic 19 origins, as described previously in other Western countries and suggestive of a large 20 international transmission network. Consistent with the low recombination rates 21 reported for HCV, only one sample revealed discordant subtypes for the two regions 22 analyzed (4d in E1 and 4a in NS5B), representing a potential new recombinant that 23 deserves further analysis. 24 INTRODUCTION 1 2 Hepatitis C virus (HCV) infection affects an estimated number of 130-170 million 3 people worldwide [START_REF] Lavanchy | The global burden of hepatitis C[END_REF]. It is a leading cause of chronic liver disease, 4 cirrhosis, and hepatocellular carcinoma, and the main indication for liver transplantation 5 in the industrialized world [START_REF] Mühlberger | 2 HCV-related burden of disease in Europe: a systematic assessment of incidence, 3 prevalence, morbidity, and mortality[END_REF]. HCV is transmitted mainly 6 through exposure to contaminated blood and blood products. With the advent of routine 7 blood screening programs, consumption of intravenous illicit drugs remains the most 8 common risk factor for HCV acquisition in developed countries. Furthermore, due to 9 common transmission routes with HIV, intravenous drug users show high rates of HIV 10 and HCV coinfection, which may affect adversely hepatitis C natural history and 11 treatment [START_REF] Sulkowski | Viral hepatitis and HIV coinfection[END_REF]. [START_REF] Antaki | The neglected 4 hepatitis C virus genotypes 4, 5 and 6: an international consensus report[END_REF] HCV is a single stranded RNA virus, classified in the genus Hepacivirus of the 13 Flaviviridae family, characterized by a high degree of genetic heterogeneity that 14 represents a major challenge for the development of both vaccines and specific targeted genotypes is still controversial, except for the sustained virological response to current 20 pegylated interferon/ribavirin therapy, with genotypes 1 and 4 presenting the lower 21 response rates [START_REF] Simmonds | Genetic diversity and evolution of hepatitis C virus-15 years on[END_REF][START_REF] Legrand-Abravanel | Influence of the HCV subtype on the 25 Page 25 of 30 John Wiley & Sons Journal of Medical Virology virological response to pegylated interferon and ribavirin therapy[END_REF]. [START_REF] Cochrane | A genetic analysis of hepatitis C 2 virus transmission between injection drug users[END_REF] HCV genotypes/subtypes present distinct distribution patterns, related mainly with 23 specific geographic areas and/or route of transmission [START_REF] Simmonds | Genetic diversity and evolution of hepatitis C virus-15 years on[END_REF]. HCV 24 genotypes 1 and 3 are widespread in Central Africa and Asia, respectively. The led to the hypothesis that subtypes 1a and 3a, globally expanded in the intravenous drug 2 user populations of industrialized countries during the 20 th century, were introduced 3 from African and Asian populations, respectively [START_REF] Pybus | The hepatitis C virus epidemic 10 among injecting drug users[END_REF]. HCV genotype 4 4 has been reported as the most prevalent in Central Africa, the Middle East and Egypt 5 [START_REF] Kamal | Hepatitis C genotype 4: What we know and what we don't 2 yet know[END_REF][START_REF] Antaki | The neglected 4 hepatitis C virus genotypes 4, 5 and 6: an international consensus report[END_REF]. This viral genotype, found at low 6 frequencies in the early 1990s in Southern Europe, has been spreading (mainly subtypes 7 4a and 4d) in the region, where the higher rates are currently found, and seems to be 8 expanding northwards, mostly associated with injection drug use and immigration 9 [START_REF] Chlabicz | High prevalence of genotype 4 among hepatitis C 21 virus-infected intravenous drug users in north-eastern Poland[END_REF] International databases. Given the economic and health burden of HCV infection, the 20 ongoing transmission among intravenous drug users, and the risk of spill over to the 21 general population, the aims of this study were (i) to determine the HCV seroprevalence 22 and active infection rate, (ii) to analyze the diversity of viral subtypes, and (iii) to screen 23 potential recombinant viruses, both in mono-infected and HIV-coinfected intravenous 24 drug users residing in the Lisbon metropolitan area. 25 Taipas) in Lisbon. Sixty six individuals, with a mean age of 31.2±6.39 years, of which 11 66.7% were male and 81.8% of Portuguese nationality, integrated Group A. Group B 12 totalized 135 individuals with a mean age of 37.6±8.5 years, of which the majority were 13 male (72.6%) and Portuguese (87.4%), 17% of whom were infected with HIV. In both 14 groups, the majority of the non-Portuguese individuals (23/29) were from Angola, Cape 15 Viral RNA was extracted from 150 µl of plasma using the INSTANT Virus RNA kit 1 (Analytik Jena, Germany) according to the manufacturer's protocol. RNA was eluted in 2 30 µl of RNase-free water and reverse transcription reactions (RevertAid™ H Minus 3 First Strand Synthesis kit, Fermentas, Canada) were performed on 5 µl of viral RNA 4 with random hexadeoxynucleotides. Subsequently, to detect the presence of HCV RNA, 5 PCR of the 5' untranslated region (5'UTR) was carried out. For subtyping, sections of 6 the envelope (E1), and the polymerase (NS5B) coding regions were further amplified 7 from HCV-RNA positive samples. The primer sets employed and reaction conditions 8 were essentially those described by [START_REF] Ray | Genetic epidemiology of 18 hepatitis C virus throughout Egypt[END_REF], modified for E1 and NS5B to 9 accommodate highly polymorphic nucleotide positions, and also included one internal 10 primer for heminested amplification of the NS5B region (Table I). Amplified DNA 11 products for the E1 and NS5B regions were purified with the QIAquick® PCR 12 Purification kit (QIAGEN, Germany) and sequenced directly with corresponding 13 internal PCR primers. 14 Reference sequences used in the E1 tree (Figure 1A): 1a (AF511950, EU256105, 1 EU362878, EU595699), 1b (AB154200, AJ000009, AJ132996, AY045702, AY587016, 2 D13558), 1c (AY651061), 1g (AM910652), 2a (AB047639, AB047642, AF169002, 3 AF177036, D00944), 2b (AB030907, DQ430817), 2c (D50409), 2i (DQ155561), 2k 4 (AB031663), 3a (AY956467, D17763, D28217, DQ430819, X76918), 3b (D49374), 3k 5 (D63821), 4a (DQ418782, DQ418788, DQ988074, DQ988079, Y11604), 4b 6 (FJ025856), 4d (DQ418786, DQ516083, EU392172, FJ462437), 4f (EU392169), 4k 7 (EU392171), 4l (FJ839870), 4p (FJ462431), 5a (AF064490), 6a (AY859526), 6b 8 (NC_009827), 6c (EF424629), 6k (D84264), 6n (AY878652), 7a (EF108306). 9

Reference sequences used in the NS5B tree (Figure 1B): 1a (AF511950, AY956469, 10 EU256105, EU362878, EU595699, EU862836), 1b (AJ000009, AJ132996, AY045702, 11 AY587016, D13558), 1c (AY051292, AY651061), 1g (AM910652), 2a (AB047639, 12 AB047642, AF169002, AF169003, AF177036, D00944), 2b (AB030907, DQ430817), 13 2c (D50409), 2i (DQ155561), 2k (AB031663), 3a (AY956467, D17763, D28217, 14 DQ430819, X76918), 3b (D49374), 3k (D63821), 4a (DQ418782, DQ418788, 15 DQ988074, DQ988079, NC_009825, Y11604), 4b (FJ025856), 4d (AJ291291, 16 DQ418786, DQ516083, EU392172, FJ462437), 4e (L29590), 4f (EU392169), 4h 17 (FJ807060), 4k (EU392171, EU392173), 4l (FJ839870), 4p (FJ462431), 5a 18 (AF064490), 6a (AY859526), 6b (NC_009827), 6c (EF424629), 6k (D84264), 6n 19 (AY878652), 7a (EF108306).

HCV seroprevalence and active infection 3

The prevalence of anti-HCV antibodies was 86.4% (57/66) for Group A and 60.7% 4 (82/135) for Group B, the former comprising only individuals infected with HIV (Table 5 II). Overall, 83.1% of the individuals infected with HIV were seropositive for HCV 6 while among the HIV-seronegative subjects only 58% presented anti-HCV antibodies, 7 indicating a strong association (p=0.000) between HIV and HCV infection amongst 8 intravenous drug users. However, this association was not maintained when the analysis 9 was restricted to Group B individuals, probably due to the low number of HIV 10 infections in this group. 11 HCV active infection, as determined by RT-PCR amplification of the 5'UTR, was 12 found in 93% and 81.7% of the HCV seropositive samples in Groups A and B, 13 respectively (Table II). Upon grouping all the HIV infected subjects, a statistically 14 significant association (p=0.000) between infection with HIV and active infection with 15 HCV was observed. In intravenous drug users infected with HIV from Group B the 16 active infection rate (88.2%) was higher than for HIV-seronegative intravenous drug 17 users (80%) (Table II), and closer to the value found in Group A (93%). However, as 18 observed for seroprevalence, HCV active infection and HIV infection were not 19 significantly related in Group B. On the whole, 91.9% of the intravenous drug users 20 infected with HIV and seropositive for HCV exhibit active infection by HCV. 21 Curiously, four samples derived from individuals infected with HIV and with a negative 22 serostatus for HCV, in Group A, revealed the presence of HCV RNA. were classified as subtype 1a (46.8%, n=58), followed by subtypes 3a (21.8%, n=27), 16 4d (12.1%, n=15), 4a (11.3%, n=14), 1b (4.8% n=6), and 2a (0.8%, n=1). Viral strains 17 belonging to HCV genotypes 5 and 6 were not detected. All, but one, of the samples 18 revealing amplicons for both regions of the HCV genome showed subtype concordance. 19

The discordant sample (R49C and R49N, in Figure 1A andB, respectively) represents a 20 potential intragenotypic recombinant strain (subtype 4d in E1 and 4a in NS5B), result 21 that awaits confirmation. Therefore, this viral strain was categorized as "indeterminate 22

subtype" together with two others (indicated by an arrow in Figure 1B) with sequences Table II shows the subtype distribution of HCV strains circulating in intravenous drug 1 users from Groups A and B, and according to the HIV status. Although separated by 2 approximately 10 years, both groups disclose similar patterns of HCV subtype 3 distribution, except for an increase in the prevalence of subtypes 1a and 4a strains and a 4 decrease in subtype 4d in the most recently collected samples. Interestingly, a major 5 contribute to the rise of subtype 4a viruses in Group B came from the subgroup of 6 individuals coinfected with HIV, in which this subtype was found in 40% of the HCV 7 strains identified (6/15). In fact, the presence of this subtype appeared statistically 8 associated (p=0.013) with HIV infection in intravenous drug users, but this association 9 should be taken cautiously due to the low number of individuals infected with HIV 10 (n=15). 11

The topology of phylogenetic trees in Figure 1 demonstrated that sequences from 12 Groups A and B, though distantly (time-span) sampled, appeared in interspersed 13 branches all along the different subtype clusters. Besides, the majority of the Portuguese 14 sequences did not segregate into well supported groups, but rather showed considerable 15 phylogenetic mixing with sequences retrieved from databases, representing viral strains 16 from diverse geographic origins. Due to graphical constraints, this branching pattern is 17 illustrated poorly in Figure 1, where the number of subtype reference sequences was 18 kept to a minimum. Additional trees for both E1 and NS5B genomic regions, expanded 19 for individual genotypes trough the introduction of larger numbers of reference 20 sequences, are provided as supplementary data. The latter demonstrate a high extent of 21 HCV transmission within international networks. Contrasting with this, a distinct, and 22 bootstrap-supported, subgroup of sequences within subtype 1b (circled in Figure 1A This study, conducted on two groups of plasma samples collected about ten years apart 3 from Portuguese injecting drug users, revealed prevalences of anti-HCV antibodies 4 between 58% and 86.4%. These are well within the ranges reported for this risk group 5 by Portuguese and most European seroprevalence studies [EMCDDA, 2009]. The 6 higher rates were observed in subgroups including HIV-positive individuals, as 7 described in similar settings. These can be explained by the common route of 8 transmission for both viruses, and the more efficient transmission of HCV (when 9 compared to HIV) through needle-sharing [START_REF] Weinstock | Hepatitis C in an 21 urban population infected with the human immunodeficiency virus[END_REF]Sulkowski and 10 Thomas, 2003;Weissenbacher et al., 2003]. In the same way, compared with HCV 11 mono-infected subjects, patients infected with HIV exhibited superior rates of HCV 12 active infection, suggesting increased difficulties in HCV clearance upon coinfection 13 with HIV [START_REF] Luetkemeyer | Clinical presentation and course of acute hepatitis virus in HIV-infected 5 patients[END_REF][START_REF] Danta | Impact of HIV on host-virus interactions during 6 early hepatitis C virus infection[END_REF]. 14

The global pattern of HCV subtypes circulating in the Portuguese intravenous drug user 15 population analyzed, with predominance of subtype 1a, followed by 3a, 4d and 4a 16 (genotype 4 totalizing >23%), is similar to those disclosed from other intravenous drug 17 user cohorts in Southern Europe, where subtype 1a predominates over 3a, and genotype 18 4 rates are high [van Asten et al., 2004; [START_REF] Esteban | The changing epidemiology of hepatitis C virus 19 infection in Europe[END_REF]. This HCV genotype 19 profile contrasts with other studies targeting different Portuguese population groups, in 20 which high rates of subtype 1b (from 30 to >50%) and low rates of genotype 4 viruses 21 (0 to 8%) were reported [START_REF] Carvalho | HCV 16 genotypes: Liver histopathology and immunologic profile in four groups of patients[END_REF][START_REF] Ramalho | Correlation of genotypes and route of transmission with histologic 15 activity and disease stage in chronic hepatitis C[END_REF]Araújo et al., 22 2005]. However, in a national survey of HCV infected patients, enrolling 80% of 23 intravenous drug users, two thirds of whom were infected with HIV, the distribution of 24 genotypes 1 and 3 matched, though without subtype discrimination, the one described in through the inclusion of numerous sequences from diverse geographic origins 9 (supplementary trees), also demonstrated phylogenetic mixing, not only for subtypes 1a 10 and 3a, but also for subtypes 4a and 4d. However, a well supported specific cluster of 11 subtype 1b sequences was identified. Given the fact that, traditionally, HCV-1b has 12 been associated with viral infection via transfusion and unsafe therapeutic injections 13 [START_REF] Magiorkinis | The global spread of hepatitis C virus 1a and 1b: a phylodynamic and 9 phylogeographic analysis[END_REF], this cluster might represent a spill over event of HCV-1b to 14 the intravenous drug users, followed by local transmission within this population group. [START_REF] Lee | Molecular epidemiology of HCV genotypes among injection drug users in 17 Taiwan: Full-length sequences of two new subtype 6w strains and a recombinant 18 form_2b6w[END_REF] Prior studies, including descriptions of viral genotype distribution in the general 16 population and clinical cohorts, suggested that HCV subtype entrance and dissemination 17 in Portugal followed the general pattern found in other Western countries [Carvalho et 18 al., genetic distances for subtypes 1a, 3a, 4a, and 4d strongly indicates that introduction of 1 the former two subtypes preceded that of genotype 4 strains, paralleling what has 2 occurred elsewhere in Europe [ [START_REF] Van Asten | Spread of hepatitis C virus among European injection drug users infected with 13 HIV: a phylogenetic analysis[END_REF]]. However, it should be pointed out that 3 ten years ago genotype 4 strains were abundant (24.6%), and that both subtypes 4a and 4 4d were already present. Intra-subtype genetic distances being quite analogous, do not 5 support speculations on the relative order of entrance of these HCV subtypes in the 6

Portuguese intravenous drug users. From 1998 to 2009, a slight increase in the 7 prevalence of subtype 1a strains was observed, but significant changes in the 8 proportions of genotypes 3 and 4 were not noticed. However, a shift was detected in the 9 relative proportions of subtypes 4a and 4d during this period. The data presented in this 10 report reveal a decrease in subtype 4d and a concomitant increase in 4a frequencies, the 11 later being significantly associated with HIV-coinfection. Nevertheless, these changes 12 should be regarded cautiously due to limitations of sampling, namely small sample size 13 and its anticipated limited representativeness relative to HCV epidemic among 14

Portuguese intravenous drug users. Furthermore, the unavailability of the most probable 15 date of infection for most individuals and the strong bias towards HIV-coinfection in 16

Group A limit possible hypothesis that may be put forward to account for the apparent 17 epidemics evolution. 18

Inter and intragenotypic, or even intra-subtype, recombinants of HCV have been 19

previously identified [reviewed in [START_REF] Lee | Molecular epidemiology of HCV genotypes among injection drug users in 17 Taiwan: Full-length sequences of two new subtype 6w strains and a recombinant 18 form_2b6w[END_REF]. However, considering the limited 20 number of reports on HCV recombination, either the exchange of genetic information 21 between HCV strains is uncommon in vivo or it has been underestimated due to the 22 generalized use of genotyping methods based on the analysis of a single genomic 23 region. Bearing this in mind, along with the fact that intravenous drug users are highly Phylogenetic trees specifically expanded to accommodate additional HCV E1 coding 17 sequences retrieved from databases for subtype 1a (1), 1b (2), 3a (3), 4a and 4d (4), and 18 additional HCV NS5B coding sequences from databases for subtype 1a (5), 1b (6), 3a 19 (7), and 4a and 4d (8). 20 21 22

  Table II. Subtype distribution of HCV strains sampled, about one decade apart, among intravenous drug users residing in the Lisbon metropolitan area, Portugal

  .8 (n=9)3.1±0.7 (n=6)The pairwise genetic distances were estimated by means of the Kimura 2-parameter method.

  (HCV) infects 2-3% of the world population and intravenous drug 4 consumption is the leading cause of transmission in industrialized countries. The 5 unavailability of data on the molecular epidemiology of HCV infection in Portugal 6 prompted the study of HCV subtypes circulating among intravenous drug users residing 7 in the Lisbon metropolitan area and sampled about ten years apart (1998-2000 and 8

  in these areas suggests long-standing endemic infection, and 1

  two groups of blood samples. One (A), dated from 1998-2000, 5 taken from individuals with a history of intravenous drug use, who at the time of blood 6 collection, were being treated for HIV infection at Hospital Prof. Doutor Fernando da 7 Fonseca (Amadora, Portugal), located in the Lisbon metropolitan area. The other (B) 8 comprised samples collected recently (2008-2009) from intravenous drug users, 9 recruited at a drug treatment centre (Centro de Acolhimento a Toxicodependentes das 10

  Verde, and Mozambique. The study protocol was approved by the Scientific Boards of 16 Instituto da Droga e Toxicodependência and Hospital Prof. Doutor Fernando da Fonseca 17 and by the Ethics Committee of Instituto de Higiene e Medicina Tropical. Subjects 18 provided written consent to be included in the study. Plasma samples were tested for 19 anti-HCV antibodies with the ImmunoFlow HCV rapid test (Core Diagnostics,

  of HCV strains were inferred from the analysis of multiple nucleotide 18 sequences aligned with MAFFT [Katoh et al., 2009]. Neighbor-Joining (NJ) trees were 19 constructed with MEGA4.0.1 [Tamura et al., 2007] using genetic distance matrixes 20 corrected with the Kimura 2-parameter (K2P) formula. The reliability of the inferred 21 topologies was evaluated by bootstrap analysis of 1,000 pseudoreplicates of the original 22 sequence data. Average intra-subtype genetic distances were calculated with default 23 parameters as established in MEGA4.0.1.

20 21 22 Statistical

 2022 Statistics analysis included, when appropriate, calculation of p values of the χ2 test or

  regions, located at the 5' and 3' ends of the HCV genome, were studied. One 4 corresponded to a ~470 nt fragment encoding mostly the N-terminal moiety of E1, 5 while the other consisted of a ~390 nt fragment covering the middle section of the 6 NS5B gene. Primer sets described in Table I allowed the efficient amplification of both 7 viral genomic regions (100% for NS5B, n=124; 98.4% for E1, n=122) in the samples 8 with detectable HCV RNA, i.e. showing a positive 5'UTR PCR amplification result. 9 Phylogenetic analysis performed with HCV sequences amplified from Portuguese 10 intravenous drug users and corresponding reference sequences available in databases 11 allowed the unequivocal genetic subtyping of all viruses. Figure1 (A and B) shows 12 typical Neighbor-Joining trees obtained from the analysis of E1 and NS5B genomic 13 regions, respectively, with groups of sequences corresponding to viral subtypes being 14 supported by high (>70%) bootstrap values. The majority of the Portuguese sequences 15

and 23 B

 23 ), including none of the reference sequences used, might represent a geographic 24 cluster. Additionally, several small groups of two sequences supported by high and with genetic distances <0.05 were also evident. These should 1 result from the local transmission of viruses between individuals related socially. Intra-2 subtype genetic distances estimated for both regions and presented in TableIIIreveal 3 lower values for NS5B, older sequences (specially for E1), and genotype 4 sequences. 4Assuming a constant evolution rate for all HCV subtypes, the genetic distance results 5 obtained for both genomic regions in Groups A and B are suggestive of an earlier 6 introduction of subtypes 1a and 3a in Lisbon intravenous drug users, while a more 7 recent seeding is expected for genotype 4 viruses.

  and a high proportion of HCV genotype 4 (18%) was already found 1[START_REF] Badura | HCV and HCV/HIV in Portugal-a multicentric cohort 13 study. 4 th International Workshop in HCV/HIV co-infection[END_REF]. 2 Typically, phylogenetic analysis of HCV-1a and 3a sequences amplified from IDU 3 communities in different countries does not show geographic clustering, as one could 4 expect from viral transmission through sharing of injection paraphernalia, and 5 suggesting the occurrence of large-scale exchanges of HCV between intravenous drug 6 users due to their high mobility[START_REF] Cochrane | A genetic analysis of hepatitis C 2 virus transmission between injection drug users[END_REF] van de Laar et al., 2005; Morice 7 et al., 2006]. The general phylogenetic profile for Portuguese HCV sequences, obtained 8

  2000; Ramalho et al., 2000; Araújo et al., 2005]. Accordingly, HCV-1b might have 19 been circulating in Portugal for a period of time long enough to give rise to geographic 20 specific clusters. However, since Portuguese HCV sequences are not generally available 21 in the databases, this assumption cannot yet be confirmed. 22 Dynamic change in the prevalence of different subtypes over time characterizes the 23 HCV epidemics in intravenous drug users [van Asten et al., 2004; Pybus et al., 2005].

Figure 1

 1 Figure 1 Phylogenetic relationships between HCV strains from Portuguese intravenous 1

available only for the NS5B region.

Regarding the Portuguese intravenous drug user population analyzed, comparison of

Fernández-Arcás N, López-Siles J, Trapero S, Ferraro A, Ibáñez A, Orihuela F,

Maldonado J, Alonso A. 2006. High prevalence of hepatitis C virus subtypes 4c and 4d


MD Xavier Carcopino

Noel Bolger

Mireille Henry

Julien Mancini

Leon Boubli

Daniel Olive

Sinead Cleary

Walter Prendiville

Catherine Tamalet

Léon Boubli

EVALUATION OF TYPE-SPECIFIC HPV PERSISTENCE AND HIGH-RISK HPV VIRAL LOAD QUANTITATION IN HPV POSITIVE WOMEN UNDER 30 WITH NORMAL CERVICAL CYTOLOGY

Keywords: Human Papillomavirus, Cervical cancer, viral load, genotyping, screening Human Papillomavirus, cervical cancer, viral load, genotyping, screening. 25

HAL is

HR-HPV: high-risk HPV; LR-HPV: low-risk HPV.

*Other high-risk HPV types initially detected: 4(2%) HPV58 and 1(0.5%) HPV82.

**Low-risk HPV types initially detected -25 patients were infected by 30 LR-HPV types (because of multiple infections): 8(4%) HPV6, 6(3%) HPV53, 4(2%) HPV66, 2(1%) HPV61, 2(1%) HPV62, 2(1%) HPV 83, 1(0.5%) HPV1, 1(0.5%) HPV11, 1(0.5%) HPV54, 1(0.5%) HPV70, 1(0.5%) HPV81 and 1(0.5%) HPV84. HR-HPV: high-risk HPV; LR-HPV: low-risk HPV.

F o r P e e r R e v i e w

* Other high risk HPV types: 3 HPV58 and 1 HPV82.

** Low risk HPV types: 6 HPV53, 4 HPV6, 2 HPV62, 2 HPV66, 2 HPV83, 1 HPV11, 1 HPV61, 1 HPV 81 and 1 HPV84. † 3 patients were diagnosed with grade 1 Cervical intraepithelial neoplasia and one with grade 2 cervical intraepithelial neoplasia. ‡ 1 patient was diagnosed with grade 1 cervical intraepithelial neoplasia.

Bold style indicates statistical significance when comparing with other HPV types. 13

F

The aim of this study was to evaluate HPV type-specific prevalence and persistence in a Infants University Hospital and the Adelaïde and Meath Hospital in Tallaght, Dublin. 17

Informed consent was obtained from all the women participating to the study. Infants University Hospital (Dublin, Ireland). Analysis were performed using ThinPrep®

22

Imaging system (Hologic Inc, Nasdaq: Holx). All slides were first screened using the Review in 20-24 and 25-29 years old women, HR-HPV prevalence is 13% and 17% respectively and 3 will decrease to 2.5 and 3.9 % in women over 30 [Cibas et al., 2007;Jacobs et al., 2000].

4

Interestingly, despite the limited number of 39 women in the key core study group, it should 5 be noted that 20 women (51.3%) presented persistent HR-HPV infection. Of these 20 women,

6
six had abnormal cytology, while 14 had sustained normal cytology after a mean interval time 7 period of 11.7 months. Thus, since the critical point is actually to know the future of this 8 persistent HPV infection in these 14 young women it will be of highest importance to monitor The Management of HPV positive young women with normal cytology remains a clinical This study was supported by a grant from the "Fondation de France". We are particularly grateful to the Tallaght's Irish Family Planning Association (IFPA) and 2 particularly to Annet Smith for their great participation to this study. We also want to thank 3 all the patients who accepted to be part of this study. Finally, we thank Fidelma Kavanagh

1423

  population of women under 30 with normal cervical cytology. In that specific population, 15 HPV 16, 18, 31 and 33 viral load, referred to as "oncogenic" HPV viral load, was evaluated 16 as a predictive factor of type-specific HPV infection persistence and of colposcopically 17 documented cervical intraepithelial neoplasia. The impact of HPV genotyping was alsoThe study design is summarized in Figure1. All women under 30 with no previous history of ) clinic in Tallaght, (Dublin, Ireland) for routine screening cervical 1 cytology were invited to participate to the study. All had a liquid-based cervical cytology 2 performed using the ThinPrep® pap test (Hologic Inc, Nasdaq: Holx) and dedicated cervical 3 brush. Women with abnormal cervical cytology were referred for colposcopic examination or 4 repeat cervical cytology according to British Society of Colposcopy and Cervical Pathology 5 (BSCCP) guidelines and were then excluded form the study. All women with normal cervical 6 cytology had a HPV test performed directly on the remaining suspension of the ThinPrep® 7 pap test vial. HPV negative women were excluded from the study and referred for routine 8 screening cytology i.e. three yearly. HPV positive women were asked to return in one-year for 9 a repeat cervical cytology and HPV test. Subsequent cervical smears were performed at the 10 Colposcopy Clinic of the Coombe Women and Infants University Hospital. All were tested 11 for HPV with systematic genotyping of HPV positive sample and quantitation of HPV 16, 18, 12 31 and 33 viral load. Women with a second abnormal smear were referred for colposcopic 13 examination. Colposcopic examinations were blinded to the results of HPV genotyping and 14 viral load quantitation. 15 Study protocol received approval from Ethics Committees of both the Coombe Women and 16

20

  All cervical smears were analyzed at the Pathology department of the Coombe Women and 21

9

  their cytological and virological follow up. What is more, given the consistent association 10 between persistent infection and increased risk of cervical precancer and cancer, persistent 11 HPV infection was the virological endpoint proposed as a relevant clinical marker in the 12 Cervarix vaccine trial [Paavonen et al., 2009](Paavonen, Lancet 2009, 374:301-14). Finally 13 the high incidence of transient HPV infection in young women could also explain the higher 14 probability of abnormal smear in young women as the cytopathic effects of HPV can be 15 detected on cytology [ACOG, 2009]. However, explanation for higher probability of 16 abnormal cytology, as observed in the present study, is probably multifactorial and includes 17 the low socioeconomic level of women attending to the IFPA clinic in Tallaght [Cotton et al., 18 2007]. 19 In the present study the initial HPV 16, 18, 31 and 33 viral load was not found to be 20 predictive of type-specific HPV persistence or the development of cervical intraepithelial 21 neoplasia although in other studies HPV viral load has been demonstrated to be correlated 22 with HPV persistence and subsequent cervical pre-malignancy [Bae et al., 2009; Dalstein et 23 al., 2003; Lai et al., 2008; Munoz et al., 2009; van Duin et al., 2002]. This correlation may et al., 2005]. It seems that no previous reports have evaluated such a 1 correlation in young women with normal smears. 2 Interestingly, mean HR-HPV viral load was relatively high. Indeed, the mean initial HR-HPV 3 viral load measured in the present study was close to the HPV 16 viral load cut-off (>2.2x10 7 4 copies/million cells) identified by Saunier et al. to allow the identification of women with 5 prevalent grade 2 or 3 cervical intraepithelial neoplasia with a high specificity [Saunier et al., 6 2008]. Thus, regardless of age, it has been shown that HR-HPV viral load increases 7 significantly with the severity of cervical lesions [Saunier et al., 2008]. But, in a previous 8 study, such significant association was only found in patients over 30 while mean HPV 16 9 and 18 viral load was already particularly high in women under 30 [Carcopino et al., 2006]. 10 These results suggest that HR-HPV viral load is commonly high in young women, possibly 11 related to the recent occurrence of HPV infection, and would therefore not be of any clinical 12 significance. 13 In this study, only HPV 16 was found to persist and associated with an increased risk of 14 cervical intraepithelial neoplasia. In comparison, LR-HPV types were significantly less likely to persist. This finding illustrates the high oncogenicity of HPV 16 and points to the potential 16 benefit of HPV genotyping in clinical practice. Indeed, HPV 16 has been already shown to 17 persist over a 8-16 months period [Ralston Howe et al., 2009]. The usefulness of HPV 16 18 testing in the management of HPV positive women with normal cervical cytology has been 19 already demonstrated [Khan et al., 2005]. In their study the detection of HPV 16 was 20 associated with a 17.2% risk of grade 3 or more cervical intraepithelial neoplasia over a 10 21 years period (95% CI: 11,5-22,9). In the present study, the absence of significant association 22 between HPV 16 infection and grade 2 or more cervical intraepithelial neoplasia occurrence 23 may be explained by the small number of HPV positive patients and because the study was 24 limited to one-year's follow-up.

4

  and all the staff of the colposcopy clinic of the Coombe Women's Hospital for their help on 5 the management of patients and samples.

F

Table 1 .

 1 Results of initial and 1-year follow up HPV testing and typing.

			HPV test
			Initial	1-year follow up
			(n=199)	(n=39)
	Number of HPV types detected n (%)
	1		41 (20.6)	17 (43.6)
	≥ 2		16 (8)	8 (20.5)
	HPV types detected n (%)
	HPV16		22 (11.1)	15 (38.5)
	HPV18		11 (5.5)	8 (20.5)
	HPV31		6 (3)	2 (5.1)
	HPV33		8 (4)	0 (0)
	Other HR-HPV types*	5 (2.5)	1 (2.6)
	LR-HPV types**	25 (12.6)	7 (17.9)
	HPV viral load (n copies / 10 6 cells) mean (SD)
	HPV16 HPV18 HPV31 HPV33	F r o	8.5.10 5 (2.04.10 6 ) 9.4.10 5 (2.87. 10 6 ) 3.66.10 5 (9.24.10 5 ) 5.27.10 5 (1.08.10 6 ) 4.57.10 6 (4.65.10 6 ) 2.11.10 8 (2.88.10 8 ) 2.37.10 6 (3.85.10 6 ) -
		P
		e
		e r
		R
		e
			v i e
			w

Table 2 .

 2 Rate of one-year HPV type persistence and cervical intraepithelial neoplasia incidence.

	Cervical intraepithelial neoplasia
	All grades	Grade 2 or 3

  Chicago, Illinois). Groups were compared using the Chi square test or the Fisher exact test for

		Carcopino et al. Carcopino et al.
	1 1	signed out by the consultant Pathologist. Cervical cytology analysis were blinded to the result determined for each reaction. Mean CV for each point of the quantification curve in HPV 16,
	2 2	of HPV genotyping and viral load quantitation. 18, 31, 33, 45 and albumin was <3%.
	3 3
	4 4	Virological analysis Statistical analysis
	5	HPV testing was performed at the department of Virology of the Timone's Hospital Statistical analysis was performed using SPSS 15.0 software for Windows (SPSS Inc,
	6 6	(Marseilles, France). All HPV tests were performed using the remaining fluid in the liquid
	7 7	based cytology vials. HPV genotyping was assessed by PCR (MY09/ MY11 primers), qualitative characteristics, and using the Mann-Whitney test for continuous ones. A two-sided
		F o r sequencing, phylogenetic analysis and cloning if necessary, as described previously [Tamalet r o F P value of less than 0.05 was considered statistically significant.
		P P
		e e
		e r e r
		R R
		e e
		v i e v i e
		w w
	21
	22	sample. The data revealed an excellent reproducibilirty of the assays as well for high, median
		or low values. The coefficient of correlation varied from 98.9% for HPV 18 to 99.7% for
		7/20 8/20

23

Scopes® followed by manual secondary screening. Negative cases were signed out by the 24 cytotechnologists and all abnormal cases (Borderline Nuclear Abnormality or greater) were 8 et al., 2010]. Quantitation of HPV 16, 18, 31 and 33 positive samples were performed using 9 quantitative duplex real-time PCR method, as reported previously [Carcopino et al., 2006].

10

HPV viral load was expressed as the number of HPV copies per million cells.

11

For quantification, we used a plasmid which contained the five target sequences of interest:

12

HPV 16,HPV 31, HPV 33,HPV 45 (each on E6 gene), HPV 18 (on E7 gene), and human 13 albumin gene (on exon12). This method allowed HPV 16 and 18, 31, 33, and 45 and albumin 14 gene copy number to be quantified in the same assay. So, HPV viral load could be expressed 15 as HPV copies per cell or per million cells. For the purpose of intra-assay reproducibility, the 16 standard curve was tested 10 times on the same plate and serial dilutions of positive samples 17 (from 107 copies to 1 copy per assay) each with a high HPV 16, 18, 31, 33, or 45 value were 18 tested in duplicate to establish the sensitivity and the reproducibility of the method. We 19 obtained a lower detection limit of 5 copies per 5 µl of sample as well for HPV 16,18, 31, 33, 20 45 as for albumin. The linearity was excellent from the highest value to 1 copy per 5 µl of 23 HPV 16. For the purpose of inter-assay reproducibility, the coefficient of variation (CV) was 5 8 9 RESULTS 10 11 Recruitment ran from September 2007 to the end of March 2009, during which 372 patients 12 participated in the study. Of these, 199 had normal cytology. 173 had an abnormal cervical 13 smear, were excluded from the study and were referred for colposcopic examination or repeat 14 cervical cytology. All of the 199 patients whose cervical cytology was normal were tested for 15

HPV. The overall HPV prevalence was 28.6% (57/199) (Table

1

  Seventeen patients were lost to follow up despite several reminder invitations by mail and 2 telephone (Figure1). The mean patient age was 24.8 years (min-max: 18-30 years). The All of the 39 patients were tested for HPV. Type-specific persistent HPV infection was 6 identified in 20 (51.3%). The mean age of patients with persistent infection and of those who Jacobs et al., 2000]. Although the rates for HR-HPV are generally lower, the 1 prevalence and natural history is comparable to overall HPV. The best evidence suggests that

			Carcopino et al. Carcopino et al. Carcopino et al.
	(min-max: 8.8-18.3 months). One patient conceived and declined a second cervical sample. Patients diagnosed with cervical intraepithelial neoplasia (any grade) were not significantly more likely to have persistent HPV infection: 3 (60%) vs. 17 (50%), respectively, though women over 30 [2 1 2
	3 3	these numbers do not allow accurate assessment of risk. Nor was the initial HPV 16, 18, 31
	4 4	mean interval between smears was comparable for HPV 16, 18, 31 and 33 positive patients as or 33 viral load predictive of cervical intraepithelial neoplasia. Initial mean HPV viral load in
	5	well as for patients who were positive for other HPV types. patients who were diagnosed with cervical intraepithelial neoplasia was 5.2x10 4 copies per
	6	million cells compared to 2.9x10 6 copies per million cells in women with a normal cervix
	7 7	(p=0.263). Also the mean age was comparable in both groups: 23.7 years vs. 24.7 years
	8 9 8	F o r cleared their infection was comparable: 24.7 years vs. 24.5, respectively. HPV 16 was significantly more likely to persist than were all other types: 11 (91.7%) vs. 9 (33.3%), respectively (p<0.001). F o r (p=0.515). Finally, compared to other HPV types, only HPV 16 infection was significantly associated with a higher probability of developing cervical intraepithelial neoplasia: 1 (3.7%) F o r
		P P P
		e e e
	13	e r e r e r
	14
	15	DISCUSSION	R R R
	17 18 19 16 18 19	(5.5%) followed by HPV 33 (4%), HPV 6 (4%), HPV 31 (3%), HPV 53, 58, 66, 61, 62 and 83 (3-1%). Single HPV infection was observed in 41 (20.6%) while 16 (8%) patients had at least e v i e w e v i e w e With a HPV and HR-HPV prevalence of 28.6% and 21.6%, respectively, the present study confirms that HPV, and particularly HR-HPV infection, is very common in women under 30 v i e years old with normal cervical cytology. Such finding concurs with the results of other w
	20 20 20	two different HPV types. The mean HR-HPV viral load for HPV 16, 18, 31 and 33 was studies. In women who reported recent coitarche and who reported just one sexual partner, the
	21 21 21	1.5x10 6 copies / million cells (Standard Deviation (SD): 3.05x10 6 ). The mean HPV 31 viral intraepithelial neoplasia was diagnosed in 4 patients who were advised to have a follow up 1-year cumulative incidence rate of HPV infection is 28.5 % (IC à 95 % : 20,6-38,6 %) and
	22 22 22	load was statistically higher than the mean HPV 16 (p=0.015) or HPV 18 viral loads colposcopy and smear within a year. One patient was diagnosed with grade 2 cervical reaches almost 50% after 3 years [Winer et al., 2008]. The overall cumulative incidence rate
	23	(p=0.019) (Figure 2). intraepithelial neoplasia, confirmed at LLETZ (large loop excision of the transformation of HPV infection in 25 year old women has been estimated to be 80 % [Winer et al., 2006].
		zone).
			9/20 10/20 11/20

). HR-HPV was identified in 16 43 (21.6%) patients. The most prevalent HPV genotypes were HPV 16 (11.1%) and HPV 18 23 After the exclusion of all HPV negative women, 57 remained for analysis. Of these, 39 24 (68.4%) patients had a second cervical sample taken within a mean interval of 11.7 months 1 5 10

Compared to HR-HPV types, low-risk HPV (LR-HPV) were significantly less likely to 11 persist: 17 (54.8%) vs. 3 (15.0%), respectively (p=0.004) (Table

2

). In HPV 16, 18, 31 and 12 33 positive patients the initial overall viral load was not predictive of type specific HPV 13 infection. Mean initial HPV viral load of women with persistent HPV 16, 18, 31 or 33 14 infection was 1.2x10 6 copies per million cells whereas viral load of women who had cleared 15 their infection was 5.3x10 6 copies per million cells (p=0.173).

16

The result of a second smear test was abnormal in 6 of the 39 patients. Three women were 17 reported as having borderline nuclear abnormality (BNA), 2 low-grade squamous 18 intraepithelial lesion (LSIL) and 1 high-grade squamous intraepithelial lesion (HSIL). After 19 colposcopic examination, one patient was diagnosed with normal cervix. Grade 1 cervical 9 vs. 4 (33.3%), respectively (p=0.025) (Table

2

). Despite the fact that the only patient 10 diagnosed with grade 2 cervical intraepithelial neoplasia was HPV 16 positive, HPV 16 11 infection was not significantly associated with higher probability of Grade 2 or 3 cervical 12 intraepithelial neoplasia: 1 (8.33%) vs. 0 (0%), respectively (p=0.3).

23

Thus, the prevalence of HPV infection in young women is very high, with a peak of 24 prevalence of 19.6% for women between 25 and 29 years. This appears to decline to 4.3% in

1

  challenge. The balance between not overtreating women with a minimal risk of progression 2 to cancer and the risk of treatment related morbidity is a fine line[Arbyn et al., 2008;Kyrgiou

	3
	4	et al., 2006]. These results suggest that the use of HPV genotyping and particularly HPV 16
	5	identification may be valuable. Because of the risk of HPV 16 persistence and the
	6	development of cervical intraepithelial neoplasia, young HPV 16 positive patients with
	7	normal smear should be monitored carefully.
	8
	9	CONCLUSION
	10
	11	This study extends the understanding of the natural history of HPV in women under 30 with
	12	normal cervical cytology. In this specific population, HPV type 16, 18, 31 and 33 viral load is
	13	often relatively high. It does not appear to predict type-specific HPV infection persistence or
	14	the development of cervical intraepithelial neoplasia. However, in the present study, HPV 16
	15	infection was more likely to persist and HPV 16 positive patients were also more likely to
	16	develop cervical intraepithelial neoplasia. The use of HPV typing may be valuable in women
	17	under 30.
	18
	19	3020 words
	20
	21	FINANCIAL SUPPORT
	22

  Khan MJ, Castle PE, Lorincz AT, Wacholder S, Sherman M, Scott DR, Rush BB, Glass AG, Sankaranarayanan R, Nene BM, Shastri SS, Jayant K, Muwonge R, Budukh AM, Hingmire S, Malvi SG, Thorat R, Kothari A, Chinoy R, Kelkar R, Kane S, Desai S, Keskar VR, Rajeshwarkar R, Panse N, Dinshaw KA. 2009. HPV screening for cervical cancer in 5 rural India. The New England journal of medicine 360:1385-1394. Saunier M, Monnier-Benoit S, Mauny F, Dalstein V, Briolat J, Riethmuller D, Kantelip B, Schwarz E, Mougin C, Pretet JL. 2008. Analysis of human papillomavirus type 16 8 (HPV16) DNA load and physical state for identification of HPV16-infected women 9 with high-grade lesions or cervical carcinoma. Journal of clinical microbiology Snijders PJ, Schrijnemakers HF, Voorhorst FJ, Rozendaal L, Nobbenhuis MA, 20 van den Brule AJ, Verheijen RH, Helmerhorst TJ, Meijer CJ. 2002. Human 21 papillomavirus 16 load in normal and abnormal cervical scrapes: an indicator of CIN 22 II/III and viral clearance. International journal of cancer 98:590-595. Winer RL, Feng Q, Hughes JP, O'Reilly S, Kiviat NB, Koutsky LA. 2008. Risk of female 1 human papillomavirus acquisition associated with first male sex partner. The Journal Winer RL, Hughes JP, Feng Q, O'Reilly S, Kiviat NB, Holmes KK, Koutsky LA. 2006. Condom use and the risk of genital human papillomavirus infection in young women. The New England journal of medicine 354:2645-2654. Wise J. 2009. Age for starting cervical cancer screening in England will not be lowered. BMJ Woodman CB, Collins S, Winter H, Bailey A, Ellis J, Prior P, Yates M, Rollason TP, Young 9 LS. 2001. Natural history of cervical human papillomavirus infection in young 10 women: a longitudinal cohort study. Lancet 357:1831-1836.

			Carcopino et al. Carcopino et al. Carcopino et al. Carcopino et al. Carcopino et al. Carcopino et al.
	1 1 1	cytologically normal cervical smears: the age-related patterns for high-risk and low-Moberg M, Gustavsson I, Wilander E, Gyllensten U. 2005. High viral loads of human who are referred for typing as a component of cervical cancer screening. American
	2 2	risk types. International journal of cancer 87:221-227. papillomavirus predict risk of invasive cervical carcinoma. British journal of cancer journal of obstetrics and gynecology 200:245 e241-247.
	3 3	92:891-894. of infectious diseases 197:279-282.
	4 4	Schiffman M. 2005. The elevated 10-year risk of cervical precancer and cancer in Munoz N, Hernandez-Suarez G, Mendez F, Molano M, Posso H, Moreno V, Murillo R,
	5 5	women with human papillomavirus (HPV) type 16 or 18 and the possible utility of Ronderos M, Meijer C, Munoz A. 2009. Persistence of HPV infection and risk of
	6 6 6	Paraskevaidis E. 2008. Perinatal mortality and other severe adverse pregnancy type-specific HPV testing in clinical practice. Journal of the National Cancer Institute high-grade cervical intraepithelial neoplasia in a cohort of Colombian women. British
	7 7 7	outcomes associated with treatment of cervical intraepithelial neoplasia: meta-97:1072-1079. journal of cancer 100:1184-1190.
	8 9 10 10	o r analysis. BMJ 337:a1284. Arbyn M, Sasieni P, Meijer CJ, Clavel C, Koliopoulos G, Dillner J. 2006. Chapter 9: Clinical applications of HPV testing: A summary of meta-analyses. Vaccine 24:S78-89. F o r F o r Koliopoulos G, Arbyn M, Martin-Hirsch P, Kyrgiou M, Prendiville W, Paraskevaidis E. F o r F o r r o F 338:b2583.
	11 11	P Bae J, Seo SS, Park YS, Dong SM, Kang S, Myung SK, Park SY. 2009. Natural history of P P P P 46:3678-3685. P
	12 12	e persistent high-risk human papillomavirus infections in Korean women. Gynecologic e e e e e
	13 13	oncology 115:75-80.	e r e r e r e r e r r e
	14	R Bosch FX, Munoz N. 2002. The viral etiology of cervical cancer. Virus research 89:183-190. Carcopino X, Henry M, Benmoura D, Fallabregues AS, Richet H, Boubli L, Tamalet C. 2006. R R R R R
			e e e e e e
	18 19	v i e w v i e w v i e w v i e v i e v i e Mayrand MH, Duarte-Franco E, Rodrigues I, Walter SD, Hanley J, Ferenczy A, Ratnam S, w w w
	20	Coutlee F, Franco EL. 2007. Human papillomavirus DNA versus Papanicolaou
	21	screening tests for cervical cancer. The New England journal of medicine 357:1579-
	22	1588.
		Moberg M, Gustavsson I, Gyllensten U. 2004. Type-specific associations of human
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15 Determination of HPV type 16 and 18 viral load in cervical smears of women referred 16 to colposcopy. Journal of medical virology 78:1131-1140.

2 8 2007. Diagnostic accuracy of human papillomavirus testing in primary cervical 9 screening: a systematic review and meta-analysis of non-randomized studies. 10 Gynecologic oncology 104:232-246. 11 Kyrgiou M, Koliopoulos G, Martin-Hirsch P, Arbyn M, Prendiville W, Paraskevaidis E. 12 2006. Obstetric outcomes after conservative treatment for intraepithelial or early 13 invasive cervical lesions: systematic review and meta-analysis. Lancet 367:489-498. 14 Lai CH, Chao A, Chang CJ, Chao FY, Huang HJ, Hsueh S, Lin CT, Cheng HH, Huang CC, 15 Yang JE, Wu TI, Chou HH, Chang TC. 2008. Host and viral factors in relation to 16 clearance of human papillomavirus infection: a cohort study in Taiwan. International 17 journal of cancer 123:1685-1692. 23 papillomavirus load with risk of developing cervical carcinoma in situ. International 24 journal of cancer 112:854-859.

2 3 4 6 7 11 Schlecht NF, Kulaga S, Robitaille J, Ferreira S, Santos M, Miyamura RA, Duarte-Franco E, 12 Rohan TE, Ferenczy A, Villa LL, Franco EL. 2001. Persistent human papillomavirus 13 infection as a predictor of cervical intraepithelial neoplasia. JAMA 286:3106-3114. 14 Tamalet C, Richet H, Carcopino X, Henry M, Leretraite L, Heid P, Leandri FX, Sancho-15 Garnier H, Piana L. 2010. Testing for Human Papillomavirus and measurement of 16 viral load of HPV 16 and 18 in self-collected vaginal swabs of women who do not 17 undergo cervical cytological screening in southern France. Journal of medical virology 18 82:1431-1437.

19 van Duin M, 3 4 5 6 8

ACKNOWLEDGMENTS

American studies on HPV testing in primary cervical cancer screening. International


Keywords: réseaux de capteur, mobilité, topologie, canaux multiples, localisation, performance de réseau

Ce papier présente des recherches effectuées sur un cas particulier de réseau de capteurs mobiles, celui où la trajectoire des mobiles est fixe et est parcourue cycliquement. L'objectif est de minimiser le délai d'acheminement des messages, le taux de perte et la consommation d'énergie. Les premiers résultats concernent une méthode pour trouver une topologie optimale associée à la trajectoire du ou des capteurs mobiles et un algorithme anticipatif de handover. L'utilisation de canaux de communication multiples permet de minimiser le taux de collision dans le cas de plusieurs capteurs mobiles. Les tests de performance sont réalisés d'abord par simulation pour évaluer l'impact sur le délai de bout en bout et la consommation d'énergie. Une plateforme expérimentale a été mise en place pour mesurer des performances réelles et obtenir le biais du simulateur.

I. INTRODUCTION

Les applications de réseaux de capteurs impliquant une mobilité d'un ou de plusieurs capteurs sont nombreuses. On peut citer le contrôle ou la surveillance de grosses machines industrielles, de véhicules de transport,… . La mobilité fait apparaître des problèmes nouveaux par rapport aux réseaux de capteurs fixes comme le changement de topologie, le routage et les conditions de la propagation radio variables en fonction de la position. Les solutions conçues pour des noeuds fixes ne peuvent pas donc être utilisées directement dans un environnement mobile. Elles engendreraient une consommation d'énergie élevée et une mauvaise qualité de service [START_REF] Akkaya | Energy and QoS Aware Routing in Wireless Sensor Networks[END_REF][2] [START_REF] Cui | Energy-efficiency of MIMO and Cooperative MIMO in Sensor Networks[END_REF]. De nouvelles solutions doivent être trouvées pour s'adapter aux limitations qui existent, ainsi qu'aux facteurs qui rentrent en jeu lors de la conception.

La capacité de changement des canaux de communication est une autre caractéristique importante des unités mobiles qui leur permet de s'adapter dans les environnements radioélectriques stochastiques [START_REF] Gao | Multiradio channel allocation in multihop wireless networks[END_REF].

Les noeuds mobiles doivent être en mesure de découvrir automatiquement, d'une manière autonome, les différents paramètres qui leur permettront de s'auto configurer [START_REF] Basagni | Distributed and mobilityadaptive clustering for multimedia support in multi-hop wireless networks[END_REF], et de s'intégrer dans l'environnement mobile pour devenir opérationnel sans intervention externe.

En plus, ces noeuds doivent acquérir les connaissances nécessaires liées aux localisations et aux contextes de fonctionnement.

Les noeuds mobiles disposent d'une énergie limitée par la capacité de leur batterie qui est parfois difficilement rechargeable en cours de déploiement [START_REF] Theoleyre | A Self-Organization Structure for Hybrid Networks[END_REF] [START_REF] Cui | Energy-constrained Modulation Optimization[END_REF]. Cette caractéristique n'est pas toujours essentielle mais elle est parfois recherchée. Pour prolonger la vie du réseau, il est alors nécessaire de chercher un moyen de réduire la consommation d'énergie [START_REF] Goldsmith | Design challenges for energy constrained Ad Hoc wireless networks[END_REF]. Les noeuds doivent ajuster dynamiquement leur puissance de transmission en fonction de la distance parcourue et de la qualité du canal de transmission. Dans la littérature, l'économie d'énergie en général se fait par anticipation du nombre d'émissions effectué par un noeud. L'algorithme de routage s'avère nécessaire pour prendre en charge la sélection des noeuds de relayage et chercher par la suite à adapter la sélection de ceux-ci en fonction de la puissance disponible.

Lors du déplacement du noeud mobile, et à cause de sa vitesse, la fréquence du signal reçu change : c'est l'effet doppler. Ce décalage de fréquence peut être positif ou négatif. Il induit des difficultés de réception et donc des pertes de messages. Une bonne connaissance de la vitesse du mobile permet de faire des adaptations.

La localisation du capteur mobile a deux objectifs, l'un applicatif et l'autre fonctionnel pour la communication. Au niveau applicatif, les données recueillies par le capteur n'ont en général d'intérêt que si la localisation de la capture de celles-ci les accompagne. A titre d'exemple, citons la surveillance de l'humidité et la température dans un champ agricole. Au niveau communication, la connaissance de la position peut aider à gérer au mieux les paramètres de transmission comme le choix du noeud destinataire des messages émis, le choix du canal de transmission, l'orientation de l'antenne si elle est directive,… Dans le cas où le noeud envoie sa position ainsi que la grandeur mesure, il faut qu'il dispose d'un moyen de connaître sa position. Nous pouvons citer plusieurs approches dans la littérature, le GPS [START_REF] Zhou | Optimal local map registration for wireless sensor network localization problems[END_REF] qui est gourmand en terme d'énergie électrique, mais qui est assez bien adapté au cas de VANETS « Vehicular ad-hoc network » [START_REF] Caliskan | Decentralized disovery of free parking places[END_REF] [START_REF] Zekri | Summarizing Sensors Data in Vehicular Ad Hoc Networks[END_REF]. D'autres approches sont basées sur la méthode de la triangulation utilisant la puissance du signal reçu « RSSI : Received Signal Strength Indication » [START_REF] Theoleyre | A Self-Organization Structure for Hybrid Networks[END_REF]. Les coordonnées relatives du mobile sont calculées en se basant sur ses distances par rapport aux capteurs voisins. Toutefois, des précautions sont nécessaires pour améliorer la plage de confiance, sachant que le calcul se fait à la base de la distance électrique et non pas de la distance géographique.

Cet article présente un travail de thèse ayant pour but de trouver une topologie optimale : le capteur mobile se déplace dans une zone assurée par un ensemble de relais « capteur fixe » dont le nombre est optimal et leur emplacement judicieux assure la couverture radio totale de la trajectoire du noeud mobile et de Le reste de cet article est organisé comme suit : la section 2 présente l'étude et une solution pour la topologie, la section 3 présente le modèle pour le handover et l'algorithme implémenté sous OPNET [12], puis nous donnons quelques résultats obtenus par simulation afin de montrer les avantages de nos algorithmes, la section 4 décrit la plateforme expérimentale montée pour obtenir des résultats plus réalistes, et finalement dans la section 5 nous donnons nos conclusions et nous proposerons quelques perspectives de ces travaux. Le nombre optimal de noeuds fixes est donc 4.

L'application de cette méthode de calcul du nombre de noeuds fixes peut être étendue pour des trajectoires à rayon plus grand.

Pour visualiser la couverture radio, nous avons développé une simulation sous Matlab qui prend en charge les caractéristiques de l'interface radio du type de noeud capteur retenu : le MicaZ de Crossbow [15]. La simulation de la couverture radio est -Une carte de niveaux de champs dite RSSI, Cette carte montre, à travers un dégradé, de couleurs, les niveaux de champs sur la zone géographique de la simulation. Elle permet d'indiquer si le design du réseau de capteur simulé permet d'assurer les niveaux de services souhaités aux endroits souhaités. Cette carte constitue l'élément le plus important d'analyse de la simulation.

-Une carte dite de « meilleur emplacement ». Cette carte montre, à travers une variété de couleurs, les zones de couverture de chaque capteur du réseau simulé. Elle permet ainsi de s'assurer de limiter les cas de dissymétrie entre la surface de couverture des capteurs qui sont souvent à l'origine des problèmes de capacité de trafic (certains capteurs captent tout le trafic et d'autres aucun). Cette carte permet également de connaître les zones de changement de cellules (handover).

Notre simulateur ne prend en charge que l'espace libre en 2D « 400m x 400m » sans obstacle et que la modulation numérique O-QPSK intégrée dans l'interface radio du capteur simulé. Dans notre cas d'étude, nous considérons une mobilité cyclique, dans une trajectoire bien définie. Nous avons exploité ces deux considérations pour élaborer un algorithme de handover anticipatif. Le noeud mobile, au cours de sa phase d'apprentissage, mémorise les positions géographiques de changement de cellules, il les utilisera par la suite pour les prochains passages, ainsi le déclenchement de handover sera basé sur ces informations, le mobile rectifiera ces informations à chaque changement de cellule. Cette approche permet entre autres d'économiser l'énergie du capteur.

PHASE PREPARATOIRE

Une fois que nous avons fixé les positions du puits et des noeuds relais et le noeud mobile, le réseau passe par quatre phases avant le déploiement : -Phase 1 : « initialisation » : le puits est configuré manuellement. Sa table de voisinage contient toutes les informations concernant la position, le nom et le numéro de canal de chaque relais. Initialement tous les capteurs sont configurés pour fonctionner avec le même canal de communication, les relais envoient en diffusion un paquet GET, pour demander au puits de leur communiquer le triplet (sa position relative, son numéro de canal, la table de voisinage).

-Phase 2 : le puits envoie le triplet à chaque noeud relais sans exception, ensuite il envoie en diffusion un paquet à destination du noeud mobile. Le paquet contient toutes les informations concernant les relais (leurs positions, numéro de canal de chacun, leurs noms).

-Phase 3 : le mobile confirme sa réception du paquet au puits. En suite le puits envoie alors un paquet, pour demander au noeud relais de fonctionner avec son numéro de canal.

-Phase 4 « apprentissage »: le mobile commence la phase d'apprentissage, il envoie le paquet HELLO, au début sur chaque canal. Dès qu'il reçoit une réponse du noeud qui a pour coordonnés (+d', 0), il enregistre cette position comme référence « cf. section 4 ». Il construit ainsi sa table de voisinage avec le quintuple (nom du relais précédant, nom du

relais suivant, coordonnées pour changer de cellule, seuil RSSI du relais précédant).

PHASE OPERATIONNELLE

Après la phase d'apprentissage, le capteur mobile commence à envoyer les informations qu'il capte, suivant le rythme décidé à la programmation de l'application, et met à jour sa table de voisinage. A chaque réception de chaque accusé de réception, il calcule la distance qui le sépare avec le noeud relais. Dans la partie qui suit, nous allons expliquer le principe pour calculer dans notre cas les coordonnées du noeud mobile.

ฮMܰ ప ሬሬሬሬሬሬሬሬԦ ฮ ଶ ൌ ฮPM ሬሬሬሬሬሬԦ ฮ ଶ ฮPܰ ூ ሬሬሬሬሬሬሬԦ ฮ ଶ െ 2. ฮPM ሬሬሬሬሬሬԦ ฮ. ฮPܰ ூ ሬሬሬሬሬሬሬԦ ฮ * cosሺPM ሬሬሬሬሬሬԦ , Pܰ ூ ሬሬሬሬሬሬሬԦ ) (10) D'où : cosሺβ ூ ሻ ൌ ୖ మ ାୢ మ ିୢ ᇲ మ ଶୖୢ ᇲ (11)
L'angle (β I ) permettra au noeud mobile de déduire l'angle α. Les résultats relatifs au délai sont donnés sur la figure 6. Au début, le délai est le même pour les deux types de noeuds mobiles, il varie aux alentours de 0,02s. C'est logique puisque les deux mobiles fonctionnent de la même façon, en utilisant le paquet HELLO pour découvrir le voisinage. Deux minutes après le lancement, on remarque que le délai de notre mobile a baissé de 0,02s vers 0,0125s. Le gain est donc significatif avec un écart de 0,0075s.

La figure 7 représente le changement de canal en fonction de l'angle α donné en ordonnée en degrés. Nous avons relié le noeud mobile à la porte d'une voiture utilisée comme mobile. Nous avons placé les noeuds fixes à des endroits déterminés par les calculs décrits dans la section 2. Les noeuds capteurs utilisés sont des MicaZ programmés avec TinyOs [START_REF]TinyOs[END_REF].

Nous avons implémenté les deux algorithmes de handover : le nôtre appelé « Wpan 1 » et celui basé sur le secrétage des canaux et la mesure du RSSI afin qu'il se connecte avec le relais offrant la meilleure RSSI appelé ici « Wpan 2 ».

Afin de réaliser une confrontation des résultats de simulation et ceux de l'expérimentation, nous avons essayé de respecter au maximum la vitesse de 80km/h de rotation ainsi de garder la voiture sur la trajectoire. algorithme et d'une moyenne de 0,009ms avec un écart type de 0,005ms sans anticipation. Il faut remarquer que ces valeurs ne sont pas centrées sur un délai moyen mais sur le délai du premier paquet, ce qui n'est pas vraiment correct mais qui est valable pour une comparaison. Celle-ci donne un avantage à notre méthode.

Figure 9 : Gigue relevée sur la plateforme expérimentale Pour la consommation, nous retrouvons avec les mesures sur la plateforme (figure 10) l'allure des résultats de simulation. Il n'y a pas d'écart important entre les deux approches, il faudra être plus précis dans les mesures pour le vérifier mais le modèle de simulation semble correct. Pourtant, le modèle de la batterie de OpenZB [13] ne prend pas en considération la consommation électrique des circuits intégrés « RAM, UC.. ». Ce sont des valeurs faibles et seules des mesures de longue durée permettront de les quantifier. Mais ceci n'est pas l'objectif principal de notre travail. Nous projetons d'étendre notre étude en utilisant des antennes directives qui devraient apporter un gain significatif puisque le mobile connaît la position de la passerelle après apprentissage. Nous développerons aussi un algorithme d'adaptation de la portée radio selon la position et l'énergie restante des noeuds et un modèle de consommation énergétique afin de faciliter la maintenance. Nous projetons également de prolonger notre étude à d'autres trajectoires de mobiles plus complexes.

  est le suivant : le ou les capteurs mobiles se déplacent en empruntant une trajectoire bien déterminée et ils doivent envoyer des mesures à un puits fixe qui les traite ou les enregistre ou qui sert de passerelle vers un autre réseau sur lequel elles sont retransmises. On suppose que les antennes sont omnidirectionnelles et on note DP le rayon de propagation ou Distance de Perception. Deux cas se présentent : 1-Si la distance D entre le capteur mobile et le puits est inférieure à DP en tout point de la trajectoire, la transmission peut toujours être directe entre le capteur et le puits ; 2-Dans le cas contraire, le capteur mobile se trouve parfois en dehors de la zone de couverture du puits comme le montre la figure 1.

Figure 1 :

 1 Figure 1 : Exemple de trajectoire avec D>DPIl est alors nécessaire de prévoir des noeuds relais fixes ou noeuds mobiles. La question qui se pose est de déterminer le nombre de relais fixes dans notre étude, qui doivent être intercalés entre le mobile et le puits pour avoir une couverture totale, c'est-à-dire un fonctionnement correct en tout point de la trajectoire. Quelle est la meilleure disposition de ces noeuds ou topologie ? L'étude effectuée est pour le moment limitée à une trajectoire circulaire et en considérant un seul mobile. Le puits est placé au centre et les noeuds relais sont fixes. Elle sera par la suite étendue à d'autres trajectoires, en divisant la surface encadrée par la trajectoire du mobile en plusieurs cercles, à chaque fois qu'on calcule la position du noeud relais, on le prend comme étant un puits pour calculer les positions des autres noeuds relais. Jusqu'à avoir une couverture radio totale.

  cartes suivantes, synthétisées sur une seule sur la figure3 :

Figure 3 :

 3 Figure 3 : Cartographie de la couverture radio 3. MECANISME DE HANDOVER Rappelons d'abord que nous avons choisi de faire fonctionner le réseau de capteurs avec plusieurs canaux, comme le permet aisément la technologie retenue. Ainsi le changement de passerelle nécessaire lorsque le mobile s'éloigne de sa passerelle actuelle et se rapproche d'une autre correspond à un changement de canal. Ce changement est connu sous le nom de handover. Le noeud mobile est muni de l'ensemble des mécanismes de handover, une propriété qui lui permet de prendre la décision de changer de passerelle. Le principe est identique à celui utilisé par un téléphone mobile lors d'un déplacement qui le fait sortir de la bonne couverture radio de la cellule où il se trouve et entrer dans une autre cellule. Ces mécanismes ne doivent pas être trop coûteux au niveau de l'énergie et pour les applications visées ne pas augmenter sensiblement le délai d'acheminement des messages. Dans ce but, nous cherchons à anticiper ce handover.Le mécanisme du handover permet de déterminer la position du noeud mobile en se basant sur la puissance du signal qu'il reçoit. La puissance du signal est considérée comme étant un critère parmi d'autres permettant d'activer le mécanisme de

4 .

 4 LOCALISATION DU MOBILE La localisation permet au noeud mobile de mémoriser la position de changement de passerelle d'une part et d'autre part d'envoyer la position associée à l'information saisie. A noter que pour la transmission du puits vers le mobile la seule localisation nécessaire est de connaître le noeud relais. Le mobile calcule l'angle β I distance selon la formule (11) On pose : PN I = d I , MN I = d' et PM=R, Avec I= {1,2,3,4} indice du noeud relais, et P : puits, M : noeud mobile et N I : le noeud relais I. β I = angle entre le rayon de la trajectoire du noeud mobile PM et la position du noeud récepteur PN I , l'angle α c'est l'angle entre PN I et PM (12).

൜

  ݔ ൌ ݀. cos ߙ ݕ ൌ ݀. sin ߙ (12) Nous avons considéré dans notre étude une trajectoire circulaire comme celle schématisée sur la figure 2. Une fois que le noeud mobile détecte le noeud relais « N 1 » qui a pour coordonnées (+d, 0) « figure 1a », il commence à calculer la distance d' 1 à partir de l'expression (9). Une fois il passe par d' 1min , il enregistre cette position et déduit la distance entre lui et le puits « R=d' 1min +d » et l'angle α = β 1 , à partir de cet instant le noeud mobile sera capable de calculer ces coordonnées polaires (x, y) en se basant sur l'angle α qui dépendra de la position du mobile.

Figure 1a :

 1a Figure 1a : M Connecté avec N1 Figure 1b : M Connecté avec N2

Figure 1c :

 1c Figure 1c : M Connecté avec N 3 Figure 1d : M Connecté avec N 4

Figure 5 :Figure 6 :

 56 Figure 5 : consommation d'énergie en joule

Figure 7 :

 7 Figure 7 : Changement de canal en fonction de α 6. PLATFORME EXPERIMENTALE

Figure 8 :

 8 Figure 8 : Plateforme expérimentale En simulation, la méthode utilisée pour calculer le délai de bout en bout des paquets est basée sur leur estampillage par une étiquette appelée temps d'envoi, grâce à la base du temps du simulateur. Quand le paquet arrive au puits, une simple soustraction entre les deux dates « date d'envoi » et « date de réception » fournit le délai, avec une meilleur précision puisque le calcul se fait sur des dates positionnées avec la même horloge. La même méthode ne peut pas être utilisée facilement sur la plateforme en raison du problème de non synchronisation des horloges de l'émetteur et du récepteur. Il existe une solution de synchronisation mais nous ne l'avons pas mise en oeuvre dans un premier temps, nous nous focalisons sur le mécanisme de handover que nous allons enrichir par la synchronisation des horloges pour une meilleure plage de confiance des résultats obtenus.

Figure 10 :

 10 Figure 10 : consommation d'énergie en joule

en (Sec) Temps en (Sec)

		Consommation d energie	en joule	0 0,1 0,2 0,3	0,000	104,175	164,181	223,687	274,172	319,203	364,166	410,042	454,185	wpan 1 wpan 2
											Time (Sec)
			0,0250
			0,0200
			0,0150
		0,0050 0,0100 Delai Wpan 2 Wpan 1
			0,0000
							0	396		828	1260	1692	2124	2556	2988	3420
	17																		C'est pourquoi nous avons choisi de mesurer uniquement la
	16 15 13 14	numero																gigue. Pour cela, le principe est de fixer la période T d'envoi des paquets. Soit Gi la valeur de la gigue pour le paquet i. Soit un compteur dans le puits. A la réception du premier paquet, ce compteur est mis à zéro et i est initialisé à 1. A la réception de chaque suivant, les opérations suivantes sont réalisées :
	12
		0	12,86	25,71	40,00	46,00	83,57	109,29	135,00	160,71	186,43	212,14	237,86	263,57	289,29	315,00	340,71	359,00	Calcul de Gi = compteur -T Sauvegarde de Gi i = 1+1
																			Remise à zéro du compteur.

La gigue mesurée (figure

9

) de cette façon varie autour d'une moyenne de 0,0016ms avec un écart type de 0,003 avec notre

de canal Ongle α en degre Wpan 1

	Wpan 1