Clinical significance of occult hepatitis B virus infection in chronic hepatitis C patients

Background/Aims

We investigated the frequency of occult hepatitis B virus (HBV) infection in anti-hepatitis C virus (HCV)-positive individuals and the effects of occult HBV infection on the severity of liver disease.

Methods

Seventy-one hepatitis B virus surface-antigen (HBsAg)-negative patients were divided according to their HBV serological status into groups A (anti-HBc positive, anti-HBs negative; n=18), B (anti-HBc positive, anti-HBs positive; n=34), and C (anti-HBc negative, anti-HBs positive/negative; n=19), and by anti-HCV positivity (anti-HCV positive; n=32 vs. anti-HCV negative; n=39). Liver biopsy samples were taken, and HBV DNA was quantified by real-time PCR.

Results

Intrahepatic HBV DNA was detected in 32.4% (23/71) of the entire cohort, and HBV DNA levels were invariably low in the different groups. Occult HBV infection was detected more frequently in the anti-HBc-positive patients. Intrahepatic HBV DNA was detected in 28.1% (9/32) of the anti-HCV-positive and 35.9% (14/39) of the anti-HCV-negative subjects. The HCV genotype did not affect the detection rate of intrahepatic HBV DNA. In anti-HCV-positive cases, occult HBV infection did not affect liver disease severity.

Conclusions

Low levels of intrahepatic HBV DNA were detected frequently in both HBsAg-negative and anti-HCV-positive cases. However, the frequency of occult HBV infection was not affected by the presence of hepatitis C, and occult HBV infection did not have a significant effect on the disease severity of hepatitis C.     

某艾滋病治疗示范区人免疫缺陷病毒感染者合并隐匿性乙型肝炎病毒感染的调查分析

目的 调查分析某艾滋病治疗示范区人免疫缺陷病毒（HIV）-1感染者中隐匿性乙型肝炎病毒（HBV）感染的情况及其影响因素.方法 采集某艾滋病治疗示范区97例经血感染HIV-1的感染者的血浆,采用酶联免疫吸附试验（ELISA）检测乙型肝炎表面抗原与抗体（HBsAg与抗HBs）、乙型肝炎e抗原与抗体（HBeAg与抗Hbe）、乙型肝炎核心抗体（抗HBc）及丙型肝炎抗体（抗HCV）;采用吸附柱法抽提HBV DNA;采用巢式聚合酶链反应（PCR）法检测HBV S区;采用流式细胞仪计数CD4＋T淋巴细胞.HBsAg阴性PCR阳性结果 者为合并隐匿性HBV感染者.合并隐匿性HBV感染者为实验组,未合并隐匿性HBV感染者为对照组.结果 97例HIV感染者中HBsAg阴性者92例（94.85%）.92例HBsAg阴性者中合并隐匿性HBV感染者27例（29.35%）,抗HCV阳性者73例（79.35%）.合并隐匿性HBV感染者和未合并HBV感染者CD4＋T淋巴细胞数、单独抗HBc阳性率分别为（212.11±133.1）和（318.9±172.2）cells/mm3、62.96%和18.46%,以上两指标两组比较差异均有统计学意义（P＜0.01）,两组间年龄、性别、是否合并HCV感染及抗HBs阳性率比较差异无统计学意义（P＞0.05）.结论 经有偿献血途径感染HIV者中存在隐匿性HBV感染;HIV阳性合并隐匿性HBV感染者中易出现单纯抗HBc阳性;CD4＋T淋巴细胞数低的HIV感染者更容易合并隐匿性HBV感染.     

Prevalence of hepatitis B,hepatitis C,and human immunodeficiency virus infections among hemophilia patients in Shiraz,south of Iran

Transfusion-transmitted viral infections, including hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiency virus (HIV) infections continue to be a major challenge in hemophilia patient. This study was conducted to assess the seroprevalence of HBV, HCV, and HIV infections among Iranian hemophilia patients. A cross-sectional study was conducted on 580 hemophilia patients (mean age ± SD = 27.11 ± 14.9) referred to the Namazi hospital, Shiraz University of medical sciences, Shiraz, Iran during July 2012 to August 2015. Blood samples were taken from the patients and tested for HBV surface antigen (HBsAg), antibody to HBV surface antigen (anti-HBs), antibody to HBV core antigen (anti-HBc), anti-HCV antibody (HCVAb), and anti-HIV antibody (HIVAb). The association of HBV, HCV, and HIV infections with age, sex, type of hemophilia (A or B), severity of hemophilia, blood group system, history of blood transfusion, and surgery were statistically analyzed using SPSS software and chi-square/Fisher exact tests. The seroprevalence of HCVAb and HBsAg were 11.4 % (66/580) and 0.2 % (1/580), respectively. None of the hemophilia patients were positive for HIV antibody. There was a significant association between HCV seropositivity and age (P < 0.001), severity of hemophilia (P < 0.0001), and history of blood transfusion (P = 0.050). It seems that strategies for prevention of HBV and HIV infections have been successful in south of Iran, but HCV is still a major infection of concern in multi-transfused patients.     

Acute hepatitis B in anti-HCV-positive patients

Seventy-six anti-HCV-positive patients with acute hepatitis B were observed. HBsAg, anti-HBc IgM, and anti-HCV were detected in the sera of all patients. During the acute phase of illness, HBV DNA was present in the sera of 90.8% patients, but not HCV RNA. The clinical picture of acute hepatitis B in anti-HCV-positive patients corresponded to HBV monoinfection with prolonged intoxication and a more benign biochemical course. Out of 10 patients observed during 2 years chronic mixed HBV/HCV hepatitis was diagnosed in 1 patient, in 8 patients only HCV infection was diagnosed, and in 6 cases HCV RNA was detected. Virus interference may be responsible for successive alternative dominant replication of HBV and HCV.     

Antibodies to hepatitis B virus and hepatitis C virus in residential detoxification clients in Jamaica

The role of non-injecting drug abuse in viral hepatitis has not been studied widely and is not well understood. A total of 301 substance abusers, residents of a detoxification/rehabilitation unit, were investigated for exposure to hepatitis B virus (HBV) and hepatitis C virus (HCV). Samples of serum were tested for anti-HCV and anti-HBc antibodies and HBsAg. All of the patients were non-injecting drug users (non-IDUs). The prevalence of anti-HCV was 1.7%; anti-HBc was found in 28.7% and HbsAg in 0.6% of patients. Anti-HCV positivity correlated with the presence of elevated aminotransferases (80%). Exposure to HBV correlated significantly with gender (p < 0.05); age (p < 0.05); and duration of substance abuse (p < 0.05). No significant correlations were found between HCV and/or HBV infection, the drug of abuse, HIV, HTLV-1 or syphilitic infection. Residential detoxification/rehabilitation provides an opportune moment to identify and treat HCV positive substance abusers in the attempt to avert the severe hepatic sequelae. Measures which exclude substance abusers from volunteer blood donation should be considered.     

Impaired response to alpha interferon in patients with an inapparent hepatitis B and hepatitis C virus coinfection

Summary.  The possibility of hepatitis B virus (HBV) infection in HBsAg-negative patients has been shown. However, an “inapparent” coinfection by HBV in hepatitis C virus (HCV)-positive patients generally is not taken into account in clinical practice. Mechanisms responsible for resistance to interferon (IFN) have not been completely clarified. The aim of this study was to investigate whether an “inapparent” coinfection by HBV in anti-HCV-positive chronic liver disease patients may influence IFN response. Fourteen anti-HCV positive, HBsAg-negative but serum HBV DNA-positive patients by PCR and 111 anti-HCV-positive, HBsAg-negative and HBV DNA (PCR)-negative patients with chronic hepatitis were treated with 3 MU of recombinant α-2a IFN 3 times weekly for 12 months. Serum HBV DNA and HCV RNA were determined before treatment, after 6–12 months and in coincidence with ALT flare-up by PCR. HBV PCR was performed using primers specific for the S region of the HBV genome and HCV PCR with primers localised in the 5′NC region of HCV genome. IgM anti-HBc was tested using IMx Core-M Abbott assay. By the end of treatment, ALT values had become normal in 4/14 HBV DNA-positive patients (28%), but all “responders” (4/4) relapsed between 2 and 5 months after therapy. All but one patient were HCV RNA-positive before treatment, 6 were also both HBV DNA and HCV RNA-positive during ALT flare-ups. In 5 patients, only HBV DNA and in 3 patients, only HCV RNA was detected when transaminase values increased. All patients remained HBsAg-negative and anti-HCV-positive. IgM anti-HBc was detected both before treatment and during ALT elevation in 3 patients and only during ALT relapse in 3 others. Of the 111 anti-HCV positive, HBsAg-negative and HBV DNA (PCR)-negative patients with chronic hepatitis, a biochemical response to IFN treatment was observed in 54% of the cases. Relapse of ALT values was observed in 47% of the cases during a follow-up of 1 year after treatment. “Inapparent” HBV/HCV coinfection may be implicated in cases of resistance to IFN treatment. In addition, HBV replication may persist in patients in whom HCV replication was inhibited by IFN treatment. The pathogenic role of HBV in liver disease was confirmed by detection of IgM anti-HBc in some cases; the appearance of these antibodies only after IFN treatment suggests that IFN may exert a selective role in favour of HBV. Further studies will show the effect of different treatment schedules. HBV DNA and/or IgM anti-HBc detection with very sensitive methods may be important both as a prognostic factor and as a tool for better understanding interviral relationships and mechanisms involved in multiple hepatitis virus infections.     

Low rates of active hepatitis B and C infections among adults and children living with HIV and taking antiretroviral therapy: A multicenter screening study in Lesotho

Lesotho presents the second-highest adult human immunodeficiency virus (HIV) prevalence globally. Among people living with HIV, data on hepatitis B virus (HBV) or hepatitis C virus (HCV) coinfection are limited. We report HBV and HCV coinfection data from a multicentre cross-sectional study among adult and pediatric patients taking antiretroviral therapy in 10 health facilities in Lesotho. Among 1318 adults screened (68% female; median age, 44 years), 262 (20%) had immunologically controlled HBV infection, 99 (7.6%) tested anti-HBs positive and anti-HBc negative, indicating vaccination, and 57 (4.3%) had chronic HBV infection. Among the patients with chronic HBV infection, 15 tested hepatitis B envelope antigen (HBeAg) positive and eight had detectable HBV viremia (median, 2 477 400 copies/mL; interquartile range, 205-34 400 000) with a mean aspartate aminotransferase-to-platelet ratio index of 0.48 (SD, 0.40). Prevalence of HCV coinfection was 1.7% (22 of 1318), and only one patient had detectable HCV viremia. Among 162 pediatric patients screened, three (1.9%) had chronic HBV infection, whereby two also tested HBeAg-positive, and one had detectable HBV viral load (210 copies/mL). Six of 162 (3.7%) had anti-HCV antibodies, all with undetectable HCV viral loads. Overall prevalence of chronic HBV/HIV and HCV/HIV coinfection among adults and children was relatively low, comparable to earlier reports from the same region. But prevalence of immunologically controlled HBV infection among adults was high. Of those patients with chronic HBV infection, a minority had detectable HBV-DNA.     

Double hepatitis B virus infection in a patient with HIV/hepatitis C virus coinfection and ‘anti-HBc alone’ as serological pattern

Reported here is a case of double hepatitis B virus (HBV) infection in an HIV/hepatitis C virus (HCV)-coinfected patient with antibodies against hepatitis B core antigen as the only serological marker (anti-HBc alone). Two different HBV genotypes were identified in this patient. A search of the medical literature indicated this report is the first to describe a multiple silent HBV infection in an HIV/HCV-coinfected-patient. The elevated incidence of the anti-HBc alone pattern in HIV-positive patients and the increasing number of silent HBV infections detected in those patients demonstrate the need to carefully examine HIV-positive patients for occult HBV infection. In addition, it appears necessary to thoroughly study such patients in order to evaluate the impact of mixed HBV infection and triple HIV/HCV/HBV infection on morbidity.     

Relevance of inapparent coinfection by hepatitis B virus in alpha interferon-treated patients with hepatitis C virus chronic hepatitis

The aim of the study was to investigate whether an “inapparent” coinfection by hepatitis B virus (HBV) in anti-HCV-positive chronic liver disease patients may influence interferon (IFN) response. Fourteen anti-HCV-positive, hepatitis B surface antigen (HBsAg)-negative but serum HBV-DNA-positive patients and 111 anti-HCV-positive, HBsAg-negative, and HBV-DNA-negative patients with chronic hepatitis were treated with 3 MU of recombinant α-2a IFN 3/week for 1.2 months. Serum HBV-DNA and HCV-RNA were determined before treatment, after 6–12 months, and at the time of alanine aminotransferase (ALT) flare-up by HBV polymerase chain reaction (PCR) and HCV PCR, respectively. IgM anti-HBc were tested using the IMx Core-M assay (Abbott Laboratories, North Chigago, IL). By the end of treatment, ALT values had become normal in 4/14 HBV-DNA-positive patients (28%), but all “responders” (4/4) relapsed. IgM anti-HBc was detected both before treatment and during ALT elevation in three patients and only during ALT relapse in another three. In the remaining 111 patients, a biochemical response to IFN treatment was observed in 54% and relapse of ALT values in 47%. “Inapparent” HBV/HCV coinfection may be implicated in cases of resistance to IFN. HBV replication and HBV-related liver damage may persist in patients in whom HCV replication was inhibited by current doses of IFN, as suggested also by the presence of IgM anti-HBc in some cases. Further studies will show the effect of different treatment schedules. HBV-DNA and/or IgM anti-HBc detection with very sensitive methods may be important both as a prognostic factor and as a tool for better understanding of intervirus relationships and mechanisms involved in multiple hepatitis virus infections. J. Med. Virol. 51:313–318, 1997. © 1997 Wiley-Liss, Inc.     

Screening for hepatitis C virus in human immunodeficiency virus-infected individuals

Immunosuppression from human immunodeficiency virus (HIV) may impair antibody formation, and false-negative hepatitis C virus antibody (anti-HCV) tests have been reported in individuals coinfected with HIV and HCV. It is unknown if the frequency of false-negative tests is sufficiently high to change screening recommendations in this setting. Thus, the prevalence of false-negative results for anti-HCV by third-generation tests was determined with samples from HIV-infected individuals. Sera from 559 HIV-infected and 944 HIV-negative prospectively followed injection drug users were tested for anti-HCV by a third-generation enzyme immunoassay and for HCV RNA by using a branched DNA assay and the HCV COBAS AMPLICOR system. Of 559 HIV-infected participants, 547 (97.8%) were anti-HCV positive. One of the remaining 12 anti-HCV-negative participants was HCV RNA positive, and she later developed detectable anti-HCV. Of the 944 HIV-negative participants, 825 (87.4%) were anti-HCV positive. One of the remaining 119 anti-HCV-negative participants was HCV RNA positive, and she also developed detectable anti-HCV at a later visit. These data indicate that HIV infection does not alter the approach to hepatitis C virus screening, which should be performed with third-generation assays for anti-HCV unless acute infection is suspected.     

Seroprevalence of hepatitis C virus in haemophiliacs in Jamaica

Haemophilic patients (n = 90) and household contacts (n = 40) were tested for serological markers of hepatitis B virus (HBV), hepatitis C virus (HCV) and elevated serum aminotransferases using commercially prepared reagents. Of the haemophiliacs 41% (37/90) tested positive for antibodies to HCV (anti-HCV); 36% (32/90) antibodies to hepatitis B core antigen (anti-HBc); 54% (49/90) antibodies to hepatitis B surface antigen (anti-HBs) and 2% (2/90) hepatitis B surface antigen. On the other hand, 29% (26/90) of the patients and 90% (36/40) of the household contacts tested negative for all of the viral markers. Anti-HCV positivity in the haemophilic patients correlated positively with anti-HBc (p < 0.025). Increasing age (odds ratio 2.09; p < 0.01), severity of disease (odds ratio 6.2; p < 0.05) and the requirement for transfusion (odds ratio 3.2; p < 0.05) were risk factors for anti-HCV positivity. The presence of anti-HBc (odds ratio 3.8; p < 0.01) and coinfection with HCV and HBV also correlated positively with age (odds ratio 2.5; p < 0.01). The provision of anti-HCV screened donor blood and virally inactivated blood products for treatment of all haemophilic patients are goals that must be achieved.     

Occult hepatitis B virus and hepatitis C virus infections

Occult HBV infection is a well-recognised clinical entity characterised by the detection of HBV-DNA in serum and/or in liver in the absence of detectable hepatitis B surface antigen (HBsAg). Occult HBV infection has been described not only in patients who have resolved an acute or chronic HBV infection but also in patients without any serological markers of a past HBV infection. Occult HBV infection in patients with chronic HCV infection may induce more severe liver disease and lower response rate to interferon treatment. The existence of occult HCV infections has been also reported more recently. Occult HCV infection is characterised by the presence of HCV-RNA in liver and peripheral blood mononuclear cells in the absence of detectable serum HCV-RNA. Occult HCV infection may occur under two different clinical situations: in hepatitis C antibody-(anti-HCV) negative and serum HCV-RNA-negative patients with abnormal liver function tests and in anti-HCV-positive patients who have no detectable serum HCV-RNA and who have normal liver enzymes. The clinical relevance of occult HCV infections is still under investigation.     

Hepatitis B virus DNA is frequently found in liver biopsy samples from hepatitis C virus-infected chronic hepatitis patients

Human hepatitis B virus (HBV) and hepatitis C virus (HCV) are two major etiologic agents of chronic hepatitis, which is closely related to the development of hepatocellular carcinoma (HCC). A possible involvement of HBV co-infection was investigated in ongoing HCV-related liver diseases in HCV-infected patients. A prevalence of anti-HBc in anti-HCV–positive/HBsAg-negative chronic hepatitis patients and a low copy number of HBV DNA were found in most of the liver biopsy samples of anti-HCV–positive/HBsAg-negative patients. The present data suggest that HBV co-infects frequently with HCV and may play an important role in the development of HCC in the anti-HCV–positive/HBsAg-negative patients with chronic hepatitis. J. Med. Virol. 54:249–255, 1998. © 1998 Wiley-Liss, Inc.     

High prevalence of HIV, HIV/hepatitis C virus coinfection, and risk behaviors among injection drug users in Chennai, India: a cause for concern

OBJECTIVE: To estimate the prevalence of HIV and hepatitis C virus (HCV) and hepatitis B virus (HBV) coinfections and current risk behaviors among HIV-positive and -negative injection drug users (IDUs) in Chennai, India. METHODS: Cross-sectional analysis of a convenience sample of 912 IDUs recruited between March 2004 and April 2005. Specimens were tested for HIV, HBV, and HCV. Adjusted prevalence ratios (PRs) were estimated using Poisson regression with robust variance estimates. RESULTS: The prevalence of HIV, hepatitis B surface antigen, and anti-HCV were 29.8%, 11.1%, and 62.1%, respectively. Among HIV-infected IDUs, prevalence of coinfection with anti-HCV and hepatitis B surface antigen/anti-HCV were 86% and 9.2%, respectively. In multivariate analysis, injecting at a dealer's place (PR: 1.57) and duration of injection drug use >or=11 years (PR: 3.02) were positively associated with prevalent HIV infection. Contrastingly, alcohol consumption >or=1 per week (PR: 0.55) was negatively associated with HIV. HIV-positive IDUs were as or more likely compared with HIV-negative IDUs to report recent high-risk injection-related behaviors. CONCLUSIONS: There is a high burden of HIV, HCV, and HBV among IDUs that needs to be addressed by improving access to therapies for these infections; furthermore, preventive measures are urgently needed to prevent further spread of HIV, HBV, and HCV in this vulnerable population.     

Clinical significance of serum hepatitis C virus (HCV) RNA as marker of HCV infection.

We have evaluated the clinical significance of hepatitis C virus (HCV) RNA determination by analyzing a group of 221 hospitalized patients with abnormal liver function tests. Serum HCV RNA was detected by "nested" PCR amplification followed by nonisotopic hybridization. Of the 200 (90.5%) patients with anti-HCV-positive enzyme-linked immunosorbent assay results, 152 (76%) were RIBA reactive, 47 (23.5%) had indeterminate results, and 1 (0.5%) was nonreactive. Of the 180 (90%) patients positive for anti-HCV and HCV RNA, 138 (76.7%) were RIBA reactive and 42 (23.3%) were RIBA indeterminate. The pattern of RIBA reactivity did not correlate with the presence of HCV RNA. Elevated alanine aminotransferase levels were associated neither with the presence of viremia nor with the RIBA pattern. Histological findings consistent with non-A non-B hepatitis correlated with the presence of HCV RNA but not with the RIBA pattern. HCV RNA was detected in 11 of 21 (52.4%) anti-HCV-negative patients. These 11 patients were either immunosuppressed or in the prodromic phase of acute hepatitis C. Circulating HCV RNA can therefore be described as being predictive of virus-induced liver damage in anti-HCV-positive patients and may be useful in the diagnosis of HCV infection in anti-HCV-negative immunosuppressed patients or in those with early acute infection.     

Prevalence of parenterally transmitted hepatitis viruses in clinically diagnosed cases of hepatitis

Hepatitis B virus (HBV) is the most important causative agent of blood borne hepatitis in humans. Hepatitis D Virus (HDV) infection occurs either as a coinfection or superinfection in HBV carriers. Hepatitis C virus (HCV) is the major cause of transfusion non-A, non-B hepatitis and continues to be a major cause of human liver disease throughout the world. The present study was conducted on 70 clinically diagnosed cases of viral hepatitis to study the prevalence of parenterally transmitted viral hepatitis. The serum samples were tested for HBsAg, HBeAg, IgM anti-HBc, anti-HBe, anti-HCV and anti-HDV using separate ELISA kits. Of the 70 serum samples tested, 28 (40%) were positive for HBsAg out of which 3 (4.28%) were positive for HBeAg also. Five (7.1%) of the HBsAg positive cases tested positive for IgM anti-HBc also. HBsAg alone was found in 17 (24.28%) cases. The prevalence of anti-HCV was 3 (4.28%) in 70 cases. Thus early screening of clinically diagnosed cases of viral hepatitis is essential for establishing diagnosis and treatment to prevent long term sequelae.     

Antibody to hepatitis C virus in cryptogenic chronic liver disease.

The prevalence of antibody to hepatitis C virus (HCV) was studied in 207 patients with chronic liver disease of unknown etiology, in relation to clinical, epidemiological and histological features. Serum antibody to C-100 epitope of HCV was detected by ELISA in 82.6% of patients, with a significant difference compared with a group of patients with primary biliary cirrhosis (10%). The presence of anti-HCV antibody in serum did not correlate with age, sex, histological diagnosis, and activity and duration of the disease, nor with serum anti-HBc, used as a marker of exposure to hepatitis B virus infection. These results strongly support the view that most cases that were previously defined as cryptogenic forms of chronic liver disease are in fact related to HCV infection. There was a correlation between serum anti-HCV antibody and history of risk for parenteral exposure or of acute hepatitis. This correlation was particularly evident for transmission by parenteral route, suggesting that HCV infection may be transmitted often by this route (36.8% among anti-HCV antibody-positive patients and 11.1% among anti-HCV-negative patients). Liver disease in patients without risk factors for parenteral transmission and with lower prevalence of anti-HCV antibody may be caused by other as yet unidentified non-A, non-B (non-C) agents or may be of nonviral origin.     

High rate of hepatitis C virus infection in an isolated community: Persistent hyperendemicity or period-related phenomena?

We investigated underlying risks for hyperendemic hepatitis C virus (HCV) infection among the 1853 inhabitants of a mountainous village in Eastern Taiwan with high prevalence of HCV and hepatitis B virus (HBV). Among the 80 selected adults, we found that having resided away from the village before 1985 was protective against HCV infection, while residing in the village after 1985 posed little risk for HCV infection to children and young adults < 30 years of age. Among the 559 school children 7 through 14 years of age, anti-HCV prevalence was 1.9%, and the HBV carrier rate was 29%. Following up 270 children 1 year later, we found that new HCV infection occurred in 0.74% and new or repeated HBV infection occurred in 6.5% of the children, indicating distinct transmission patterns between HBV and HCV. Children of anti-HCV-positive mothers were either anti-HCV-negative or were infected by distinct genotypes of HCV from those infecting their mothers; most married couples in whom both were infected, were infected by HCV of discordant genotypes, indicating negligible importance of sexual or vertical HCV transmission. A case-control study comparing 13 anti-HCV-positive and 53 anti-HCV-negative children showed that having received parenteral medication in local clinics was a significant risk for HCV infection. Our data indicate that, unlike the case of HBV, HCV transmission by vertical or sexual route, or through casual contact are extremely inefficient, and our data further suggest that HCV hyperendemicity is unlikely to persist as a result of the more stringent practice of parenteral precautions in nearly all aspects of daily life. J. Med. Virol. 52:370–376, 1997. © 1997 Wiley-Liss, Inc.