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High incidence of extrahepatic manifestations in an HCV hyperendemic area
Hepatology Research 22 (2002) 27 – 36
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High incidence of extrahepatic manifestations in an HCV hyperendemic area Yumiko Nagao a,b,*, Junko Tanaka c, Toshio Nakanishi d, Takashi Moriya c, Keiko Katayama c, Junko Kumagai c, Yutaka Komiya c, Yuji Itoh e, Yoshinari Myoken f, Megumu Fujihara g, Michio Sata a,h, Hiroshi Yoshizawa c a
Research Center for Inno6ati6e Cancer Therapy, Kurume Uni6ersity School of Medicine, Kurume, Fukuoka 830 0011, Japan b Department of Oral Surgery, Kurume Uni6ersity School of Medicine, Kurume, Fukuoka 830 0011, Japan c Department of Hygiene, Hiroshima Uni6ersity School of Medicine, Hiroshima, Japan d First Department of Medicine, Hiroshima Uni6ersity School of Medicine, Hiroshima, Japan e Clinical Laboratory, Kurume Uni6ersity School of Medicine, Kurume, Fukuoka, Japan f Department of Oral Surgery, Hiroshima Red-Cross and Atomic Bomb Sur6i6ors Hospital, Hiroshima, Japan g Department of Pathology, Hiroshima Red-Cross and Atomic Bomb Sur6i6ors Hospital, Hiroshima, Japan h Second Department of Medicine, Kurume Uni6ersity School of Medicine, Kurume, Fukuoka, Japan Received 21 December 2000; received in revised form 07 March 2001; accepted 18 April 2001
Abstract We previously investigated the incidence of extrahepatic manifestations including oral precancerous disease among the inhabitants in a hepatitis C virus (HCV) hyperendemic area in Fukuoka in Japan. The present study design was based on a prospective cohort at the other HCV hyperendemic area. One oral surgeon examined the oral lesions of 59 adult inhabitants (21 men, 38 women; mean age of 70.7 years), of a hyperendemic area of HCV infection. Furthermore, all subjects were interviewed regarding the natural history of extrahepatic manifestations. All sera were examined for antibodies to HCV (anti-HCV), serum HCV RNA, HCV genotype, antinuclear antibody (ANA), rheumatoid factor (RF) activity, and anti-SS-A and-B antibodies. Anti-HCV or HCV RNA was detected in sera from 59 (100%) or 57 (96.7%) of all subjects. Oral lichen planus (OLP), leukoplakia with leukoedema, or only leukoedema was observed in 8 (8.5%), 1 (1.7%), or 2 (3.4%) subjects, respectively. The incidence of all subjects with one or more HCV-related extrahepatic manifestation was 66.1% (39/59). The subjects with dry mouth were 25.4% (15/59). There was no relation among these autoantibodies, symptoms of dry mouth, or prevalence of HCV-associated extrahepatic manifestations. These findings demonstrate that the inhabitants with HCV infection showed various extrahepatic manifestations, and was not always limited to specific HCV areas. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hepatitis C virus (HCV); Oral lichen planus (OLP); Extrahepatic manifestation; Autoantibody; HCV hyperendemic area Abbre6iations: HCV, Hepatitis C virus; OLP, Oral lichen planus; RT-PCR, reverse transcription polymerase chain reaction, ANA, antinuclear antibody; RF, rheumatoid factor; ELISA, enzyme-linked immunosorbent assay; DM, diabetes mellitus. * Corresponding author. Tel.: +81-942-317746; fax: + 81-942-317747. E-mail address: [email protected] (Y. Nagao). 1386-6346/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 0 1 ) 0 0 1 1 4 - 0
Y. Nagao et al. / Hepatology Research 22 (2002) 27–36
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1. Introduction
2. Subjects and methods
Hepatitis C virus (HCV) infection not only causes chronic liver diseases but shows extrahepatic manifestations and immunological disorders which include membranoproliferative glomerulonephritis, cryoglobulinemia, autoimmune thyroiditis, Sjo¨ gren’s syndrome, malignant lymphoma, and lichen planus [1– 3]. We previously reported that the incidence of OLP in subjects with HCV infection was significantly higher than in those without HCV using the mass screening of 685 inhabitants of a hyperendemic area, in H town, for HCV infection [4]. After 6 years, we investigated the incidence or malignant transformation of OLP and the natural history of extrahepatic manifestations among random inhabitants from the same area [5]. In addition to OLP, the prevalences of anti-HCV in the other extrahepatic manifestations were higher than those without HCV. H town [6,7], located in the Fukuoka prefecture in northern Kyushu, Japan, has an adult population of approx. 7400. In the present investigation, we extended the study to the other HCV hyperendemic area of O town, which has a population of approx. 3900 in the northwest of Hiroshima prefecture in Honshu, Japan, to clarify the rate of OLP and the other extrahepatic manifestations. An epidemiologic study of HCV infection had been followed in O town since 1993 [8].
2.1. Subjects A total of 59 adult inhabitants, as HCV carriers, from O town, a hyperendemic area of HCV infection, participated in the present study, 21 men and 38 women (mean age9 S.D.: 70.79 7.2; Table 1). Oral membrane diseases in all subjects were observed by one oral surgeon for each inhabitant. A topographic map of the oral mucosa was exactly classified into 56 areas according to Roed-Petersen et al.’s classification [9] (Fig. 1) and examination of the oral mucosa was based on ‘Guide to epidemiology and diagnosis of oral mucosal diseases and conditions. World Health Organization (WHO)’ [10]. The diagnosis was made on the basis of clinical features and based on WHO criteria. Informed consent was obtained from all subjects, and color photographs of their oral cavity were taken. Furthermore, to study the natural history of extrahepatic manifestations, all study subjects were interviewed in person by two trained interviewers. The following signs were questioned: skin disease, renal disease, rheumatoid arthritis, abnormal thyroid function, heart disease, hypertension, diabetes mellitus (DM), and extrahepatic malignant tumor. We recorded history of smoking, use of denture wear, condition of periodontitis, dry mouth, and oral health care, oral mucosal diseases, information on previous
Table 1 Characteristics of oral membrane lesions in adult inhabitants with HCV infection Total
Subjects Age (yr) (mean 9S.D.) Sex (M/F) Anti-HCV (+) (%) HCV RNA (+) (%) a
59 70.797.2 21/38 59 57
Oral membrane lesion Oral lichen planus
Leukoplakia and leukoedemaa
Leukoedema
5 (8.5) 74.8 9 5.2 1/4 5/59 (8.5) 5/57 (8.8)
1 (1.7) 65 9 0 1/0 1/59 (1.7) 1/57 (1.8)
2 (3.4) 62 9 3.0 2/0 2/59 (3.4) 1/57 (1.8)
The patient with leukoplakia in the left upper gingiva and leukoedema in the bilateral buccal mucosa.
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Fig. 1. Assessment form for oral mucosal diseases modified WHO guide [10] and topography of the oral mucosa modified by Roed-Petersen et al. [9]. A form for the recording of information on oral mucosal diseases and oral habits was reproduced. Oral areas were classified into 56 areas. The numbers for locations refer to the drawing in the figure. Several location code spaces were used to record lesions which extend over more than one site.
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Y. Nagao et al. / Hepatology Research 22 (2002) 27–36
Fig. 1. (Continued)
treatment, treatment requirements, and the other extrahepatic manifestations on our original recording form by modified Roed-Petersen et al.’s classification [9] (Fig. 1). All findings were reported to the individual inhabitants. The inhabitants with OLP or oral leukoplakia were introduced to the Department of Oral Surgery, Hiroshima Red-Cross and Atomic Bomb Survivors Hospital, Hiroshima, and were biopsied, and treated.
2.2. Examination of anti-HCV and HCV RNA in serum The sera of all inhabitants were tested for antibodies to HCV (anti-HCV) by second-generation, enzyme-linked immnosorbent assay (Abbott HCV PHA 2nd Generation, Dainabot Co., Ltd., Tokyo, Japan) according to the manufacturer’s instructions [11,12]. HCV RNA in the sera was determined by reverse transcription-nested polymerase chain reaction (RT-nested PCR) using the Amplicore HCV test (Nippon Roche, Tokyo, Japan) as described previously [13]. Ultrasonographic examination was performed on all inhabitants.
2.3. Typing HCV by RT-PCR The HCV genotype was determined based on the sequence of the core region by modification of the method of Okamoto et al. [14]. With this method, type I [1a]-specific DNA fragments were observed as a 57-base pair (bp) band, whereas type II [1b]-, III [2a]-, and IV [2b]-specific fragments were found as 144-, 174-, and 123-bp bands, respectively [15].
2.4. E6aluation by testing for ANA, RF, and anti-SS-A and B antibody in serum Antinuclear antibody (ANA), rheumatoid factor (RF), anti-SS-A antibody, and anti-SS-B-antibody were tested by immunofluorescence (FLUORO HEPANA test, MBL, Nagoya, Japan), latex agglutination (N-Latex RF Kit II, Dade Behring, Tokyo, Japan), enzyme-linked immunosorbent assay (ELISA) (Mesacup™ SS-A/ Ro, MBL, Nagoya, Japan), and ELISA; Mesacup™ SS-B/La, MBL, Nagoya, Japan), respectively
Y. Nagao et al. / Hepatology Research 22 (2002) 27–36
3. Results Anti-HCV antibodies and HCV RNA were detected in the sera from 59 (100%) and 57 (96.7%) of 59 inhabitants, respectively (Table 1). The other two inhabitants (3.4%) were negative for HCV RNA because the therapeutic response was judged after interferon therapy as complete response. HCV genotypes in 57 subjects included 45 cases of 1b (45/57, 78.9%), six of 2a (6/57, 10.5%), five of 2b (5/57, 8.8%) and one of 1b and 2b (1/57, 1.8%). The prevalence of OLP in all subjects was 8.5% (5/59), while that of OLP in HCV RNA positive subjects was 8.8% (5/57), are shown in the Table 1. The clinical appearances of five OLP subjects were three with reticular and two with erosive. Three subjects with OLP had no pain. Most of the sites of OLP was buccal mucosa (Table 2). The diagnosis of all OLP subjects was made on the basis of clinical features. However, all OLP subjects were biopsied and subsequently diagnosed as OLP histopathologicaly. Although none had oral cancer, the incidences of leukoplakia with leukoedema or only leukoedema in the subjects with HCV infection was 1.7% (1/59), and 3.4% (2/59), respectively (Table 1). All leukoedema were located in the buccal mucosa, and leukoplakia in the gingiva. One patient had leukoplakia in the left upper gingiva and Table 2 Topographical classification of the oral membrane disease Oral membrane disease
No. of patients
No. of the sites
Oral lichen planus
1
15, 25, 37, 14, 20, 30 20 28, 19, 19, 19,
2 3 4 5 Oral leukoplakia 6 Oral leukoedema 6 7 8
16, 27, 39, 19, 45
19, 20, 21, 23, 28, 31, 33, 34, 40, 47, 51, 52 20
34 20 20 20
No. 6 patient had leukoplakia in the left upper gingiva and leukoedema in the bilateral buccal mucosa.
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leukoedema in the bilateral buccal mucosa. All subjects with leukoedema have a history of smoking. The diagnosis of leukoplakia was made on the basis of clinical features, biopsy and subsequent histopathological examination. We also examined the relation between macroscopic findings of HCV-associated oral membrane disease (OLP, leukoplakia, and leukoedema) and HCV genotype but no significant relation was observed. HCV genotypes in OLP subjects were all 1b. Of 59 subjects, glabrous or cleft tongue, dry mouth, or uncomfortable dry feeling of oral cavity was 16.9% (10/59), 10.2% (6/59), or 13.6% (8/59), respectively. The subjects with one or more of these symptoms were 25.4% (15/59). The positive% of ANA, RF, anti-SS-A, or anti-SS-B was 30.5% (18/59), 8.5% (5/59), 13.6% (8/59), 3.4% (2/59), respectively (Table 3). There was no significant differences among these autoantibodies, symptoms of dry mouth, or prevalence of HCVassociated extrahepatic manifestations. The 15 subjects with symptoms of dry mouth used partial or full dentures. In addition to OLP, the prevalences of hypertension, heart disease, DM, rheumatoid arthritis, skin disease, renal disease, extrahepatic malignant tumor, or abnormal thyroid function was 28.8% (17/59), 10.2% (6/59), 15.3% (9/59), 1.7% (1/59), 5.1% (3/59), 1.7% (1/59), 11.9% (7/59), 0%, respectively (Fig. 2). The diagnosis of seven extrahepatic malignancies were carcinoma of the uterus (3), stomach (3), and malignant lymphoma (1). The incidence of the subjects with not less than one extrahepatic manifestation of HCV was 66.1% (39/59). The incidences of inhabitants with one, two, or three manifestations were 45.76% (27/59), 13.56% (8/59), or 6.78% (4/59), respectively. The incidence of 1b, 2a, 2b, and 1b+2b of HCV genotypes in subjects with extrahepatic manifestations was 71.8% (28/39), 15.4% (6/39), 5.1% (2/39), and 2.6% (1/39), respectively, while that of 1b or 2b in subjects without manifestations was 85% (17/20) or 15% (3/20). Two subjects (2/39, 5.1%) with extrahepatic manifestations were negative for HCV RNA. There was no significant difference in HCV genotypes regardless of the presence or absence of extrahepatic manifestations.
32 Y. Nagao et al. / Hepatology Research 22 (2002) 27–36 Fig. 2. Prevalence (%) of extrahepatic manifestations in adult inhabitants with HCV infection. Diagnosis of oral membrane lesions include OLP (8.5%, 5/59), leukoplakia with leukoedema (1.7%, 1/59), and leukoedema alone (3.4%, 2/59). The prevalence of the oral membrane lesions, hypertension, heart disease, DM, rheumatoid arthritis, skin disease, renal disease, extrahepatic malignant tumor, or abnormal thyroid function was 13.6% (8/59), 28.8% (17/59), 10.2% (6/59), 15.3% (9/59), 1.7% (1/59), 5.1% (3/59), 1.7% (1/59), 11.9% (7/59), 0%, respectively.
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Table 3 Characteristics of autoantibodies in adult inhabitants with HCV infection
4. Discussion Mass screening for oral precancerous lesions and registration of detected patients in a referral center are important components of an adult oral health program. However, the incidence of oral cancer and its precancerous lesions such as OLP and leukoplakia among the general population with HCV is still unclear. A complete history and physical examination by a multidisciplinary group of health care providers uncovers common problems, especially mucosal lesions of lichen planus are found in the oral cavity, esophagus, conjunctiva, bladder, nose, larynx, stomach, and anus. In Japan, OLP was reported to be found in 0.5% [16].
We previously investigated the incidence of precancerous lesions among the inhabitants in an HCV hyperendemic area (H town in Fukuoka prefecture) [4]. The prevalence of oral membrane disease (OLP, leukoplakia, and leukoedema) and other extrahepatic manifestations in the subjects with HCV infection was significantly higher than that in those without HCV [5]. In H town, OLP was observed in 12 (6.3%, 12/190) subjects. AntiHCV was observed in 21.1% (40/190) of the subjects and HCV RNA in 16.3% (31/190). The incidence of OLP in those subjects with serum HCV RNA-positive (16.1%, P B 0.05 vs. the OLP-HCV RNA negative group) was significantly higher than those without HCV. Besides OLP, the prevalences of anti-HCV in the other extrahepatic manifestations were higher than those without
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Y. Nagao et al. / Hepatology Research 22 (2002) 27–36
HCV. The prevalence of OLP in the habitants with HCV infection was significantly higher than that of OLP in the general population (0.5%). In the present study, we performed an epidemiologic examination of another HCV hyperendemic area (O town in Hiroshima prefecture) and found the presence of HCV-associated extrahepatic manifestations. This finding demonstrates that the phenomenon, that the inhabitants with HCV infection had various extrahepatic manifestations in H town, was not always limited to a specific HCV area. Epidemiology is concerned with the study of factors that affect the health of groups of people. Thus, the oral observer was an individual with long training and experience in the diagnosis of lesions of the oral mucosa. A systematic and standard approach system for recording of different conditions of the oral mucosa is very important. The classification proposed by Roed-Petersen and Renstrup [9] is convenient for the present epidemiologic purpose and is used in the guide of WHO [10]. Data of French, Japanese, Italian and Spain patients do not suggest any association between a specific HCV genotype and the development of oral or skin lichen planus [17– 20]. We reported that HCV seropositive patients with OLP were more commonly infected by genotype 1b [18]. In the present study, genotypes in OLP subjects were 1b. Moreover most of genotype was 1b regardless of the presence or absence of extrahepatic manifestations. These may simply reflect the high prevalence of genotype 1b in the Japanese population. Cacoub et al. reported a high prevalence of both clinical and biologic extrahepatic manifestations in a study of 1614 patients who had chronic HCV infection [21]. Several immunologic abnormalities were reported to be observed, including cryoglobulines (40%), ANA (10%), anti-smooth muscle antibodies (7%). In their prospective multicenter study of 321 patients, 38% (122/321) of patients presented at least one clinical extrahepatic manifestation [22]. The incidence of manifestations (66.1%, 39/59) in the present study was higher than that of Cacoub’s study. It was reported that 10–20% of HCV patients have sicca
symptoms of either the mouth or eyes [23,24]. There was no Sjo¨ gren’s syndrome in the subjects from O town, but 25.4% (15/59) of HCV subjects had symptoms of dry mouth, 42.4% (25/59) had at least one of the autoantibodies, including ANA, RF, anti-SS-A, or anti-SS-B. The incidence of autoantibodies in females (19/25, 76%) was significantly higher than that in males (6/25, 24%). We found a high incidence of autoantibodies and dry mouth. However, we could not find a relation among autoantibodies, symptoms of dry mouth, and prevalence of HCV-associated extrahepatic manifestations. Sikuler et al. evaluated the relationship between HCV infection and extrahepatic malignancies, and reported that extrahepatic malignancies were detected in 15 of 103 patients who were positive for HCV antibody (14.6%) [25]. We previously reported that the incidence of HCV infection was also high in oral cancer or OLP patients [26– 28], and that the incidence of various extrahepatic manifestations including OLP was also high in HCV infected subjects [4,5,29]. Moreover, the incidence of HCV infection is high in patients with double cancer consisting of oral cancer and nonHodgkin’s lymphoma [30,31]. Numerous cases of non-Hodgkin’s lymphoma have been reported in association with HCV [32,33]. In the present study one of 59 HCV infected subjects developed a malignant lymphoma. It was noted that HCV may proliferate not only in hepatocytes but also in various cells [34– 38]. Both positive and negative HCV RNA strands in OLP, oral cancer tissue, and myocardium with myocarditis and cardiomyopathy were detected [39–41]. These findings may indicate that HCV persists and replicates in these lesions, suggesting a pathological role of HCV, although the mechanisms are unclear. Chronic HCV infection may be associated with a risk of development of lymphoma. These observations suggested that attention should be focused on the development of extrahepatic manifestations in patients with HCV infection. OLP patients with reticular and papular types were often unaware of the oral lesions because some patients have no pain [42]. Therefore, we recommend the examination of patients with HCV infection by a oral medical specialist.
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Acknowledgements This study was supported, in part, by grants as a project for establishing new high technology research centers, by a Grant-in-Aid for Scientific Research (C) (No. 11670548) and by a Grant-inAid for Encouragement of Young Scientists (No. 12771260) from the Ministry of Education, Science, Sports and Culture of Japan. We thank Mr. S. Nakamura and Mr. K. Masuda for assisting with the interviews, Ms. M. Nagao for her assistance with English, and Dr. M. Kage for helpful comments.
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High incidence of extrahepatic manifestations in an HCV hyperendemic area Yumiko Nagao a,b,*, Junko Tanaka c, Toshio Nakanishi d, Takashi Moriya c, Keiko Katayama c, Junko Kumagai c, Yutaka Komiya c, Yuji Itoh e, Yoshinari Myoken f, Megumu Fujihara g, Michio Sata a,h, Hiroshi Yoshizawa c a
Research Center for Inno6ati6e Cancer Therapy, Kurume Uni6ersity School of Medicine, Kurume, Fukuoka 830 0011, Japan b Department of Oral Surgery, Kurume Uni6ersity School of Medicine, Kurume, Fukuoka 830 0011, Japan c Department of Hygiene, Hiroshima Uni6ersity School of Medicine, Hiroshima, Japan d First Department of Medicine, Hiroshima Uni6ersity School of Medicine, Hiroshima, Japan e Clinical Laboratory, Kurume Uni6ersity School of Medicine, Kurume, Fukuoka, Japan f Department of Oral Surgery, Hiroshima Red-Cross and Atomic Bomb Sur6i6ors Hospital, Hiroshima, Japan g Department of Pathology, Hiroshima Red-Cross and Atomic Bomb Sur6i6ors Hospital, Hiroshima, Japan h Second Department of Medicine, Kurume Uni6ersity School of Medicine, Kurume, Fukuoka, Japan Received 21 December 2000; received in revised form 07 March 2001; accepted 18 April 2001
Abstract We previously investigated the incidence of extrahepatic manifestations including oral precancerous disease among the inhabitants in a hepatitis C virus (HCV) hyperendemic area in Fukuoka in Japan. The present study design was based on a prospective cohort at the other HCV hyperendemic area. One oral surgeon examined the oral lesions of 59 adult inhabitants (21 men, 38 women; mean age of 70.7 years), of a hyperendemic area of HCV infection. Furthermore, all subjects were interviewed regarding the natural history of extrahepatic manifestations. All sera were examined for antibodies to HCV (anti-HCV), serum HCV RNA, HCV genotype, antinuclear antibody (ANA), rheumatoid factor (RF) activity, and anti-SS-A and-B antibodies. Anti-HCV or HCV RNA was detected in sera from 59 (100%) or 57 (96.7%) of all subjects. Oral lichen planus (OLP), leukoplakia with leukoedema, or only leukoedema was observed in 8 (8.5%), 1 (1.7%), or 2 (3.4%) subjects, respectively. The incidence of all subjects with one or more HCV-related extrahepatic manifestation was 66.1% (39/59). The subjects with dry mouth were 25.4% (15/59). There was no relation among these autoantibodies, symptoms of dry mouth, or prevalence of HCV-associated extrahepatic manifestations. These findings demonstrate that the inhabitants with HCV infection showed various extrahepatic manifestations, and was not always limited to specific HCV areas. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hepatitis C virus (HCV); Oral lichen planus (OLP); Extrahepatic manifestation; Autoantibody; HCV hyperendemic area Abbre6iations: HCV, Hepatitis C virus; OLP, Oral lichen planus; RT-PCR, reverse transcription polymerase chain reaction, ANA, antinuclear antibody; RF, rheumatoid factor; ELISA, enzyme-linked immunosorbent assay; DM, diabetes mellitus. * Corresponding author. Tel.: +81-942-317746; fax: + 81-942-317747. E-mail address: [email protected] (Y. Nagao). 1386-6346/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 0 1 ) 0 0 1 1 4 - 0
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1. Introduction
2. Subjects and methods
Hepatitis C virus (HCV) infection not only causes chronic liver diseases but shows extrahepatic manifestations and immunological disorders which include membranoproliferative glomerulonephritis, cryoglobulinemia, autoimmune thyroiditis, Sjo¨ gren’s syndrome, malignant lymphoma, and lichen planus [1– 3]. We previously reported that the incidence of OLP in subjects with HCV infection was significantly higher than in those without HCV using the mass screening of 685 inhabitants of a hyperendemic area, in H town, for HCV infection [4]. After 6 years, we investigated the incidence or malignant transformation of OLP and the natural history of extrahepatic manifestations among random inhabitants from the same area [5]. In addition to OLP, the prevalences of anti-HCV in the other extrahepatic manifestations were higher than those without HCV. H town [6,7], located in the Fukuoka prefecture in northern Kyushu, Japan, has an adult population of approx. 7400. In the present investigation, we extended the study to the other HCV hyperendemic area of O town, which has a population of approx. 3900 in the northwest of Hiroshima prefecture in Honshu, Japan, to clarify the rate of OLP and the other extrahepatic manifestations. An epidemiologic study of HCV infection had been followed in O town since 1993 [8].
2.1. Subjects A total of 59 adult inhabitants, as HCV carriers, from O town, a hyperendemic area of HCV infection, participated in the present study, 21 men and 38 women (mean age9 S.D.: 70.79 7.2; Table 1). Oral membrane diseases in all subjects were observed by one oral surgeon for each inhabitant. A topographic map of the oral mucosa was exactly classified into 56 areas according to Roed-Petersen et al.’s classification [9] (Fig. 1) and examination of the oral mucosa was based on ‘Guide to epidemiology and diagnosis of oral mucosal diseases and conditions. World Health Organization (WHO)’ [10]. The diagnosis was made on the basis of clinical features and based on WHO criteria. Informed consent was obtained from all subjects, and color photographs of their oral cavity were taken. Furthermore, to study the natural history of extrahepatic manifestations, all study subjects were interviewed in person by two trained interviewers. The following signs were questioned: skin disease, renal disease, rheumatoid arthritis, abnormal thyroid function, heart disease, hypertension, diabetes mellitus (DM), and extrahepatic malignant tumor. We recorded history of smoking, use of denture wear, condition of periodontitis, dry mouth, and oral health care, oral mucosal diseases, information on previous
Table 1 Characteristics of oral membrane lesions in adult inhabitants with HCV infection Total
Subjects Age (yr) (mean 9S.D.) Sex (M/F) Anti-HCV (+) (%) HCV RNA (+) (%) a
59 70.797.2 21/38 59 57
Oral membrane lesion Oral lichen planus
Leukoplakia and leukoedemaa
Leukoedema
5 (8.5) 74.8 9 5.2 1/4 5/59 (8.5) 5/57 (8.8)
1 (1.7) 65 9 0 1/0 1/59 (1.7) 1/57 (1.8)
2 (3.4) 62 9 3.0 2/0 2/59 (3.4) 1/57 (1.8)
The patient with leukoplakia in the left upper gingiva and leukoedema in the bilateral buccal mucosa.
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Fig. 1. Assessment form for oral mucosal diseases modified WHO guide [10] and topography of the oral mucosa modified by Roed-Petersen et al. [9]. A form for the recording of information on oral mucosal diseases and oral habits was reproduced. Oral areas were classified into 56 areas. The numbers for locations refer to the drawing in the figure. Several location code spaces were used to record lesions which extend over more than one site.
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Fig. 1. (Continued)
treatment, treatment requirements, and the other extrahepatic manifestations on our original recording form by modified Roed-Petersen et al.’s classification [9] (Fig. 1). All findings were reported to the individual inhabitants. The inhabitants with OLP or oral leukoplakia were introduced to the Department of Oral Surgery, Hiroshima Red-Cross and Atomic Bomb Survivors Hospital, Hiroshima, and were biopsied, and treated.
2.2. Examination of anti-HCV and HCV RNA in serum The sera of all inhabitants were tested for antibodies to HCV (anti-HCV) by second-generation, enzyme-linked immnosorbent assay (Abbott HCV PHA 2nd Generation, Dainabot Co., Ltd., Tokyo, Japan) according to the manufacturer’s instructions [11,12]. HCV RNA in the sera was determined by reverse transcription-nested polymerase chain reaction (RT-nested PCR) using the Amplicore HCV test (Nippon Roche, Tokyo, Japan) as described previously [13]. Ultrasonographic examination was performed on all inhabitants.
2.3. Typing HCV by RT-PCR The HCV genotype was determined based on the sequence of the core region by modification of the method of Okamoto et al. [14]. With this method, type I [1a]-specific DNA fragments were observed as a 57-base pair (bp) band, whereas type II [1b]-, III [2a]-, and IV [2b]-specific fragments were found as 144-, 174-, and 123-bp bands, respectively [15].
2.4. E6aluation by testing for ANA, RF, and anti-SS-A and B antibody in serum Antinuclear antibody (ANA), rheumatoid factor (RF), anti-SS-A antibody, and anti-SS-B-antibody were tested by immunofluorescence (FLUORO HEPANA test, MBL, Nagoya, Japan), latex agglutination (N-Latex RF Kit II, Dade Behring, Tokyo, Japan), enzyme-linked immunosorbent assay (ELISA) (Mesacup™ SS-A/ Ro, MBL, Nagoya, Japan), and ELISA; Mesacup™ SS-B/La, MBL, Nagoya, Japan), respectively
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3. Results Anti-HCV antibodies and HCV RNA were detected in the sera from 59 (100%) and 57 (96.7%) of 59 inhabitants, respectively (Table 1). The other two inhabitants (3.4%) were negative for HCV RNA because the therapeutic response was judged after interferon therapy as complete response. HCV genotypes in 57 subjects included 45 cases of 1b (45/57, 78.9%), six of 2a (6/57, 10.5%), five of 2b (5/57, 8.8%) and one of 1b and 2b (1/57, 1.8%). The prevalence of OLP in all subjects was 8.5% (5/59), while that of OLP in HCV RNA positive subjects was 8.8% (5/57), are shown in the Table 1. The clinical appearances of five OLP subjects were three with reticular and two with erosive. Three subjects with OLP had no pain. Most of the sites of OLP was buccal mucosa (Table 2). The diagnosis of all OLP subjects was made on the basis of clinical features. However, all OLP subjects were biopsied and subsequently diagnosed as OLP histopathologicaly. Although none had oral cancer, the incidences of leukoplakia with leukoedema or only leukoedema in the subjects with HCV infection was 1.7% (1/59), and 3.4% (2/59), respectively (Table 1). All leukoedema were located in the buccal mucosa, and leukoplakia in the gingiva. One patient had leukoplakia in the left upper gingiva and Table 2 Topographical classification of the oral membrane disease Oral membrane disease
No. of patients
No. of the sites
Oral lichen planus
1
15, 25, 37, 14, 20, 30 20 28, 19, 19, 19,
2 3 4 5 Oral leukoplakia 6 Oral leukoedema 6 7 8
16, 27, 39, 19, 45
19, 20, 21, 23, 28, 31, 33, 34, 40, 47, 51, 52 20
34 20 20 20
No. 6 patient had leukoplakia in the left upper gingiva and leukoedema in the bilateral buccal mucosa.
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leukoedema in the bilateral buccal mucosa. All subjects with leukoedema have a history of smoking. The diagnosis of leukoplakia was made on the basis of clinical features, biopsy and subsequent histopathological examination. We also examined the relation between macroscopic findings of HCV-associated oral membrane disease (OLP, leukoplakia, and leukoedema) and HCV genotype but no significant relation was observed. HCV genotypes in OLP subjects were all 1b. Of 59 subjects, glabrous or cleft tongue, dry mouth, or uncomfortable dry feeling of oral cavity was 16.9% (10/59), 10.2% (6/59), or 13.6% (8/59), respectively. The subjects with one or more of these symptoms were 25.4% (15/59). The positive% of ANA, RF, anti-SS-A, or anti-SS-B was 30.5% (18/59), 8.5% (5/59), 13.6% (8/59), 3.4% (2/59), respectively (Table 3). There was no significant differences among these autoantibodies, symptoms of dry mouth, or prevalence of HCVassociated extrahepatic manifestations. The 15 subjects with symptoms of dry mouth used partial or full dentures. In addition to OLP, the prevalences of hypertension, heart disease, DM, rheumatoid arthritis, skin disease, renal disease, extrahepatic malignant tumor, or abnormal thyroid function was 28.8% (17/59), 10.2% (6/59), 15.3% (9/59), 1.7% (1/59), 5.1% (3/59), 1.7% (1/59), 11.9% (7/59), 0%, respectively (Fig. 2). The diagnosis of seven extrahepatic malignancies were carcinoma of the uterus (3), stomach (3), and malignant lymphoma (1). The incidence of the subjects with not less than one extrahepatic manifestation of HCV was 66.1% (39/59). The incidences of inhabitants with one, two, or three manifestations were 45.76% (27/59), 13.56% (8/59), or 6.78% (4/59), respectively. The incidence of 1b, 2a, 2b, and 1b+2b of HCV genotypes in subjects with extrahepatic manifestations was 71.8% (28/39), 15.4% (6/39), 5.1% (2/39), and 2.6% (1/39), respectively, while that of 1b or 2b in subjects without manifestations was 85% (17/20) or 15% (3/20). Two subjects (2/39, 5.1%) with extrahepatic manifestations were negative for HCV RNA. There was no significant difference in HCV genotypes regardless of the presence or absence of extrahepatic manifestations.
32 Y. Nagao et al. / Hepatology Research 22 (2002) 27–36 Fig. 2. Prevalence (%) of extrahepatic manifestations in adult inhabitants with HCV infection. Diagnosis of oral membrane lesions include OLP (8.5%, 5/59), leukoplakia with leukoedema (1.7%, 1/59), and leukoedema alone (3.4%, 2/59). The prevalence of the oral membrane lesions, hypertension, heart disease, DM, rheumatoid arthritis, skin disease, renal disease, extrahepatic malignant tumor, or abnormal thyroid function was 13.6% (8/59), 28.8% (17/59), 10.2% (6/59), 15.3% (9/59), 1.7% (1/59), 5.1% (3/59), 1.7% (1/59), 11.9% (7/59), 0%, respectively.
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Table 3 Characteristics of autoantibodies in adult inhabitants with HCV infection
4. Discussion Mass screening for oral precancerous lesions and registration of detected patients in a referral center are important components of an adult oral health program. However, the incidence of oral cancer and its precancerous lesions such as OLP and leukoplakia among the general population with HCV is still unclear. A complete history and physical examination by a multidisciplinary group of health care providers uncovers common problems, especially mucosal lesions of lichen planus are found in the oral cavity, esophagus, conjunctiva, bladder, nose, larynx, stomach, and anus. In Japan, OLP was reported to be found in 0.5% [16].
We previously investigated the incidence of precancerous lesions among the inhabitants in an HCV hyperendemic area (H town in Fukuoka prefecture) [4]. The prevalence of oral membrane disease (OLP, leukoplakia, and leukoedema) and other extrahepatic manifestations in the subjects with HCV infection was significantly higher than that in those without HCV [5]. In H town, OLP was observed in 12 (6.3%, 12/190) subjects. AntiHCV was observed in 21.1% (40/190) of the subjects and HCV RNA in 16.3% (31/190). The incidence of OLP in those subjects with serum HCV RNA-positive (16.1%, P B 0.05 vs. the OLP-HCV RNA negative group) was significantly higher than those without HCV. Besides OLP, the prevalences of anti-HCV in the other extrahepatic manifestations were higher than those without
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HCV. The prevalence of OLP in the habitants with HCV infection was significantly higher than that of OLP in the general population (0.5%). In the present study, we performed an epidemiologic examination of another HCV hyperendemic area (O town in Hiroshima prefecture) and found the presence of HCV-associated extrahepatic manifestations. This finding demonstrates that the phenomenon, that the inhabitants with HCV infection had various extrahepatic manifestations in H town, was not always limited to a specific HCV area. Epidemiology is concerned with the study of factors that affect the health of groups of people. Thus, the oral observer was an individual with long training and experience in the diagnosis of lesions of the oral mucosa. A systematic and standard approach system for recording of different conditions of the oral mucosa is very important. The classification proposed by Roed-Petersen and Renstrup [9] is convenient for the present epidemiologic purpose and is used in the guide of WHO [10]. Data of French, Japanese, Italian and Spain patients do not suggest any association between a specific HCV genotype and the development of oral or skin lichen planus [17– 20]. We reported that HCV seropositive patients with OLP were more commonly infected by genotype 1b [18]. In the present study, genotypes in OLP subjects were 1b. Moreover most of genotype was 1b regardless of the presence or absence of extrahepatic manifestations. These may simply reflect the high prevalence of genotype 1b in the Japanese population. Cacoub et al. reported a high prevalence of both clinical and biologic extrahepatic manifestations in a study of 1614 patients who had chronic HCV infection [21]. Several immunologic abnormalities were reported to be observed, including cryoglobulines (40%), ANA (10%), anti-smooth muscle antibodies (7%). In their prospective multicenter study of 321 patients, 38% (122/321) of patients presented at least one clinical extrahepatic manifestation [22]. The incidence of manifestations (66.1%, 39/59) in the present study was higher than that of Cacoub’s study. It was reported that 10–20% of HCV patients have sicca
symptoms of either the mouth or eyes [23,24]. There was no Sjo¨ gren’s syndrome in the subjects from O town, but 25.4% (15/59) of HCV subjects had symptoms of dry mouth, 42.4% (25/59) had at least one of the autoantibodies, including ANA, RF, anti-SS-A, or anti-SS-B. The incidence of autoantibodies in females (19/25, 76%) was significantly higher than that in males (6/25, 24%). We found a high incidence of autoantibodies and dry mouth. However, we could not find a relation among autoantibodies, symptoms of dry mouth, and prevalence of HCV-associated extrahepatic manifestations. Sikuler et al. evaluated the relationship between HCV infection and extrahepatic malignancies, and reported that extrahepatic malignancies were detected in 15 of 103 patients who were positive for HCV antibody (14.6%) [25]. We previously reported that the incidence of HCV infection was also high in oral cancer or OLP patients [26– 28], and that the incidence of various extrahepatic manifestations including OLP was also high in HCV infected subjects [4,5,29]. Moreover, the incidence of HCV infection is high in patients with double cancer consisting of oral cancer and nonHodgkin’s lymphoma [30,31]. Numerous cases of non-Hodgkin’s lymphoma have been reported in association with HCV [32,33]. In the present study one of 59 HCV infected subjects developed a malignant lymphoma. It was noted that HCV may proliferate not only in hepatocytes but also in various cells [34– 38]. Both positive and negative HCV RNA strands in OLP, oral cancer tissue, and myocardium with myocarditis and cardiomyopathy were detected [39–41]. These findings may indicate that HCV persists and replicates in these lesions, suggesting a pathological role of HCV, although the mechanisms are unclear. Chronic HCV infection may be associated with a risk of development of lymphoma. These observations suggested that attention should be focused on the development of extrahepatic manifestations in patients with HCV infection. OLP patients with reticular and papular types were often unaware of the oral lesions because some patients have no pain [42]. Therefore, we recommend the examination of patients with HCV infection by a oral medical specialist.
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Acknowledgements This study was supported, in part, by grants as a project for establishing new high technology research centers, by a Grant-in-Aid for Scientific Research (C) (No. 11670548) and by a Grant-inAid for Encouragement of Young Scientists (No. 12771260) from the Ministry of Education, Science, Sports and Culture of Japan. We thank Mr. S. Nakamura and Mr. K. Masuda for assisting with the interviews, Ms. M. Nagao for her assistance with English, and Dr. M. Kage for helpful comments.
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