Dr Silvia Meschi

Monica Sané Schepisi

Emanuele Nicastri

Nazario Bevilacqua

Concetta Castilletti

Maria Rosaria Sciarrone

Maria Grazia Paglia

Robert Fumakule

Jape Mohamed

Andrew Kitua

Inmi L Spallanzani

Ivo De Carneri

Di Caro

Francesco Vairo

Sañé Schepisi

Paglia

Caro A Di

The prevalence of antibodies to human herpesvirus 8 and hepatitis B virus in patients in two Hospitals in Tanzania

Keywords: seroprevalence, immunization, PCR, hepatitis, antibody titer Seroprevalence, immunization, PCR, hepatitis, antibody titer

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Pemba Island and Tosamaganga from 336 outpatients and sent to the virology laboratory in Rome, 5

Italy for testing. HHV-8 DNA and HBV DNA were amplified by two in-house molecular methods, 6 anti-HHV-8 antibody titers were determined by an immunofluorescence assay (IFA), and anti-7

HCV, HBsAg, anti-HBs, and anti-HBc were evaluated by microplate enzyme immunoassay 8 (MEIA). The seroprevalence of HHV-8 was 30.7% (96/313). In Pemba Island, the prevalence was 9 lower than in Tosamaganga (14.4% vs. 46.3%). A higher prevalence of low titers of HHV-8 IgG 10 (<1:80, 81%) was found among those under five years of age. HHV-8 DNA was detected in 6 11 seropositive patients (6.7%). The prevalence of HBsAg, anti-HBs, and anti-HBc was 4.3%, 37.6% 12 and 29.3% respectively. Out of 277 patients, 70 had had a previous infection (25.3%). One case of 13 occult hepatitis was found. The cover of hepatitis B vaccination was higher among children born 14 after 2002 (66.7%) than in patients born before 2002. HHV-8 infection is endemic in Tanzania and 15 the seroprevalence rate was higher in the mainland than on Pemba Island. The 3.9% percentage of 16 HBsAg in children younger than 4 years of age suggests that increased efforts are required in order 17 to achieve universal and compulsory immunization of children against HBV. 18 Introduction 2

Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi's sarcoma (KS), primary effusion B-3 cell lymphoma and multicentric Castleman's disease of the plasma-cell type [START_REF] Cesarman | Kaposi's sarcoma-associated 14 herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas[END_REF]4 Chang et al., 1994;[START_REF] Soulier | Kaposi's sarcoma-associated herpesvirus-like DNA 5 sequences in multicentric Castleman's disease[END_REF]. In industrialized countries, HHV-8 infection is acquired 5 most frequently in late adolescence or in adult life, i.e., after the beginning of sexual activity. The 6 sexual route of transmission is supported by the presence of HHV-8 DNA in semen and by 7 increased risk of HHV-8 infection among homosexual men infected with the human 8 immunodeficiency virus (HIV), as compared to others at risk of HIV infection [START_REF] Pauk | 7 Mucosal shedding of human herpesvirus 8 in men[END_REF]. 9

Conversely, HHV-8 infection is relatively common in early childhood in developing countries, 10 particularly in Africa where the seroprevalence of HHV-8 in children increases with age [Serraino 11 et al., 2001]. The risk factors for acquiring HHV-8 infection are still uncertain, although saliva 12 exchange is likely to play a major role [START_REF] Andreoni | Primary Human Herpesvirus 8 Infection in Immunocompetent Children[END_REF][START_REF] Mbulaiteye | Human 7 herpesvirus 8 infection within families in rural Tanzania[END_REF]. Although 13 few studies have investigated the prevalence of anti-HHV-8 antibody titers in the population of 14 countries where a high rate of infection occurs, one study shows that anti-HHV-8 antibody titers in 15 blood donors from different regions of Italy mirror the incidence of Kaposi 's sarcoma in these 16 regions [Whitby et al., 1998]. Among black South African patients with cancer, the highest anti-17 HHV-8 antibody titers were observed in KS patients [Sitas et al., 1999]. Finally, high anti-HHV-8 18 antibody titers in males aged 40 years or older parallel the increase in the incidence of endemic KS 19 in older African men [START_REF] Plancoulaine | High titers of anti-human herpesvirus 8 9 antibodies in elderly males in an endemic population[END_REF]. 20 Hepatitis B is a serious global public health problem. In sub-Saharan Africa, where the 21 seroprevalence of hepatitis B virus (HBV) is particularly high [START_REF] Olubuyide | Prevalence and epidemiological characteristics of hepatitis B and C 5 infections among doctors and dentists in Nigeria[END_REF], chronic 22 HBV infection affects an estimated 50 million people with a prevalence of ≥8% [Burnett et al., 23 2005]. The general population is at risk of acquiring the virus in areas where it is endemic and the 24 major route of transmission is horizontal, with most infections established during childhood by 25 inter-family contacts [START_REF] Jacobs | Sexual 12 transmission of hepatitis B in Mwanza, Tanzania[END_REF]. In East Africa, post puberty transmission also occurs, and 26 clearly demonstrated [START_REF] Bile | Late 8 seroconversion to hepatitis B in a Somali village indicates the important role of venereal 9 transmission[END_REF][START_REF] Hudson | Risk factors for the spread of AIDS in rural Africa: evidence from a 9 comparative seroepidemiological survey of AIDS, hepatitis B and syphilis in southwestern 10 Uganda[END_REF][START_REF] Ter Meulen | Evaluation of seroepidemiological associations between HIV-infection, hepatitis B and 10 other sexually transmitted diseases in African patients[END_REF]Van de Perre et 2 al., 1987]. In this setting, the only logical approach for preventing transmission of hepatitis B virus 3 in early childhood is mass immunization of newborns and infants [Burnett et al., 2005]. The World 4 Health Organization (WHO) recommended that all countries with a prevalence of HBsAg of at least 5 8% should have the hepatitis B vaccine incorporated into their national Expanded Programme on 6 Immunization (EPI) by 1995. Tanzania initiated universal hepatitis B vaccinations in March 2002 7 [START_REF] Mphahlele | Epidemiology 9 and control of hepatitis B: implications for eastern and southern Africa[END_REF], but few studies have examined whether this vaccination campaign was 8 effective. 9

In African populations, the overall prevalence of hepatitis C virus (HCV) infection has been 10 estimated to range from 3 to 5% [START_REF] Madhava | Epidemiology of chronic hepatitis C virus infection in 25 sub-Saharan Africa[END_REF]. The prevalence of HCV infection in a 11 population-based, cross-sectional study carried out in a rural population of Southwest Tanzania in 12 1996 was 2% [START_REF] Puato | Does HCV 14 infection have a more favourable outcome in Tanzanian people? Data from the Lugalawa study[END_REF]. 13 The seroprevalence of HHV-8, HBV and HCV and the immunization against HBV in patients with 14 febrile illness admitted to two rural hospitals in Tanzania were investigated to determine the 15 prevalence of HHV-8, HBV and HCV infection and the coverage of the immunization program 16 against HBV. 17 the Chake Chake and Tosamaganga hospitals. Outpatients involved in the present survey were also 2 included in a study which evaluated malaria infection in the same Tanzanian areas [Nicastri et al., 3 2009]. Inclusion criteria were age of ≥ 1 year and a body temperature of ≥38° for a period of less 4 than 10 days. Exclusion criteria were the presence of signs of severe disease. Patients affected by 5 mental illness or with a clinical picture of measles, chickenpox, otitis, infected wounds, chest X-ray 6 documented pneumonia or with a previous antimalarial treatment were excluded. 7 Ethical Committee. Ethical approval was obtained from the "L. Spallanzani" National Institute of 8

Infectious Diseases in Italy, the National Institute for Medical Research in Tanzania and the 9 Zanzibar Ministry of Health and Social Welfare. 10 Protocol procedure. An easy-to-read, friendly questionnaire was provided for the collection of 11 epidemiological and clinical data. A physical examination with a clinical note of any clinical signs 12 or symptoms, including a final diagnosis, was requested from the local medical officer for each 13 enrolled patient. The protocol procedures and the study collaborators did not change the current 14 standard of care of each facility. After receiving complete information in Kiswahili about the 15 collection of clinical samples for research purposes, all consenting adult patients and parents or 16 legal guardians of minors signed an informed consent form. 17 Laboratory Investigations. Two specimens of venous blood (5ml into a serum-separator tube, 5ml 18 into a vial containing EDTA) and five drops of venous blood which were absorbed on filter paper 19 collection cards (S&S 903™, Schleicher and Schuell, Dassel, Germany) were collected from each 20 enrolled patient. All blood samples were dispatched at the appropriate temperatures to "L. 21

Spallanzani" National Institute of Infectious Diseases. 22 Venous blood and serum samples were processed for detection of HHV-8 DNA and HBV DNA, 23 respectively. 125 microliters of blood and 250 microliters of serum were used to extract DNA using 24 BioRobot® MDx and QIAamp® DNA Blood BioRobot® MDx Kit (Qiagen, Hilden, Germany). 25 For 18 patients no blood aliquot were available and nucleic acid extraction was performed from 26 dried blood spots using DNA with QIAamp DNA Mini Kits (Qiagen, Hilden, Germany). 1 Amplification and quantification of human herpesvirus 8 were carried out with real-time PCR on an 2 ABI Prism® 7700 instrument (Applied Biosystem, Foster City, CA) thermal cycler as previously 3 described [START_REF] Lallemand | Quantitative analysis of 20 human herpesvirus 8 viral load using a real-time PCR assay[END_REF]. All samples were analyzed in triplicate. A qualitative HBV 4 nested PCR was performed, as described previously [START_REF] Lindh | Detection of hepatitis B virus 22 precore TAG mutant by an amplification-created restriction site method[END_REF] in HbsAg positive samples, 5 and in all samples positive for anti-HBc and negative for the other HBV markers. A PCR for β-6 globin was conducted in parallel on both blood and serum samples to assess suitability for 7 amplification of the extracted DNA [START_REF] Saiki | Enzymatic 20 amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of 21 sickle cell anemia[END_REF]. The sizes of the products were estimated by 8 electrophoresis on 2% agarose gel stained with ethidium bromide. 9

To determine HHV-8 antibody titers, sera were tested at 1:40 dilution in a "in house" 10 immunofluorescence assay (IFA) using latently infected body-cavity-based lymphoma 1 (BCBL-1) 11 cell lines untreated and treated chemically with tetradecanoyl phorbol ester acetate (TPA). Punctate 12 nuclear staining in an untreated BCBL-1 cell line was considered a positive result for latency-13 associated nuclear antigen (LANA), whereas the entire cell fluorescence in approximately 20% of 14 TPA-treated cells was considered as positive for lytic-phase Kaposi's sarcoma-associated 15 herpesvirus (KSHV) antigens [START_REF] Kedes | The seroepidemiology of 14 human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus): distribution of infection in KS 15 risk groups and evidence for sexual transmission[END_REF]. 16 HBsAg, anti-HBs, anti-HBc and antibodies to HCV in sera were detected using a microparticle 17 enzyme immunoassay (MEIA) (Abbott Diagnostics Division, Weisbaden, Germany) according to 18 the manufacturer's instructions. Immunoblot Deciscan HCV Plus (Biorad, Marnes-la-Coquette, 19

France) was carried out to confirm anti-HCV results. HBV markers were performed in all available 20 samples. Antibodies to HCV were performed in a selected group of 101 patients from both sites. 21 Data analysis. Data were entered, processed and analyzed using SPSS software package (SPSS 15. During the study period, 1,800 outpatients were seen to at the two sites: 960 patients (53.3%) 1 were screened at the Tosamaganga Hospital and 840 patients (46.7%) were screened at the Chake 2 Chake Hospital; 1,463 patients (81.3%) were excluded because they did not meet the study 3 enrollment criteria; 337 patients (18.3%) were eligible and 336 patients (18.7%) were recruited 4 (figure 1). One hundred -seventy patients were adults (50.6%), represented equally in the two study 5 sites. 92 out of 336 patients (27.4%) were children under five years of age and had a higher 6 representation in the Chake Chake Hospital (60 patients, 65.2%). In both sites there were slightly 7 more female patients than males. The symptoms reported most frequently were headache (46.7%), 8 cough (41.4%), joint pain (35.4%) and vomiting (22.3%). 9

Anti-lytic antibodies to HHV-8 were tested in 313 out of 336 (93.2%) recruited patients, while 10 HHV-8 DNA was tested in the all 336 patients recruited. Overall, 96 of 313 (30.7%) patients had 11 antibodies for HHV-8 lytic-phase antigens. No differences in the prevalence of HHV-8 seropositive 12 individuals were found between men (39 of 131, 29.8%) and women (57 of 182, 31.3%). The 13 seroprevalence of HHV-8 decreased from 28.4% (21 of 74) among children under age five to 22.5% 14 (16 of 71) among subjects aged 5 to 15 but increased to 35.1% (59 of 168) in adults. The 15 seroprevalence of HHV-8 (22 of 153, 14.4%) was significantly lower (p<0.001) among patients 16 screened at the Chake Chake Hospital than those patients screened at the Tosamaganga Hospital (74 17 of 160, 46.3%) (figure 2). In the Pemba Hospital the prevalence of anti-lytic HHV-8 antibodies 18 among children under age five (8 of 45, 17.8%) was higher than that observed in subjects aged 5 to 19 15 years (3 of 36, 8.3%) or in adults (11 of 72, 15.3%). In the Tosamaganga Hospital, the age-20 specific seroprevalence presented the same trend observed in the entire study population. A 21 significant difference in the seroprevalence of HHV-8 between Pemba Hospital and Tosamaganga 22 Hospital was noted in all age categories: under five (p=0.024), 5 to 15 (p=0.009) and adults 23 (p<0.001). The percentage of patients with anti-lytic HHV-8 antibodies titer >1:640 was higher in 24 older patients than in those aged 5-15 and <5 years (χ2 for trend: p=0.01) (figure 3). All samples 25 were tested for HHV-8 DNA independently of the presence of anti-HHV-8 antibodies, considering 26 DNA. Among 96 HHV-8 seropositive patients, 6 (6.3%) patients (all from the Tosamaganga 2 Hospital), were positive for HHV-8 DNA (Table I). Three patients had detectable but not 3 quantifiable HHV-8 viral load (<250 copies/ml) and the remaining three patients had a higher 4 HHV-8 viral load. The only child under the age of five who was positive for HHV-8 DNA (viral 5 load of 2,880 copies/ml) had a high anti-lytic HHV-8 antibody titer. All six patients had febrile 6 syndrome, with prevalent high respiratory tract involvement in 4 cases and gastrointestinal 7 involvement in the remaining two cases. 8

HBV markers were examined in 282 patients. The prevalence of HBsAg, anti-HBc and anti-HBs 9 was 4.3% (12 of 277), 29.3% (82 of 280) and 37.6% (106 of 282), respectively. Among the 12 10 patients with active HBV infection, eight (66.6%) had at least one gastrointestinal symptom. The 11 prevalence of HBsAg, anti-HBc and anti-HBs was lower in Pemba than in Tosamaganga: 1.7% (2 12 of 120) vs. 6.4% (10 of 157), 15.9% (20 of 123) vs. 39.5% (62 of 157) and 32.3% (40 of 124) vs. 13 41.8% (66 of 158 p<0.001) (figure 4). 14 Among the 13 patients with anti-HBc only, one person (7.7%) was positive for HBV DNA in sera, 15 whereas among the 12 patients with positive HBsAg, seven cases (58.3%) were positive for HBV 16 DNA. 17 Among the children born after 2002, the prevalence of persons with anti-HBs seroprotective titer 18 (>10 UI/ml) was higher than in patients born before 2002: 66.7% (34 of 51) vs. 44% (10 of 226), 19 respectively (p<0.001) (figure 5). 20

No patients had active co-infection with HHV-8 and HBV. Conversely, in 33 patients, 94% in 21 Tosamaganga, had antibodies to HHV-8 and to hepatitis B core antigen: seven (21.2%) were under 22 five years of age, two (6.1%) were aged 5 to 15 and 24 (72.7%) were adults. The seroprevalence of 23 HHV-8 among anti-HBc positive subjects (33 of 82, 40.2%) was slightly higher than among 24 patients without previous HBV infection (58 of 198, 29.3%) (p=0.1). 25 Antibodies to HCV were undetectable in all analyzed patients. 26 This study on the differential diagnosis of febrile syndromes, conducted in two hospitals in 3 Tanzania in 2007, found a 30.7%, 4.3% and 0% seroprevalence rate of HHV-8, HBsAg and HCV 4 with no significant differences in age and gender. Among 96 HHV-8 seropositive persons, six 5 patients (6.3%) from Tosamaganga Hospital with respiratory tract infections (four patients) and 6 gastrointestinal symptoms (two cases) were positive for HHV-8 DNA. HHV-8 DNA was not 7 detected among patients without antibodies to HHV-8. 8

The overall prevalence rate of HHV-8 found in this study is consistent with previous data from 9 different African countries. A review of 28 seroepidemiological studies of HHV-8 in adult 10 populations from 16 African countries reported that between 1996 and 2002, most African countries 11 (namely Botswana, Cameroon, Democratic Republic of Congo, Egypt, Gambia, Ghana, Ivory 12 Coast, Nigeria, South Africa, Tanzania, Uganda, Zambia, and Zimbabwe) had high seroprevalence 13 rates of HHV-8 ranging from 26% to 86%, with the exception of Central African Republic, Eritrea 14 and Senegal which had relatively lower seroprevalence rates (up to 25%) [START_REF] Dedicoat | Review of the distribution of Kaposi's sarcoma-associated 20 herpesvirus (KSHV) in Africa in relation to the incidence of Kaposi's sarcoma[END_REF]. A 15 39.8% prevalence rate was reported in 5 to 10 year-old children in northern Cameroon [Rezza et al., 16 2000], 36% in Ghana [START_REF] Nuvor | Higher prevalence of antibodies to 22 human herpesvirus 8 in HIV infected individuals than in the general population in Ghana, west 23 Africa[END_REF] and approximately 50% in Zambia [Olsen et al., 1985] 17 and Uganda [START_REF] Mayama | Prevalence and transmission of Kaposi's sarcoma-associated herpesvirus (human 5 herpesvirus 8) in children and adolescents[END_REF]]. Among families in rural Tanzania, the seroprevalence of HHV-18 8 increased from 3.7% in infants to 58.1% in 3 and 4-year-old children [START_REF] Mbulaiteye | Human 7 herpesvirus 8 infection within families in rural Tanzania[END_REF]. 19 No difference in the seroprevalence of HHV-8 was described between men and women in the 20 different countries. Similar HHV-8 prevalence rates was reported in children (44%) and adults 21 (66%) from North Mara District in Tanzania during the period of 1972 through 1975, the period 22 prior to the onset of the AIDS-related epidemic of Kaposi's sarcoma in East Africa [ de-Thé et al., 23 1999]. 24 These seroepidemiological studies suggest that HHV-8 is transmitted mainly among family 25 members and close contacts by a horizontal, non-sexual route. Sexual transmission may be an 26 Recently, in a cross-sectional study conducted in Ghana, the seroprevalence of HHV-8 was 8 significantly lower in HIV-seronegative healthy blood donors than in asymptomatic persons 9 infected with HIV (23.7% and 65.6% respectively, p<0.005) [START_REF] Adjei | Seroprevalence of 2 HHV-8, CMV, and EBV among the general population in Ghana,West Africa[END_REF]. Consistent with 10 the previous report [START_REF] Plancoulaine | High titers of anti-human herpesvirus 8 9 antibodies in elderly males in an endemic population[END_REF], in this study, an age-related increasing anti-HHV- 8 11 antibody titer was found: this seroprevalence trend parallels the increase of the incidence of 12 endemic KS in elderly African men [START_REF] Taylor | Kaposi's sarcoma in Uganda: geographic and ethnic 7 distribution[END_REF]. 13 In this study, the 4.3% prevalence rate of HBsAg was found in the general population receiving 14 hospital care for febrile syndromes is consistent with the 4.2% prevalence rate reported recently in 15 the Moshi urban area in Tanzania among women of childbearing age [START_REF] Msuya | Seroprevalence of hepatitis 12 B and C viruses among women of childbearing age in Moshi Urban, Tanzania[END_REF]. Similar 16 seroprevalence rates of HBsAg were found in Tosamaganga (6.4%) and in Pemba Island (1.4%). As 17 mentioned previously for HHV-8, the low prevalence of HBV infection in Pemba Island may be 18 related to geographical restriction and to social behavior. 19 In 2002, HBV vaccination was included in the Tanzanian Expanded Programme on Immunization 20 [START_REF] Mphahlele | Epidemiology 9 and control of hepatitis B: implications for eastern and southern Africa[END_REF]. Anti-HBs titer (>10 UI/ml) among children born before or after 2002 21 increased significantly from 4.4% to 66.7% with no substantial differences between the two 22 surveyed areas or between gender. Despite this considerable result obtained in only 5 years, further 23 public health efforts are needed to protect Tanzanian children (approximately 1/3) and prevent 24 susceptible children who are likely to acquire HBV in early infancy from becoming chronic carriers 25 and potential transmitters of the virus. In this setting, the only logical approach to preventing the 26 Consistent with previous data on the prevalence of occult chronic hepatitis B in patients from sub-3 Saharan African countries [START_REF] Gibney | Isolated core antibody hepatitis B in sub-Saharan 3 African immigrants[END_REF][START_REF] Hübschen | 5 Exceptional genetic variability of hepatitis B virus indicates that Rwanda is east of an emerging 6 African genotype E/A1 divide[END_REF], HBV DNA was detected in 4 sera in 7.7% of patients with only anti-HBc. The definitive diagnosis of occult chronic hepatitis B 5 depends on the detection of HBV DNA in the liver, so the nature of the tested specimen for the 6 detection of HBV DNA (the serum alone in this study) will underestimate the prevalence of occult 7 chronic hepatitis B by up to 40% [Mariscal et al., 2004]. A possible explanation for this diagnostic 8 pattern includes a false positive serological test result, the "window phase" of acute HBV infection 9 or resolved HBV infection [Knoll et al., 2006]. HBV DNA was also detected in 58.3% of patients 10 with HBsAg. These apparently surprising data can be explained by different factors: 1. the 11 occurrence of false positive results in the HBsAg immunoassay, as reported in African countries 12 [START_REF] Pirillo | Seroprevalence of hepatitis B and C viruses among HIV-infected pregnant 12 women in Uganda and Rwanda[END_REF]; 2. the occurrence of a false negative PCR, most likely due to the presence of 13 inhibitors in the extracted samples or if the sampling, shipping or storing were not performed 14 accurately. Nevertheless, in the present study, ß-globin PCR was used as a positive control to 15 monitor inhibition, and the extracted amount of DNA from clinical samples was suitable for 16 amplification. 17

Differently from other epidemiologic studies [START_REF] Madhava | Epidemiology of chronic hepatitis C virus infection in 25 sub-Saharan Africa[END_REF][START_REF] Puato | Does HCV 14 infection have a more favourable outcome in Tanzanian people? Data from the Lugalawa study[END_REF] wich 18 reported a higher prevalence of HCV among elderly subjects, no positive samples were found in 19 this study. It could be related to the lower age of enrolled patients (median, 20 years; IQR 3-32), 20 and to the low exposure of this rural population to well-known risk factors for hepatitis C. 21

Among the 33 patients with previous exposure to HHV-8 and HBV, a higher seroprevalence was 22 detected in adult patients, suggesting that sexual transmission was an additional factor. 23

There are a number of limitation to this study. First, this is a cross-sectional study, and neither 24 longitudinal follow-up nor the clinical outcome of febrile patients were included in the study 25

  hepatitis B virus (HBV) infection in febrile patients in two districts of the 3 United Republic of Tanzania. Between February and March 2007, blood samples were collected in 4

  has been suggested, its role in the spread of the infection has not been 1

  The study was conducted in two peripheral hospitals in Tanzania: in the Chake Chake 20 Hospital (on Pemba Island) the study was carried out from February 9, 2007 to March 12, 2007 21 jointly with the "Ivo De Carneri" Public Health Laboratory (PHL-IdC). In the Tosamaganga 22 Missionary Hospital, (Iringa region) the study was conducted from March 3, 2007 to March 29, 23 2007 jointly with CUAMM-Doctors with Africa (University College for Aspiring Missionary 24 Doctors), an Italian non-governmental organization (NGO). 25 A cross-sectional observational study was conducted in the outpatient departments of 1

0 22 for

 22 Windows, Chicago, IL). Frequencies were compared with the Yates corrected χ2 test. The 23 seroprevalence of HHV-8 was compared with the χ2 test for trend.

  the acute phase of disease. No HHV-8-seronegative patients were positive for HHV-8 1

  transmission in endemic population in Africa [Eltom et al., 2002; Volpi et al., 1 2004]. 2 A higher seroprevalence of HHV-8 was found in Tosamaganga (46.3%) than in the Pemba Island 3 (14.4%). The low HHV-8 circulation among the general population of Pemba may be related either 4 to the intrinsic geographical isolation, Muslim-based religious behavior with extensive social and 5 ethical control and the low HIV prevalence rate of Pemba Island in the adult population compared 6 to the Tanzanian mainland (0.3% vs. 5.8%) [National Bureau of Statistics (NBS) Tanzania, 2008]. 7

  in early childhood is to implement mass immunization of newborns and 1 infants[Burnett et al., 2005]. 2

4 KaposiFigure 1 .Figure 2 . 3 Figure 3 . 7 Yates corrected χ2 test. 8 Figure 4 .Figure 5 .Figure 1 .Figure 2 .Figure 3 .

 412337845123 Figures legend 1

Figure 4 .Figure 5 .

 45 Figure 4. Prevalence of HBV markers. 167x74mm (96 x 96 DPI)

Table I .

 I Patients with HHV-8 reactivation or with HBV infection. 1 § All patients had fever ≥38° C.

	2
	Code	Age (years)	Sex	Clinical symptoms §	HHV-8 DNA (copies/ml)	HHV-8 antibodies titers	HBV infection status
	T056	23	F Cough, joint pain	40,800	1:320	Previous infection
	T113	30	F	Difficulty eating/ drinking, headache, chest pain, joint pain	15,440	1:320	Previous infection
	T126	1	M	Difficulty eating/drinking, cough, crepitation+++	2,880	≥1:1280	Previous infection
	T158	28	M	Difficulty eating/drinking, vomiting, joint pain	positive, not quantifiable	1:640	Previous infection
	T074 T131 T133 T130	19 44 46 17	F Headache, cough, joint pain F Headache, vomiting, joint pain M Headache, diarrhoea, joint pain F o M Headache, joint pain r	positive, not quantifiable positive, not quantifiable negative negative	1:160 1:80 1:640 1:320	Previous infection Not previous infection Active infection, HBV DNA+ Active infection, HBV DNA+
	T146	2.2	F	Cough, vomiting, abdominal pain P	negative	1:40	Active infection, HBV DNA+
	P073 T101 T105	13 6 50	M Chills, headache, vomiting F Difficulty eating/drinking, headache, vomiting F Headache, joint pain e negative negative negative e r	<1:40 <1:40 <1:40	Active infection, HBV DNA+ Active infection, HBV DNA+ Active infection, HBV DNA+
	T143 T136 T093 P085 T031 T147 T156	21 9 1.7 40 33 53 25	F M Headache, vomiting Chest pain, vomiting, abdominal pain, joint pain F Cough, vomiting M Difficulty eating/drinking, chills, fits or convulsions, vomiting, abdominal pain F Headache, joint pain F Headache, cough M joint pain Chest pain, abdominal pain,	negative negative negative negative negative negative negative R e v <1:40 <1:40 1:40 <1:40 <1:40 <1:40 <1:40 i e w	Active infection, HBV DNA+ Occult hepatitis, HBV DNA+ Active infection Active infection Active infection Active infection Active infection
	3

Journal of Medical Virology

Acknowledgments 7

This work has been supported by grants from the Italian Cooperation, Italian Ministry of Foreign 8 Affairs (Project: AID 8282). 9

John Wiley & Sons

Journal of Medical Virology


Thomas Iftner
email: thomas.iftner@med.uni-tuebingen.de

Sonja Eberle

Angelika Iftner

Barbara Holz

Norbert Banik

Wim Quint

Anja-Natascha Straube

Prevalence of Low-Risk and High-Risk types of Human Papillomavirus and other risk factors for HPV infection in Germany Within Different Age Groups in Women up to 30 Years of Age: An Epidemiological Observational Study

Keywords: HPV prevalence, screening, comparison of tests, HPV vaccination 43 HPV prevalence, screening, comparison of tests, HPV vaccination 44 Abbreviations: 45 HPV, Human papillomavirus, CIN, cervical intraepithelial neoplasia, WHO, World Health Organisation, 46

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Persistent infection with human papillomavirus (HPV) is a necessary risk factor for cervical cancer 49 [Munoz et al., 1992;[START_REF] Schiffman | Epidemiologic evidence showing that human papillomavirus infection causes 115 most cervical intraepithelial neoplasia[END_REF][START_REF] Bosch | The causal relation between human 14 papillomavirus and cervical cancer[END_REF], which is the second most prevalent tumour 50 in women worldwide. In Germany, the incidence of cervical cancer is one of the highest compared to 51 other Western countries, with about 6,200 newly diagnosed cases and more than 1,600 deaths per 52 year. Among women younger than 50 years, it is still the second most common cause of mortality in 53

Germany [START_REF] Koch-Institute | Robert Koch-105 Institute & Gesellschaft der epidemiologischen Krebsregister[END_REF]]. HPV DNA is detectable in almost all cervical tumours 54 [START_REF] Walboomers | Human papillomavirus is a necessary cause of invasive cervical cancer worldwide[END_REF]]. Cervical cancer progresses slowly from cervical intraepithelial neoplasia 55 (CIN) to invasive cancer, and precursor lesions can be detected by cytological screening. The 56 mainstay of screening programs has been the Pap smear. However, considerable numbers of false-57 negative Pap smears may occur with the traditional Pap technique, due mostly to sampling errors 58 [Nanda et al., 2002]. The prevalence of HPV in Europe is estimated to be between 8-15% throughout 59 all age groups, but is considerable higher among younger women aged 20-24 years, with up to 50. 2% 60 in Denmark [De [START_REF] Sanjosé | Worldwide 42 prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal 43 cytology: a meta-analysis[END_REF][START_REF] Bosch | Chapter 3: Epidemiology of human 16 papillomavirus infections and associations with cervical cancer: New opportunities for prevention[END_REF], Kjaer et al., 2008]. Infection occurs mostly in 61 adolescence and early adulthood: 74% of all infections are diagnosed in women aged 15 to 24 years 62 [START_REF] Weinstock | Sexually transmitted diseases among American youth: 131 incidence and prevalence estimates, 2000[END_REF]. Estimations in Finland and in the USA give a lifetime incidence of 70-80% 63 [START_REF] Syrjänen | 125 Prevalence, incidence, and estimated life-time risk of cervical human papillomavirus infections in a 126 nonselected Finnish female population[END_REF][START_REF] Myers | Mathematical model for the natural 90 history of human papillomavirus infection and cervical carcinogenesis[END_REF]. However, in the majority (70-90%) of cases, the infection is 64 transient and HPV DNA is not detectable 1 to 2 years after infection [START_REF] Ho | 52 Persistent genital human papillomavirus infection as a risk factor for persistent cervical dysplasia[END_REF]Schiffman et al., 65 2007]. With increasing age, prevalence rates decline but the relative contribution of persistent 66 infections increase.' 67 Among more than 100 HPV types, approximately 13 genotypes have been identified as carcinogenic 68 (high-risk [HR] types), whereas others are considered to be low-risk (LR) types or HPV types of 69 unknown risk. According to the WHO, the following HPV types are classified as class I carcinogenic : 70 HPV 16,18,31,33,[START_REF] Chan | Epidemiologic risk profile of infection 36 with different groups of human papillomaviruses[END_REF][START_REF] Cogliano | Carcinogenicity of human 40 papillomaviruses[END_REF]45,[START_REF] Ho | 52 Persistent genital human papillomavirus infection as a risk factor for persistent cervical dysplasia[END_REF]52,[START_REF] Kjaer | Type specific persistence of high risk human papillomavirus 58 (HPV) as indicator of high grade cervical squamous intraepithelial lesions in young women: 59 population based prospective follow up study[END_REF]58,59 and 66 [START_REF] Cogliano | Carcinogenicity of human 40 papillomaviruses[END_REF]. Among these, the 71 prevalence of HPV 16 in cervical cancer cells is predominant. HPV 16 is detectable in 50-60% of all F o r P e e r R e v i e w 5 cervical tumour samples, HPV 18 in 10-20%, and HPV16/18 in approximately 70% of all cervical 73 cancer cases [START_REF] Munoz | which is due to the rather insensitive methods that have been 366 combined in meta-analyses[END_REF]2004]. At present, the Hybrid Capture 2 (HC2) assay (Qiagen,74 Hildesheim, Germany) is one of two tests approved by the US Food and Drug Administration for the 75 detection of HPV DNA in cervical cancer screening. The HC2 assay uses an RNA probe cocktail to 76 detect 13 high-risk and five low-risk HPV types, but in fact detects many more types through cross 77 reactivity [START_REF] Castle | Restricted cross-reactivity of hybrid capture 2 with nononcogenic human 31 papillomavirus types[END_REF]. However, the test does not distinguish individual genotypes and 78 cannot identify infection with multiple genotypes. Several PCR based genotyping tests for HPV types 79 are available, but the tests differ with respect to sensitivity for certain types, and there is currently 80 no "gold standard" [START_REF] Klug | Comparison of the performance of different HPV genotyping methods for detecting 72 genital HPV types[END_REF]. 81 Since March 2007, the German Standing Committee on Vaccination (STIKO) has recommended 82 vaccination against HPV types 16 and 18 for all female adolescents aged 12 to 17 years. 83 Unfortunately, data on the incidence of genital HPV infections and HPV prevalence in younger age 84 groups in Germany are scarce. However, such information is vital for estimating the burden of HPV 85 infection, the HPV-type distribution and the putative impact of HPV vaccination. [START_REF] Muñoz | Against 87 which human papillomavirus types shall we vaccinate and screen? The international perspective[END_REF] An epidemiological, observational study was performed in 77 centres throughout Germany to 87 determine the prevalence of LR-HPV and HR-HPV in young women up to 30 years of age undergoing 88 routine consultation in office-based gynaecological practices. Women were sampled at pre-defined 89 age intervals in a stratified manner. After a routine Pap smear, a cervical swab was taken for HPV 90 detection and genotyping. Finally, the women were interviewed regarding their medical history and 91 lifestyle factors. The cervical swabs were tested for high-risk (HR)-and low-risk (LR)-HPV by means of 92 Hybrid Capture 2 (HC2), and genotyping was performed for HC2 positive samples using both the PCR-93 based SPF 10 /LiPA 25 test and the PapilloCheck  test. The primary objective of the study was to assess 94 the distribution of the age-specific HPV types in young women up to 30 years of age in Germany. To 95 assess the impact of the following additional risk factors on HPV prevalence, women completed a 96 questionnaire on pregnancy, smoking behaviour, contraceptives, vaccination against HPV, history of 97

MATERIALS AND METHODS 102

Study Population 103

Women were recruited at office-based gynaecologists when they attended for routine screening. 104 Subjects were eligible for inclusion in the study if they were under 30 years of age, had not 105 undergone hysterectomy and signed an informed consent form. A routine Pap smear was taken 106 before the additional cervical sample was collected. In the case of minors, informed consent had to 107 be given by a parent or legal representative. In addition, minors were provided with an adequate 108 age-based informed consent form for signature. The study was approved by the Ethics Committee of 109 the State Medical Chamber of Bavaria. 110

The sample size for this study was based on obtaining a representative sample of subjects in all 111 predefined age strata. To achieve a high level of accuracy for prevalence estimators and with an 112 expected prevalence of 5% -25% it was planned to include 2,500 women in the study, 500 per age 113 group (≤ 16 years, 17 to ≤19 years, 20 to ≤22 years, 23 to ≤26 years and 27 to ≤30 years). The 114 subjects were planned to be recruited in approximately 100 gynaecological practices spread all over 115 Germany, in total 25 women per practice and 5 women per age group. Administrative difficulties in 116 recruiting centres led to a smaller sample size than was planned originally. 117

Between February 2007 and August 2008, 1,803 women with informed consent from 79 participating 118 centres in Germany were recruited to the study and included in the safety analysis implying 119 documentation of adverse events due to the collection of cervical samples. One thousand six 120 hundred and ninety-two of those subjects with valid HC2 tests from 77 centres were included in 121 statistical analyses (evaluable subjects)(Fig. 1). 122

Study Objectives 123

The primary objective of the study was to assess the distribution of the age-specific 24 HPV types: 13 124 high-risk (HR-) HPV types (HPV 16,18,31,33,[START_REF] Chan | Epidemiologic risk profile of infection 36 with different groups of human papillomaviruses[END_REF][START_REF] Cogliano | Carcinogenicity of human 40 papillomaviruses[END_REF] HPV types using the HC2 test [START_REF] Castle | Restricted cross-reactivity of hybrid capture 2 with nononcogenic human 31 papillomavirus types[END_REF] in girls and young women up to 30 years of age in 132 Germany. In addition, the impact of the following additional risk factors on HPV prevalence was 133 determined: pregnancy, smoking behavior, contraceptives, vaccination against HPV, history of 134 cervical cancer, immunosuppressive disease, immunosuppressive drugs, sexually transmitted 135 diseases, and the number of sexual partners. 136

Cytological Material 137

After routine Pap smear for cytological evaluation using the Second Munich Cytological Classification, 138 an additional cervical scrape was taken for HPV detection and genotyping. The second scrape was 139 obtained using a cervical sample device (Qiagen, Hildesheim, Germany) and suspended in 1 ml 140 specimen transport medium (STM; Qiagen, Hildesheim, Germany) for HPV DNA testing. HPV 141 detection was performed without knowledge of the cytological diagnosis. 142

Cytological Diagnosis 143

All Pap smears were examined at the routine cytological laboratories normally used by the office-144 based gynaecologists. The laboratories were not informed of the study and the slides and 145 accompanying forms did not contain any mention of the study. The Pap smears were assessed 146 according to the Second Munich Cytological Classification [Bundesaerztekammer, 1994]. 147

HPV Detection 148 After the amplification reaction, an additional 15 cycles of 30 sec at 95 °C and 45 sec at 72 °C were 177 performed to achieve a single-stranded product for the following hybridization step. The PCR was 178 carried out on a MJ Thermocycler PTC200 (Bio-Rad Laboratories, Munich, Germany). Five microlitres 179 of the PCR product and 30 µl hybridization buffer were used for the hybridization on the low-density 180 microarray plastic HTA12 chip. A volume of 25 µl of the PCR hybridization mix was applied to every 181 well on the chip and incubated for 15 min at room temperature in a humid atmosphere. Twelve 182 hybridization reactions can be performed simultaneously on one chip. Hybridization is followed by 183 three washing steps (the first for 10 sec at room temperature; the second for 60 sec at 50 °C; the 184 third for 10 sec again at room temperature). The chip was dried subsequently under a stream of 185 compressed air. The chip was scanned automatically and analysed using CheckScanner and 186 CheckReport software (Greiner Bio-One, Frickenhausen, Germany) respectively. 187 SPF 10 -LiPA 25 Assay: A 5-ul aliquot of extracted DNA was used for each SPF10 PCR. The SPF10 PCR 188 primer set was used to amplify at least 54 HPV genotypes, as described earlier [START_REF] Kleter | Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital 65 human papillomaviruses[END_REF] Thermocycler PTC 200 (Bio-Rad Laboratories, Munich, Germany). The SPF 10 PCR line probe array v1 199 test amplifies a short (65 bp) fragment in the L1 region using four forward and two reverse primers. A 200 10-µl aliquot of the PCR reaction was taken, denatured and hybridized at 49 °C for 60 min to one 201 detection strip, followed by multiple washing steps. Detection was performed using the substrate 202 solutions provided and bands were read manually and interpreted according to the manual. 203

Statistical Analysis 204

All data were analyzed descriptively using summary statistics for categorical and continuous data. 205

These calculations were performed for all evaluable subjects as well as for age groups and the HPV 206 HPV type 18, the second most common subtype in cervical cancer, was 3.2% (95% CI: [1.4-5.0]). 270

(

271

The SPF 10 assay showed a positive result for 94.7% [95% CI: 92.4 -97.0] of subjects with positive HC2 272 findings. Among 377 HPV-positive women, the prevalence of HPV 16 measured by SPF 10 was 29.4% 273 (95% CI: [24.8-34.1]), followed by HPV 51 (21.0%), HPV 31 (17.0%), HPV 52 (13.5%) and HPV 66 (HR; 274 12.2%). HPV 18 was detected in 8% (95% CI: [5.2-10.7]) samples (Figure 4). 275 276

Correlation Between the HC2 Results and risk Factors 277

A larger proportion of pregnant women at the time was recorded among the HPV-positive subjects 278 (9%) than in women with a negative test result (5.6%) (Table I). 279

Nearly half (48.5%) of HPV-positive women were current smokers compared to 35.4% of subjects 280 with negative HC2 test results (HR-HPV only: 50.2%, LR-HPV only: 37.0%, HR & LR: 51.2%). The 281 proportion of women taking contraceptives was similar between the HC2-positive and negative 282 subjects, namely 83.6% and 82.6% respectively. Regarding the occurrence of self-reported cervical 283 cancer and immunosuppressive diseases and the use of immunosuppressive drugs, only minor 284 differences between HC2-positive and HC2-negative subjects were found. As expected, the 285 proportion of subjects who had already been vaccinated against HPV was smaller in subjects with 286 positive HC2 results than in those with negative findings (11.1% vs. 20.3%). 14.8% of subjects with 287 low HPV risk only, 10.9% of subjects who had high HPV risk only, and 9.5% with LR & HR HPV types 288 had been vaccinated. 289

A larger proportion of subjects with a higher number of sexual partners were recorded in subjects 290 with a positive HC2 test result than in subjects with a negative test result (figure 5a and5b). 291 and higher compared to subjects without detection of HPV according to the HC2 result (HC2-positive: 293 7.2% vs. HC2-negative: 0.7%). 294

The influence of age on the occurrence of a positive HC2 test result was investigated by logistic 295 regression. The univariate logistic model showed a significant correlation between age and positive 296 HC2 results (odds ratio: 1.028; 95% CI: [1.003-1.054]). According to this univariate model, higher age 297 was significantly associated with higher risk of HPV infection. In order to adjust the age effect for 298 possible confounders, a multivariate analysis was performed including pregnancy, smoking, 299 hormonal contraception, HPV vaccination and the number of sexual partners besides age. Based on 300 this model, the effect of age was no longer statistically significant (odds ratio: 0.981; 95% CI: [0.952-301 1.011]). Variables showing statistical significance were numbers of sexual partners, HPV vaccination 302 and smoking. Due to the strong positive correlation between age and the number of sexual partners, 303 the significant age effect observed in the univariate model is a result due to confounding. The risk of 304 HPV detection increased with "smoking" (yes vs. never) (OR: 1.508; 95% CI: [1.155-1.968]) and 305 "number of sexual partners" (OR: 1.105; [1.069-1.142]) and declined significantly with "vaccination 306 against HPV" (OR: 0.589; [0.398-0.872]) (table III). 307 308

Agreement of Test Results 309

The level of agreement between the different laboratory methods (i.e. HC2, PapilloCheck  and SPF 10 ) 310 was analysed in 377 subjects with positive HC2 findings. In the HC2 test, 54 subjects (14.3%) showed 311 LR-HPV types only and 239 subjects (63.4%) only HR-HPV types. 84 subjects (22.3%) were infected 312 with both, LR-and HR-HPV types. 88.6% of subjects were positive both by HC2 and PapilloCheck  , 313 whereas 11.4% of HC2-positive subjects had negative results when tested by PapilloCheck  . 86.6% of 314 subjects with HPV-type HR only according to the HC2 test showed infection with an HR-HPV (only) 315 type using the PapilloCheck  screening, whereas 11.3% showed negative results. Of the LR-HPV 316 IV). 318 By comparing HPV detection using HC2 and SPF10, the replication rate of HC2-positive findings with 319 SPF 10 was 94.7%. Twenty samples (5.3%) were positive when examined with HC2 and negative when 320 examined with SPF 10 .. Of the subjects with LR-HPV (only) according to HC2, 31.5% had negative test 321 results with SPF 10 . It is noteworthy that the LR-HPV positive results (LR only) with HC2 were only 322 confirmed in 14.8% of subjects when tested by SPF 10 (Table V). 323

On comparison, both methods (SPF 10 and PapilloCheck  ) were found to correspond to a large extent 324 as regards HR-HPV types (98.1% with PapilloCheck  as reference, 81.3% with SPF 10 as reference). 325 However, the degree of correspondence in the detection of LR-HPV types was considerably smaller 326 (39.4% and 45.3% respectively). 327 328 A total of six adverse events in six subjects (0.3%), which occurred during the collection of cervical 329 smears, were reported, five of which were minor bleedings and one narrow cervical canal. 330

DISCUSSION 331
Epidemiological data especially in young women on the prevalence of HPV and the distribution of 332 HPV types in Germany is lacking. HPV vaccination has become available recently, which may 333 influence the prevalence of certain HPV types in the future. Therefore, it is important to obtain a 334 picture of the status quo before the HPV vaccine coverage becomes high in the population. 335

This German epidemiological study in young women shows that more than one fifth (22%) of the 336 subjects were HPV positive according to the HC2 test. There was an age-dependent prevalence, 337

showing the highest prevalence for women at the age of 20-22 years, and then declining with 338 increasing age. These findings are in accordance with a recent American study on HPV prevalence 339 [START_REF] Dunne | Prevalence 45 of HPV infection among females in the United States[END_REF], in which 27% of women were HPV positive, with the highest prevalence (45%) in 340 women aged 20-24 years. It is estimated that 74% of all infections are diagnosed in women aged 341 between 15 and 24 years [START_REF] Weinstock | Sexually transmitted diseases among American youth: 131 incidence and prevalence estimates, 2000[END_REF]. In this study 95.8% of women did not show signs 342 of any cervical lesion. Due to the fact that in this observational study women visiting their 343 gynaecologist only for routine reasons were included a possible selection bias might be taken into 344

account. 345

To reduce the rate of infection with HPV, it is reasonable to vaccinate women prophylacticly against 346 HPV, preferentially as young girls before they become sexually active. This is reflected by the 347 recommendations of the German Standing Committee on Vaccination (STIKO), which advises 348 vaccination against HPV types 16 and 18 for all females aged 12 to 17 years in Germany. In 349 accordance with this recommendation, this study shows the lowest HPV prevalence in the age group 350 10 to 16 years, in which half of the women were immunized with at least one vaccination dose, 351 followed by the age group 17-19 years, with one third of women immunized. At the present time, 352 HPV vaccination is not a replacement for cervical screening. Routine testing by Pap smears has the 353 disadvantage of low sensitivity in the range of 50-60% for a single test [START_REF] Nanda | Accuracy of 93 the Papanicolaou test in screening for and follow-up of cervical cytologic abnormalities: a systematic 94 review[END_REF]354 Wentzensen et al., 2008]. To detect persistent HPV infections, which are a necessary risk factor for 355 cervical cancer and to identify and monitor those high-risk women, the use of genotyping tests is 356 being discussed currently. This study revealed that 85.7% of the HPV-positive women were infected 357 with high-risk types according to the HC2 test. Genotyping by PapilloCheck  and SPF 10 confirmed 358 approximately 90% of these high-risk types. In contrast, HC2 low-risk results were confirmed by 359 PapilloCheck  and SPF 10 genotypingto a less extent. This underlines the importance of reliable 360 genotyping tests, since HC2 often detects erroneously an infection with low-risk-types when a high-361 risk infection is present or vice versa. 362

The genotyping tests in this study revealed the highest prevalence for the HR-HPV types 16, 31, 51, 363 52 and 56 as well as HPV LR subtype 42, which was the second most common subtype detected by 364 the PapilloCheck  assay. In other studies, different HPV types have been described to be prevalent 365 Risk factors for HPV infection were smoking and the number of sexual partners. Smoking as a risk 393 factor for HPV infection has also been described in recent epidemiological studies in China and Brazil 394 [START_REF] Chan | Epidemiologic risk profile of infection 36 with different groups of human papillomaviruses[END_REF][START_REF] Sarian | 109 Increased risk of oncogenic human papillomavirus infections and incident high-grade cervical 110 intraepithelial neoplasia among smokers: experience from the Latin American screening study[END_REF]]. An association between sexual behaviour and the risk of HPV 395 infection has been reported in other European countries [START_REF] Lenselink | 77 Sexual behaviour and HPV infections in 18 to 29 year old women in the pre-vaccine era in the 78 Netherlands[END_REF]

1 2

 1 Prevalence of Low-Risk and High-Risk types of Human Papillomavirus and other risk 1 factors for HPV infection in Germany Within Different Age Groups in Women up to 30 Years of Age: An Epidemiological Observational Study

  immunosuppressive disease, immunosuppressive drugs, sexually transmitted 98 diseases and the number of sexual partners. The results of the HC2 assay and the two HPV 99 genotyping tests (SPF 10 /LiPA 25 and PapilloCheck  ) were also compared to ascertain the degree to 100 which they corresponded in terms of multiple infection and individual HPV type detection. 101

9 HC2

 9 test. The HC2 test (Qiagen, Hildesheim, Germany), using the high-risk and low-risk probe 149 cocktail, was performed on the second cervical smear taken for all women included in the study. A 150 positive result refers to a subject who is positive for one or more of the 13 high-risk and 5 low-risk 151 HPV types included. Samples were considered positive if they attained or exceeded the Food and 152 Drug Administration approved threshold of 1.0 pg HPV DNA/ml, which corresponds to a 1.0 relative 153 light units coefficient (RLU/CO). 154 HPV genotyping. All samples positive by HC2 were genotyped using DNA extracted from the 155 denatured pretested HC2 sample. Two different genotyping methods for the detection of genital HPV 156 types were used to compare sensitivity and specificity for HPV-positive, high-risk HPV types: 157 PapilloCheck  (Greiner Bio-One GmbH, Frickenhausen, Germany) and the SPF 10 HPV LiPA 25 , version 1 158 (Labo Bio-Medical Products, Rijswijk, The Netherlands). 159 DNA extraction using MagNA Pure LC (MPLC) (Roche Diagnostics, Mannheim, Germany) and all pre-160 and post-PCR procedures were carried out in separate rooms and cabinets. Buffer and blank controls 161 were included at every 30th position to obtain sufficient numbers of negative controls to monitor 162 contamination events. All samples were tested for integrity of DNA by using the PapilloCheck  HPV 163 genotyping test sample control function. 164 The PapilloCheck     HPV genotyping assay recognizes the following HPV types: 6, 11, 16, 18, 31, 33, 165 35, 39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82. Thus the test recognizes all 166 13 HPV types recently classified as class-I carcinogenic with respect to cervical cancer by the WHO 167 (Cogliano et al., 2005), six HPVs (6, 11, 40, 42, 43, 44/55) considered as low-risk types, and five types 168 (53, 68, 70, 73, 82) with proposed high-risk or unclear risk potential. Additionally, the human ADAT1 169(t-RNA specific adenosine desaminase1) gene is amplified and used as a control for the integrity of 170 the purified DNA. The PapilloCheck  chips and all reagents were provided by Greiner Bio-One GmbH 171 (Frickenhausen, Germany). All PCRs were performed with 5 µl input DNA in the presence of 172 nucleotides with a fluorescent label in a final volume of 25 µl using the AmpliTaq Gold hot start 173 Molecular Systems, Inc, Somerville, USA). An initial activation step of 95 °C for 10 174 min was followed by 40 cycles of 30 sec of denaturation at 95 °C, by 25 sec primer annealing at 55 °C 175 and 45 sec of primer extension at 72 °C. The average size of the amplified product is 350 bp. 176

  -smear results showed that more HC2-positive subjects were classified as PAP III 292

  infected subjects tested by HC2, 68.5% were confirmed by PapilloCheck  and 24.1% had 317 negative results For the LR & HR-group the results matched in 52.4% of cases (Table

  shown significant effects of HPV vaccination on the occurrence of cervical disease 391[START_REF] Harper | Efficacy of a bivalent L1 virus-like particle vaccine in prevention of 49 infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial[END_REF][START_REF] Villa | Prophylactic quadrivalent human papillomavirus (types 6, 11, 16, and 138 18) L1 virus-like particle vaccine in young women: a randomised double-blind placebo-controlled 139 multicentre phase II efficacy trial[END_REF][START_REF] Paavonen | Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted 102 vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final 103 analysis of a double-blind, randomised study in young women[END_REF][START_REF] Brown | The impact of quadrivalent human papillomavirus (HPV; types 6, 11, 16, and 18) L1 virus-like 24 particle vaccine on infection and disease due to oncogenic nonvaccine HPV types in generally HPV-25 naive women aged 16-26 years[END_REF]. 392

Figure 3 :

 3 Figure 3: HPV genotyping according to the PapilloCheck  test (24 HPV types) among women testing

Figure 4 :

 4 Figure 4: HPV genotyping according to the SPF 10 assay (26 HPV types) among women testing HPV-

Figure 5 :

 5 Figure 5: Number of sexual partners in women with (a) negative and (b) positive HC2 test

  ; 189[START_REF] Kleter | Development and clinical evaluation of a highly sensitive PCR-reverse 68 hybridization line probe assay for detection and identification of anogenital human papillomavirus[END_REF]. Briefly, this primer set amplifies a small fragment of 65 bp from the L1 region of 190 HPV. Reverse primers contain a biotin label at the 5' end. The SPF 10 amplimers were used to identify 191 the HPV genotype by reverse hybridization on line probe assay (LiPA 25 ) containing probes for 25 HPV 192 at 52 °C and 45 sec of primer extension at 72 °C. The PCR was carried out on a MJ 198

		of primer annealing
		F o r
		P
		e
		e r
		R
		e
		v i e
		w
	193	genotypes . The LiPA 25 detects HPV types 6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52,
	194	53, 54, 56, 58, 59, 66, 68/73, 70, and 74.
	195	All PCRs were performed with 5 µl input DNA and biotinylated primers in a final volume of 50 µl using
	196	the AmpliTaq Gold hot start polymerase (Roche Molecular Systems, Somerville, USA) and an initial
	197	activation step of 94 °C for 9 min, followed by 40 cycles of 30 sec of denaturation at 94 °C, by 45 sec
		11

  HC2 test result). Overall and age specific prevalence with asymptotic 95% confidence interval (CI) 207 was estimated for all three HPV tests. Calculations according to HC2 test were based on evaluable 208 subjects (women with a valid laboratory test), prevalence estimation due to the two genotyping 209 assays and comparisons between the different testing methods were based on those evaluable 210 subjects showing a positive HC2 test result. 211 Univariate and adjusted multivariate Odds Ratios (OR) with 95% CI were obtained by logistic 212 regression models assessing the impact of possible risk factors (especially age) on the HC2 test result. 213 Adverse events reported in the context of taking the cervical sample were categorized and described 214 by counts and percentages. 215 All statistical analyses were performed using SAS  9.1 software (SAS Institute Inc., Cary, NC, USA) 216 Twenty-two subjects (1.3%) had suffered previously from a sexually transmitted 244 disease, including 11 subjects with chlamydial infection and six with anogenital warts. 245According to the Pap smear, 1,621 out of 1,692 subjectss (95.8%) showed no signs of cervical 246 abnormalities (PAP I and PAP II). PAP III, PAP IIID, and PAP IVa were documented in six (0.4%), 28 247 95% CI: [75.7-79.7]) had negative findings (TableII). The proportion of subjects with 253 positive HPV findings steeply increased from 11.6% in the 10-16 year range to a maximum of 28.3% 254 in the 20-22 year-old group. The prevalence of HPV then declined with increasing age (TableII). 255According to these findings, the distribution of HR-and LR-HPV positive subjects showed the lowest 256 proportion in the age group 10-16 years (HR only: 7.2%, LR only:2.4%, LR & HR: 2.1%) and the highest 257 proportion in the age groups 20-22 years (HR only: 15.3%, LR only: 4.2%, LR & HR: 8.8%) and 23-26 258 years (HR only: 17.4%, LR only: 4.0%, LR & HR: 4.6%). In all groups there were more HR-HPV than LR-259 HPV types detected. TableIIshows the number of subjects with a positive HC2 test result in each age 260 group related to the number of subjects included in each age group. In contrast figure 2 depicts the 261 distribution of subjects with a negative LR, HR or both ─ HC2 test result per age group in relation to 262 all women with a negative LR, HR or both ─ HC2 test result, respectively. 263

	248 268 269	predominant in this study, amounting to 23.9% (95% CI: [19.6-28.2]). Other frequent HPV types were cervical cancer. (1.7%) and two (0.1%) subjects, respectively. Sixteen Pap smears (0.9%) were not suitable for HPV 42 (LR; 19.1%), HPV 51 (HR; 16.7%), HPV 56 (HR; 10.3%) and HPV 31 (HR; 8.8%). The rate of HR-
	249	diagnosis, 19 (1.1%) without specification of PAP status.
	250
	251 252	F o r F o r Prevalence of HPV Types In total, 377 subjects (22.3%; 95% CI: [20.3-24.3]) showed a positive HC2 test result, and 1,315 F o r
	217 264	RESULTS subjects (77.7%; Cervical samples of the 377 women with a positive HC2 test result were genotyped with the P e e r R e v i e w P e e r R e v i e w P e e r R e v i e w
	218	Demography and risk factors
	265	PapilloCheck  HPV screening test and the SPF 10 HPV LiPA 25 assay. HPV genotyping by PapilloCheck 
	219 266	A total of 1,803 women attending gynaecological practices for routine consultation were enrolled confirmed 88. 6% (95% CI: [85.4-91.8]) of the positive HC2 findings. As shown in Figure 3, the
	220 267	into the study. Of these subjects, 1,692 women (93.8%) with appropriate cervical samples for HPV prevalence of HR-HPV type 16 measured by PapilloCheck  (among 377 HC2-positive subjects) was
		13 14

TABLE I :

 I Risk factors versus HC2-laboratory results

					Journal of Medical Virology
		7	46	2.7	10	2.7	2	3.7	6	2.5	2	2.4
		8	39	2.3	16	4.2	5	9.3	6	2.5	5	6.0
	396 397	2008; Nielsen et al., 2008]. 9 10 age, mean ± SD >10 contraceptives highest graduation Hauptschule secondary school university none hormonal* current pregnancy yes no Self-reported cervical cancer yes no vaccination against HPV yes no immunosuppressive disease yes no Immunosuppressive drugs yes no smoking yes no never smoked previous sexual transmitted disease other* PAP smear result not suitable for diagnosis PAPI PAPII PAPIII PAPIIID PAPIVa *multiple response	; Ammatuna et al., F o r P e e r R e v i e w Total (N=1692) Positive (N=377) Low Risk only (N=54) 13 0.8 4 1.1 0 0.0 63 3.7 30 8.0 3 5.6 21.5 ± 4.6 22.0 ± 4.0 21.4 ± 4.3 48 2.8 22 5.8 0 0.0 395 1145 134 23.3 67.7 7.9 107 240 27 28.4 63.7 7.2 14 34 6 25.9 63.0 11.1 117 6.9 29 7.7 4 7.4 1421 84.0 315 83.6 42 77.8 108 1584 6.4 93.6 34 343 9.0 91.0 7 47 13.0 87.0 5 1687 0.3 99.7 1 376 0.3 99.7 0 54 0.0 100.0 309 1383 18.3 81.7 42 335 11.1 88.9 8 46 14.8 85.2 3 1689 0.2 99.8 2 375 0.5 99.5 0 54 0.0 100.0 17 1675 1.0 99.0 5 372 1.3 98.7 3 51 5.6 94.4 649 272 771 38.4 16.1 45.6 183 61 133 48.5 16.2 35.3 20 12 22 37.0 22.2 40.7 178 10.5 37 9.8 8 14.8 16 185 1436 6 28 2 0.9 10.9 84.9 0.4 1.7 0.1 2 40 307 2 23 2 0.5 10.6 81.4 0.5 6.1 0.5 1 9 42 0 2 0 1.9 16.7 77.8 0.0 3.7 0.0 F o r P e e r R e v i e w	High Risk only (N=239) 3 21 22.3 ± 4.1 20 71 150 15 17 201 18 221 1 238 26 213 1 238 2 237 120 35 84 25 1 20 198 2 16 2	1.3 8.8 8.4 29.7 62.8 6.3 7.1 84.1 7.5 92.5 0.4 99.6 10.9 89.1 0.4 99.6 0.8 99.2 50.2 14.6 35.1 10.5 0.4 8.4 82.8 0.8 6.7 0.8	1 High & Low Risk 6 (N=84) 2 21.6 ± 3.5 22 56 6 8 72 9 75 0 84 8 76 1 83 0 84 43 14 27 4 0 11 67 0 5 0	1.2 7.1 2.4 9.5 26.2 85.7 66.7 7.1 10.7 89.3 0.0 100.0 9.5 90.5 1.2 98.8 0.0 100.0 51.2 16.7 32.1 4.8 0.0 13.1 79.8 0.0 6.0 0.0
		yes	22	1.3	12	3.2	1	1.9	10	4.2	1	1.2
		no	1666	98.5	365	96.8	53	98.1	229	95.8	83	98.8
		number of sexual partners
		0	67	4.0	2	0.5	0	0.0	2	0.8	0	0.0
		1	480	28.4	43	11.4	8	14.8	24	10.0	11	13.1
		2	291	17.2	45	11.9	6	11.1	33	13.8	6	7.1
		3	230	13.6	58	15.4	12	22.2	33	13.8	13	15.5
		4	151	8.9	55	14.6	7	13.0	35	14.6	13	15.5
		5	165	9.8	65	17.2	8	14.8	40	16.7	17	20.2
		6	62	3.7	25	6.6	3	5.6	15	6.3	7	8.3
						20 21
						John Wiley & Sons

Table II :

 II HPV prevalence by age strata according to the HC2-, PapilloCheck  -and SPF 10 -Test includes also possible HR-HPV types or types with unclear potential † positive without feasible specification or types 34 or 74 (unknown risk, SPF 10 only), respectively

	-unknown risk †	1	0.3 [0.0-0.8]	0	-	1	1.2 [0.0-3.7]	0	-	0	-	0	-
	SPF 10	377		34		82		100		91		70
	(only HC2 positive women)
	-positive	357	94.7 [92.4-97.0]	31	91.2 [81.1-100.0]	78	95.1 [90.4-99.9]	96	96.0 [92.1-99.9]	86	94.5 [89.7-99.3]	66	94.3 [88.7-99.9]
	-low risk only	11	2.9 [1.2-4.6]	2	5.9 [0.0-13.8]	3	3.7 [0.0-7.7]	4	4.0 [0.2-7.8]	1	1.1 [0.0-3.2]	1	1.4 [0.0-4.2]
	-high risk only*	254	67.4 [62.6-72.1]	17	50.0 [33.2-66.8]	56	68.3 [58.2-78.4]	70	70.0 [61.0-79.0]	62	68.1 [58.6-77.7]	49	70.0 [59.3-80.7]
	age strata % [95% CI] 17-19 N 23.4 [19.0-27.9] 3.1 [1.3-5.0] 14.9 [11.1-18.6] 5.4 [3.1-7.8] 354 100 15 54 31 20.7 [12.0-29.5] 2.4 [0.0-5.8] 19 3 F o r P e e r R e v i e w total 10-16 20-22 % [95% CI] N % [95% CI] N % [95% CI] 22.3 [20.3-24.3] 3.2 [2.4-4.0] 14.1 [12.5-15.8] 5.0 [3.9-6.0] 293 34 7 21 6 11.6 [7.9-15.3] 2.4 [0.6-4.1] 7.2 [4.2-10.1] 2.1 [0.4-3.7] 350 82 11 52 19 28.3 [23.5-33.0] 4.2 [2.1-6.3] 15.3 [11.5-19.0] 8.8 [5.8-11.7] 19.9 [15.9-23.9] 4.5 [2.4-6.6] 10 2 29.4 [14.1-44.7] 5.9 [0.0-13.8] 17 2 19.0 [11.3-26.7] 3.0 [0.0-6.4] r N 1692 377 54 239 84 75 17 F HC2 -positive -low risk only -high risk only -low & high risk -low & high risk -unknown risk † o P e PapilloCheck     (only HC2 positive women) 377 34 82 100 e r	N 350 91 14 61 16 20 3 91	23-26 % [95% CI] 26.0 [21.4-30.6] 4.0 [2.0-6.1] 17.4 [13.5-21.4] 4.6 [2.4-6.8] 22.0 [13.5-30.5] 3.3 [0.0-7.0]	N 345 70 7 51 12 9 7 70	27-30 % [95% CI] 20.3 [16.0-24.6] 2.0 [0.5-3.5] 12.9 [5.0-20.7] 10.0 [3.0-17.0] 14.8 [11.0-18.5] 3.5 [1.5-5.4]
	-positive -low risk only -high risk only* -low & high risk	334 50 234 49	88.6 [85.4-91.8] 13.3 [9.8-16.7] 62.1 [57.2-67.0] 13.0 [9.6-16.4] R 29 8 17 4 e 85.3 [72.8-97.8] 23.5 [9.3-37.8] 50.0 [33.2-66.8] 11.8 [0.9-22.6] v i e	74 9 53 11 22	90.2 [83.7-96.8] 11.0 [4.2-17.7] 64.6 [54.3-75.0] 13.4 [6.0-20.8]	96 17 59 20	96.0 [92.1-99.9] 17.0 [9.6-24.4] 59.0 [49.4-68.6] 20.0 [12.2-27.8]	79 9 60 10	86.8 [79.7-93.9] 9.9 [3.8-16.0] 65.9 [56.2-75.7] 11.0 [4.6-17.4]	56 7 45 4	80.0 [70.4-89.6] 10.0 [3.0-17.0] 64.3 [53.1-75.5] 5.7 [0.3-11.2]
					w
			23

*

TABLE III :

 III Univariate and multivariate analysis showing the dependence of HC2 -positivity on selfreported/risk factors

			Univariate	multivariate
	Self-reported Factor		Odds Ratio	95% CI	Odds Ratio	95% CI
	Age		1.028	1.003 -1.054	0.981	0.952 -1.011
	Current pregnancy (yes vs. no)		1.662	1.089 -2.538	1.559	0.996 -2.442
	Smoking behavior (no vs. never smoked)	1.387	0.986 -1.950	1.114	0.778 -1.594
	Smoking behavior (yes vs. never smoked) Hormonal contraception (yes vs. no) Vaccination against HPV (yes vs. no) F o r	1.884 0.960 0.492	1.462 -2.427 0.704 -1.308 0.348 -0.697	1.508 0.995 0.589	1.155 -1.968 0.715 -1.385 0.398 -0.872
	Number of sexual partners	P	1.120	1.086 -1.155	1.105	1.069 -1.142
		e
		e r
			R
			e
				v i e
				w
			24

TABLE IV :

 IV Degree of correspondence in HPV high-risk and low-risk types obtained from HC2 and PapilloCheck  . Only HC2 positive subjects were tested.

				HC2
	PapilloCheck 
		Low risk only High risk only Low & high risk
		N	Col %	N	Col %	N	Col %
	Negative	13	24.1	27	11.3	3	3.6
	Low risk only	37	68.5	1	0.4	12	14.3
	High risk only* Low & high risk Unknown †	2 2 0	3.7 207 3.7 3 0.0 1 F o r	86.6 1.3 0.4	25 44 0	29.8 52.4 0.0
				P
				e
					e r
						R
						e
							v i e
							w
						25

TABLE V :

 V Degree of correspondence in HPV high-risk and low-risk types obtained from HC2 and SPF 10 . Figure 1: Flow of participants enrolled in the study Figure 2: Percental distribution of age groups with negative and positive HC2 test results. Subjects with positive HC2 test results are subdivided in low risk, high risk, and both low and high risk groups.The figure shows the distribution of subjects with a negative LR, HR or both ─ HC2 test result per age group in relation to all women with a negative LR, HR or both ─ HC2 test result, respectively.

	Only HC2 positive women were tested.
				HC2
	SPF 10	Low risk only High risk only Low & high risk
		N	Col %	N	Col %	N	Col %
	Negative	17	31.5	3	1.3	0	0.0
	Low risk only	8	14.8	1	0.4	2	2.4
	High risk only* Low & high risk 15 5 Unknown † 9	9.3 197 27.8 32 16.7 6 F o r	82.4 13.4 2.5	52 28 2	61.9 33.3 2.4
				P
				e
					e r
						R
						e
							v i e
							w

John Wiley & SonsJournal of Medical Virology

ACKNOWLEDGMENTS

The study was sponsored by GlaxoSmithKline Inc.

T.I. has received speaker honorarium from GlaxoSmithKline GmbH & Co KG.

We thank Heike von Krempelhuber for writing assistance of this article. Page 33 of 40 John Wiley & Sons Journal of Medical Virology


Maria Luisa Mateos

Gustavo Gabilondo

Beatriz ; Romero

Oihane S De

La Maza

Teresa Perez-Gracia

Maria ; Mateos

Maria Luisa

Mateos Lindemann

Oihane Martín

S De La Maza

Maria Teresa Pérez-Gracia

Maria Luisa Mateos

Servicio De Microbiología

Low prevalence of hepatitis E

Keywords: hepatitis E, pregnant women, viral hepatitis, imported diseases hepatitis E, HEV, pregnant women, imported diseases, viral hepatitis

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in developing countries.

In Europe many cases of autochthonous hepatitis E have been recognized recently in patients without any evidence of travel to endemic areas; this has been suggested as a distinct clinico-epidemiological entity and the name of ¨indigenous to developed countries hepatitis E¨ (IDC HE) has been proposed (Teo,2006). Acute infection by HEV generally causes mild and self-limiting hepatitis but it can also result severe clinical fulminant hepatic failure in pregnant women and is associated with very high mortality particularly during the third trimester in endemic areas [START_REF] Mushahwar | Molecular virology, clinical features, diagnosis, transmission, epidemiology and prevention[END_REF].

Interestingly, this feature has never been reported in industrialized countries where genotype 3 is the prevalent genotype in contrast to genotypes 1 and 2 which are found most frequently in developing countries. The reasons for the differences in the morbidity and outcome of HEV in pregnant women in individual countries remain unclear. Prevalence studies conducted in Spain have reported a wide range of prevalence rates from 0.6% in pregnant women in the north of Spain [START_REF] Gonzalez | Prevalencia de inmunidad frente a los virus de la hepatitis en gestantes del Ärea Sanitaria de Gijón[END_REF] to 7.3 % in the general population of Catalonia [START_REF] Buti | Prevalence of hepatitis E Virus infection in children in the northeast of Spain[END_REF].

There has been a large influx of immigrants to Madrid in recent years with figures in June, 2009 as high as 17.1% of the total population and 27.3 % in some of the poorer neighbourhoods (Consejeria de Inmigración, 2009). Since a large number of pregnant women in Spain come from endemic countries and hepatitis E is more severe in this group of patients, it is important to determine the characteristics of HEV infection in this risk group.

The aim of this study was to investigate the seroprevalence of HEV infection in a representative sample of pregnant women and determine the clinical features in relation to the presence of IgM anti-HEV antibodies in the blood of the patients.

PATIENTS AND METHODS

Serum samples from 1040 pregnant women, who attended the Obstetric Department of the Ramon and Cajal Hospital (Madrid, Spain) for routine screening were included in this study. Serum was taken from the patient during the first trimester of pregnancy during routine antenatal check-ups. One hundred and eighty one patients were studied in 2007, 588 patients in 2009 and 271 patients in 2010. Habitat (rural or urban), age, ALT and AST and clinical symptoms were assessed in all patients. The sample size for an expected prevalence of IgG HEV antibodies of 2.9% in Madrid [START_REF] Mateos | Hepatitis E virus:relevance in blood donors and risk groups[END_REF], an alpha error of 5% and a precision of + 0.001 was 1,046 pregnant women. IgG anti-HEV antibodies were detected in serum by a commercial enzyme immunoassay (EIA) (HEV Ab, DiaPro Diagnostic Bioprobes, Milan, Italy) following the manufacturer's instructions. All positive samples were studied further for the presence of IgM anti-HEV antibodies (HEV IgM, Dia.Pro Diagnostic Bioprobes, Milan, Italy). A result was considered positive by both tests when the ratio of the sample optical density and the cut off value was higher than 2. Positive results by EIA were confirmed by Western blot analysis (RecomBlot HEV IgG/IgM, Mikrogen, Martinsried, Germany). In addition, HEV RNA was amplified by reverse transcriptase (RT)-nested PCR (Fernández Barredo et al, 2006) in all serum samples with IgM anti-HEV.

The Institutional Ethic Committee approved the study and written informed consent was obtained from all the patients.

RESULTS

All the patients included in this study were living in urban areas in Madrid. The age range was 14 to 45 years (media: 30.6 years). None of the patients presented clinical symptoms associated with hepatitis when the blood sample was drawn. IgG anti-HEV antibodies were found in 2.1% (5/181) of the 2007 group, in 4.08 % (24/588) of the 2009 group and in 3.3% (9/271) of the 2010 group. The overall prevalence was 3.6% (38/1040). IgM anti-HEV antibodies were detected in 0.67 % (7/1040) suggesting acute or recent infection. ARN HEV was negative in all 7 samples IgM anti-HEV positive tested. None of them presented clinical symptoms related to viral acute hepatitis. ALT and AST had normal values in all the patients who presented IgG and IgM anti-HEV in serum. Six oh those patients were Spanish and 1 was from Egypt.

DISCUSSION

HEV circulates in Spain. The virus has been isolated from sewage [START_REF] Pina | HEV identified in serum from humans with acute hepatitis and in sewage of animal origin in Spain[END_REF] and it is considered a zoonotic disease and the most probable reservoir for the human population is swine (Fernández [START_REF] Barredo | Detection of hepatitis E virus shedding in feces of pigs at different stages of production using reverse transcription-polymerase chain reaction[END_REF]. HEV has been the cause of sporadic acute hepatitis E in Spain [START_REF] Perez-Gracia | Detection of hepatitis E virus in patients sera in southern Spain[END_REF] and is considered as an occupational disease [START_REF] Perez-Gracia | Autochthonous hepatitis E infection in a slaughterhouse worker[END_REF]. However in industrialized countries, the transmission routes for autochthonous HEV infections remain unknown although food and environmental routes are considered the most important [START_REF] Teo | Much meat, much malady: changing perceptions of the epidemiology of hepatitis E[END_REF]. Since all the patients included in this study were living in urban settings, direct animal contact was not a probable transmission route. Obviously, exposure to food in the kitchen to raw pork or poultry cannot be excluded neither can shellfish which is consumed frequently in Spain. The prevalence of IgG anti-HEV antibodies was 2.1 %, 4.08% and 3.3 % in the group of patients studied in 2007, 2009 and 2010 respectively. The overall prevalence was 3.6%. In studies conducted in 1999 in the same area, a prevalence of 2.9% was found [START_REF] Mateos | Hepatitis E virus:relevance in blood donors and risk groups[END_REF]. The odds ratio was 0.8 and the 95% CI was 0.67-1.73. This difference was not statistically significant (p< 0.810). Interestingly, seven (0.67 %) patients presented IgM anti-HEV antibodies in blood. All of them were asymptomatic cases of hepatitis E occurring during the first trimester of pregnancy.

Unfortunately it was not possible to test any further blood samples since these patients were healthy during pregnancy. No risk factors for transmission were found by interviews held with these infected patients. In contrast, pregnant women in endemic areas suffer very frequently severe hepatitis and even death. The reasons for the difference in the outcome of HEV in different geographical areas remain unclear but it could be the result of early childhood exposure to HEV, producing long-lasting immunity and/or modified subsequent responses of exposure to the virus. Alternatively, the predominant HEV genotypes in Spain could be less virulent than those in developing countries. The HEV genotypes circulating in endemic areas, mainly genotype 1 and 2, are very uncommon in industrialized countries. In conclusion, the data obtained during this study demonstrate that asymptomatic hepatitis E occurs in pregnant women in non-endemic countries at a very low rate (0.67 %). The HEV genotypes and subtypes circulating in different countries can be responsible for the different geographical morbidity in pregnant women [START_REF] Navaneethan | Hepatitis E in pregnancy: understanding the pathogenesis[END_REF]; seroprevalence of IgG HEV antibodies has not increased in Spain in these recent years (3.6 %), considering the seroprevalence studies conducted in the same area in 1999 (2.9%) [START_REF] Mateos | Hepatitis E virus:relevance in blood donors and risk groups[END_REF]. Discrepancies between the prevalence rates found in Spain by other authors (0.6%-7.3%) (Suarez [START_REF] Gonzalez | Prevalencia de inmunidad frente a los virus de la hepatitis en gestantes del Ärea Sanitaria de Gijón[END_REF][START_REF] Buti | Prevalence of hepatitis E Virus infection in children in the northeast of Spain[END_REF]) may be due to epidemiological characteristics of the patients studied (rural or urban areas, gender, age, profession), methodology and lack of confirmation with other tests such as Western blot. The geographical situation inside the country can also have a role since a very high prevalence has been reported in south west France [START_REF] Mansuy | High prevalence of anti-hepatitis E virus antibodies in flood donors from south west France[END_REF]. The good hygienic measures and public health conditions in Spain may be the most

  maintaining the low prevalence rate of hepatitis E which is not considered a major health problem at the moment. Nonetheless if the number of clandestine immigrants from endemic countries continues to increase in Spain and the route of transmission of HEV is not established completely in order to prevent the spread of HEV, this favourable epidemiological situation may change in future years.

Conflict of interests

The authors declare that there is not any conflict of interest in the present report.


Elena Chiappini

Luisa Galli

Alberta Azzi

Massimo Resti

Francesca Bonsignori

Maurizio De Martino
email: maurizio.demartino@unifi.it

Prof Maurizio De Martino

Lymphocytopenia as a

Keywords: lymphocytopenia, influenza A (H1N1), children children, influenza A (H1N1), lymphocytopenia

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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

Introduction

Lymphocytopenia has been reported to be a marker for pandemic influenza A/H1N1 2009 virus infection in adults [START_REF] Cunha | Diagnostic importance of relative lymphopenia as a marker of swine influenza (H1N1) in adults[END_REF]. This is consistent with previous studies reporting the occurrence of lymphocytopenia in adults and children with other types of influenza A virus infections [START_REF] Liem | Clinical features of human influenza A (H5N1) infection in Vietnam: 2004-2006[END_REF][START_REF] Lupovitch | White cell differential count and influenza A[END_REF]. Influenza-mediated lymphocytopenia has been also investigated as a possible cause of increased susceptibility to bacterial super-infection [START_REF] Stegemann | Increased susceptibility for superinfection with Streptococcus pneumoniae during influenza virus infection is not caused by TLR7-mediated lymphopenia[END_REF]. Data from children with flu-like illness that had been referred to a paediatric Infectious Disease Unit between July and November 2009 were analyzed; nasopharyngeal swabs samples were taken and tested for pandemic influenza A/H1N1 2009 virus by polymerase chain reaction PCR), and white blood cell (WBC) counts were also measured. The data collected allowed the for the evaluation of the occurrence of lymphocytopenia and its possible role as a marker for pandemic influenza A/H1N1 2009 virus infection in children.

Methods

Study children

Data were collected retrospectively from children referred to the Infectious Disease Unit, Anna et al., 2010).

Statistical analysis

Age, WBC count and differential cell count, red blood cell count, CRP levels, haemoglobin concentration, platelet count and time elapsed between onset of disease and PCR assay were expressed as median and interquartile range (IQR). In children, lymphocyte count results should be interpreted on the basis of age-related reference values [START_REF] Weerkamp | Age-related changes in the cellular composition of the thymus in children[END_REF]. The z-score of individual lymphocyte count indicates how much each absolute lymphocyte measurement varies from the mean of the reference group in each age class, and may be useful to interpret study results in children of different ages [START_REF] Cao | National Influenza A Pandemic (H1N1) 2009 Clinical Investigation Group of China. 2009. Clinical features of the initial cases of 2009 pandemic influenza A (H1N1) virus infection in China[END_REF]. Thus, the z-score of individual absolute lymphocyte count, referring to reference values for each age class, was calculated [START_REF] Nicholson | Chapter 710. Reference Ranges for Laboratory Tests and Procedures[END_REF].

Receiver operating characteristic (ROC) curve analysis was conducted to determine the best absolute lymphocyte count and lymphocyte count z-score thresholds in determining the presence of positive RT-PCR assay, and corresponding sensitivity and specificity were reported. The area under the ROC curve (AUC) with 95% confidence interval (CI) was also calculated. In order to assess the difference in proportions between groups, the χ 2 test or the Fisher test were used where appropriate. The Mann-Whitney-U test was used to compare continue variables.

Statistical analysis was performed using the statistical software SPSS for Windows, version 14.0. P <0.05 was considered statistically significant.

Results

Among 76 children enrolled into the study, 37 (48.7%) had a positive RT-PCR assay for pandemic influenza A/H1N1 2009 virus in nasopharyngeal swab. The characteristics of these children are reported in Table I.

When comparing data from these latter children with that from 39 children with uncomplicated flulike illness and negative pandemic influenza A/H1N1 2009 virus PCR, no difference in disease duration, median age, red blood cell count, haemoglobin concentration, CRP values, and absolute neutrophil count was observed, whereas significant differences were observed with respect to WBC count, absolute and relative lymphocyte counts, absolute lymphocyte count z-score, and platelet count (Table II). ROC analysis revealed that the best cut-points for absolute lymphocyte count and absolute lymphocyte count z-score were 2,256/µL and -0.89, respectively, sensitivity being 0.81 (95%CI: 0.68-0.94), specificity 0.87 (95%CI: 0.77-0.98), positive predictive value 0.85 (95%CI: 0.74-0.97), and negative predictive value 0.83 (95%CI: 0.71-0.94). Absolute lymphocyte count and respective z-score below the abovementioned cut-offs were observed in 30/37 (81.1%) infected children vs. 7/34 (20.6%) controls (P<0.0001). (Figure 1).

Discussion

The authors reported lymphocytopenia as a possible marker for pandemic influenza A (H1N1) virus infection in adults [START_REF] Cunha | Diagnostic importance of relative lymphopenia as a marker of swine influenza (H1N1) in adults[END_REF][START_REF] Coşkun | Relative lymphopenia and monocytosis may be considered as a surrogate marker of pandemic influenza a (H1N1)[END_REF]. In one study, relative lymphocytopenia (defined as <21% of WBC count) was present in approximately 90% of adults, but only in 3/16 (18.7%) of infected children [START_REF] Cunha | Diagnostic importance of relative lymphopenia as a marker of swine influenza (H1N1) in adults[END_REF]. The authors concluded that, although useful in adults, lymphopenia appeared not to be a marker for pandemic influenza A/H1N1 2009 virus infection in children. By contrast, lymphocytopenia (defined as an absolute lymphocyte count < 3000/µL) was observed in 91% of Chinese children with pandemic influenza A/H1N1 2009 infection (Cao B et al., 2009).

The present data not only confirm that lymphocytopenia is found commonly in children infected with pandemic influenza A/H1N1 2009 virus infection, but also provide evidence that practical cutoff points (absolute lymphocyte count <2,556 cells/µL or z-score <-0.86) may help to discriminate children at higher risk of infection with 81% sensitivity and 87% specificity.

The finding of lower median platelet count in children with pandemic influenza A/H1N1 2009 virus infection compared to controls has been previously observed [START_REF] Cunha | Diagnostic importance of relative lymphopenia as a marker of swine influenza (H1N1) in adults[END_REF]. However, in both groups of children, platelet count was within the normal reference values and no child displayed thrombocytopenia. Platelet count therefore appears not to be a useful marker for pandemic influenza A/H1N1 2009 infection in children.

A potential limitation of the study described above is that no other specific virologic assay other than PCR for pandemic influenza A/H1N1 accessed November 16th, 2009). Finally, due to the limited dataset results should be interpreted with a degree of caution.

In conclusion, the data strongly suggest that lymphocytopenia may be used as a marker for influenza A/H1N1 2009 infection in children, suggesting that it may help to discriminate children with higher risk for infection during epidemics. From a practical point of view, absolute lymphocyte count <2,556 cells /µL or z-score< -0.89 may be useful cut-offs for adoption in children.

  present study based on children with flu-like illness examined during a pandemic period, a significantly lower median absolute lymphocyte count and absolute lymphocyte count z-score were observed in children with documented pandemic influenza A/H1N1 2009 infection compared to controls. Lymphocytopenia was been previously documented in 61% of adult patients with influenza A/H1N1 2009 during the late March 2009 outbreak in Mexico (Perez-Padilla et al., 2009). Other

  characteristic (ROC) plot is shown, illustrating sensitivity and specificity of absolute lymphocyte count and absolute lymphocyte count z-score in predicting pandemic influenza A (H1N1) infection in 76 children with influenza-like illness during a pandemic period. Area under the ROC curve was 0.852 (95%IC: 0.756-0.948) considering absolute lymphocyte count and 0.872 (95%IC: 0.783-0.961) for absolute lymphocyte count z-score; P<0.0001 vs. the identity line (diagonal line).

  2009 virus was performed, not allowing for specific diagnosis in children flu-like symptoms but negative for pandemic influenza A/H1N1 2009 virus PCR. This kind of analysis was however not the objective of this study. It is plausible theoretically

	Season, September 29, 2009. website: http://www.cdc.gov/h1n1flu/guidance/diagnostic_tests.htm,
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	that false negatives may have been generated in a minor proportion of children with negative
	influenza A/H1N1 2009 PCR assay. Nevertheless, the qualitative real-time PCR assay employed
	7

here , and performed according to the Centres for Disease Control and Prevention recommendations, has been demonstrated to be both highly sensitive and very specific (Interim Recommendations for Clinical Use of Influenza Diagnostic Tests During the 2009-10 Influenza

Table I .

 I These findings have significant practical implications for the near future, in view of the fact that that the pandemic virus is highly likely to continue to circulate in the next winter season (European Centre for Disease Prevention and Control. 2010) White blood cell count, relative and absolute lymphocyte counts and absolute lymphocyte count z-score, calculated considering age-related reference values (6), in 37 children referring to the Infectious Disease Unit, Department of Paediatrics, Florence University, Italy, with pandemic influenza A (H1N1) infection between July and November 2009.

	Case	Age		WBC* count	Relative lymphocyte	Absolute	Absolute
	identifier	(years)	(cells/µL)	count (% of WBC)	lymphocyt	lymphocyte
						e count	count
						(cells/µL)	z-score
	1	0.08		10,320	74.0	7,637	0.66
	2	0.08		8,200	10.8	886	-1.62
	3 4 5 6 7	0.08 0.08 0.08 0.17 0.17	F o r F o r F o r 6,250 2,710 4,700 7,280 15,510 F o r	35.2 18.9 19.2 60.1 36.9	2,200 512 902 4,375 5,723	-1.18 -1.75 -1.61 -0.44 0.01
	8 9 10 11 12 13 14	0.33 0.50 0.92 2 3 4 5		P e e r P e e r P e e r 9,220 34.6 6,680 15.5 9,720 35.5 P 11,950 21.1 9,440 41.7 e 4,760 46.7 10,300 15.6 e r	3190 1,035 3,451 2,521 3,937 2,223 1,607	-0.84 -1.57 -0.75 -0.88 -0.07 -1.05 -1.41
	15 16 17 18 19 20 21 22 23 24 25	6 6 6 8 9 11 11 12 12 12 12		5,150 5,290 12,640 2,850 6,330 4,750 4,110 6,230 5,940 5,860 8,280	R e v i e w R e v i e w R e v i e w 10.7 35.8 6.7 R 12.4 14.2 e 14.3 20.4 29.9 29.7 v i e 11.1 6.1 w	551 1,894 847 353 899 679 838 1,863 1,764 651 505	-2.01 -1.24 -1.84 -2.12 -1.81 -1.94 -1.85 -1.26 -1.32 -1.95 -2.04
	26	12		8,440	5.4	456	-2.07
	27	13		2,320	25.6	594	-1.99
	28	13		6,220	14.2	883	-1.82
	29	13		3,710	33.6	1,246	-1.61
	30	13		7,040	13.0	915	-1.80
	31	13		19,710	59.3	11,688	4.37
	32	14		8,180	8.6	704	-1.92
	33	14		5,190	26.1	1,354	-1.55
	34	14		6,010	31.3	1,881	-1.25
	35	16		6,990	5.4	377	-2.11
	36	16		5,600	20.2	1,131	-1.68
	37	16		13,100	5.9	773	-1.88
	Note. WBC: White blood cell count.

Table II .

 II Data from 76 children with flu-like illness with or without pandemic influenza A (H1N1) infection.

	Haemoglobin	12.5 (11.0-14.0)		12.6 (11.8.-1350)	0.629
	concentration* (g/L)
	Platelet count*	Children 252 (207-346)	with	Children with viral 338 (286-431)	P 0.003
	(1,000/µL)	Influenza A H1N1	respiratory infection
	C reactive protein*	infection 0.66 (0.34-1.80)		(n=39) 0.62 (0.40-1.29)	0.817
	(mg/dL)	(n=37)
	Males (n;%)	20 (54.1%)		18 (46.2%)	0.491
	Note: * Median and interquartile range
	Age* (years) Time period*between disease onset and	9.0 (0.3-13.0) 1.9 (1.2-3.7) F o r		9.1 (1.1-14.2) 1.7 (1.5-4.2)	0.896 0.641
	influenza PCR assay	P
	(days)	e
	WBC count* (cells/µL) 6,330 (5,170-9,330) e r 9040 (7450-11100)	0.002
	Relative lymphocyte count* (%) Absolute lymphocyte count* (cells/µL)	20.2 (11.7-34.9) 1,035 (691-2,211)	47.7 (33.8-56.1) R 3,751 (2884-4832) e v i e	<0.0001 <0.0001
	Absolute lymphocyte	-1.61 (-1.90 -1.11)	-0.30 (-0.67 0.02) w	<0.0001
	count z-score*
	Relative neutrophil	60.8 (47.4-74.6)		40.8 (27.6-55.6)	<0.0001
	count* (%)
	Absolute neutrophil	4,155 (2,721-5,770)	3,549 (2,470-5,194)	0.427
	count* (cells/µL)
	Red blood cell count*	4,540 (3,840-4,945)	4,745 (4,445 -5,140)	0.062
	(1,000 cells/µL)
					12

Journal of Medical Virology