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Parenteral nutrition (PN) products are regulated in the United States by the Food and Drug Administration (FDA). The FDA regulates PN products as "drugs," and the approval of a new product depends on there being "substantial evidence" of safety and efficacy obtained from "adequate and well-controlled studies." The processes by which the FDA approves PN products in the United States are designed to protect patients from unsafe or ineffective products, but they may also contribute to delaying and preventing access to certain valuable new products. This tutorial is designed to inform the PN community about FDA processes with regard to PN products, including the structure of FDA review teams, requirements for investigational studies, applications for marketing approval, postapproval clinical trial requirements, and requirements for studies in pediatric populations. Knowledge of how the FDA process works and how the FDA regulates PN products can help the PN community work together in advancing the development of new products.

Many drugs approved by the US Food and Drug Administration (FDA) for use in adults lack adequate data on safety and efficacy in pediatric patients, a potential source of unintended harm to pediatric patients. Through a series of laws, regulations, and guidance documents, the US Congress and FDA have created a program both to encourage and mandate clinical studies in pediatric patients to develop evidence-based dosing, safety, and efficacy information. A "Pediatric Study Plan" (PSP) is required for every new drug. FDA provides incentives for the voluntary conduct of clinical trials in pediatric patients, including opportunities for added marketing exclusivity and for obtaining a "priority review voucher." FDA also mandates that clinical studies for new drugs be conducted in each pediatric age group (newborns, infants, children, and adolescents), except in circumstances where a waiver or a deferral of studies can be justified. Sometimes this mandate can be met by extrapolation from studies in adults, or from patients in one pediatric age group to another, for evidence of efficacy. However, separate studies of safety and dosing are usually required for each pediatric age group. The package insert for each new drug now must address the use in pediatric patients. In addition, the FDA website displays all changes in drug labeling related to pediatric patients (excerpted from the labels for easy access), summaries of all pediatric studies that have led to labeling changes, links to FDA medical reviews of pediatric studies, summaries of all pediatric safety issues presented to the FDA Pediatric Advisory Committee (with links to the meeting materials and transcripts), and details of deferred pediatric studies with their timelines and progress. These measures reflect the increasing attention by FDA and the medical community to the importance of clinical studies in pediatric patients.

The Food and Drug Administration Amendments Act of 2007 has improved the transparency of the clinical drug development landscape in the United States in many ways. Some of the important changes require: (a) increased public disclosure of information on clinical trials both while being conducted and during the subsequent public disclosure of the results of the trial; (b) changes to the required labeling including the development of a Risk Evaluation and Mitigation Strategy (REMS) to ensure that the benefits of the product outweigh its risks in the target population; (c) a requirement to do additional drug safety studies when indicated based on the available safety data; and (d) a requirement for the performance of pediatric studies when the drug may have a potential to provide better treatment options for pediatric subjects and subsequent revision of the labeling based on pediatric studies. The act has also increased FDA's authority to mandate necessary changes.

SEN virus (SEN-V) is a blood-borne, single-stranded, nonenveloped DNA virus. Although its prevalence varies by geographic region, it has been detected in as many as 30 percent of postoperative transfusion recipients, compared to 3 percent of postoperative patients who did not receive transfusions. A significant association has been observed between transfusion volume and the occurrence of SEN-V infection. Transmission by transfusion also has been confirmed by the detection of greater than 99 percent homology between SEN-V in donor and recipient sera. Concurrent infections with SEN-V and hepatitis B virus, hepatitis C virus, or human immunodeficiency virus type 1 have been documented, and these observations probably reflect the blood-borne transmission of these viruses as well as SEN-V. Although SEN-V was discovered as part of a search for causes of posttransfusion hepatitis, there is no firm evidence so far that SEN-V infection either causes hepatitis or worsens the course of coexistent liver disease. Nevertheless, SEN-V appears to be transmitted by transfusion, and further studies may reveal more about its role in the future.

A few hepatitis B virus (HBV) infections are characterized by the presence of HBV DNA in serum or liver tissue, or both, in the absence of detectable hepatitis B surface antigen (HBsAg) in serum. However, such infections have rarely been described previously in North American patients. In the present study, 31 hepatocellular carcinoma (HCC) patients from the United States and Canada who had no detectable HBsAg in their serum were studied. In these 31 HBsAg-negative HCC patients, HBV DNA was detected in HCC and/or in adjacent nontumorous liver tissue using nested polymerase chain reaction (PCR) in 5/9 (56%) patients from the United States and in 12/22 (55%) from Canada. The 17 HBV DNA-positive/HBsAg-negative patients from the United States and Canada included 9 without any serological markers for HBV and 8 with detectable antibodies to hepatitis B core antigen. In these patients, HBV genotype C was the most prevalent genotype (11/17; 64%). HBV genotypes have not been previously reported in HCC patients from North America. Replicative intermediate forms of HBV (covalently closed circular HBV DNA) were detected in 2/17 (12%) HBV DNA-positive/HBsAg-negative patients, indicating that at least two of these patients had actively replicating HBV infections. The use of tests to detect HBV DNA permitted the identification of HBV infections in HBsAg-negative HCC patients from North America. Among these patients, those with antibody to hepatitis C virus (HCV) would otherwise have been designated “HCV-associated HCCs” based on serological tests alone. These findings provide a new perspective on determining the possible viral etiologies of HCCs in North America. J. Med. Virol. 70: 20–26, 2003. © 2003 Wiley-Liss, Inc.

Serial serum samples were obtained over a 27-year period from a hepatitis C virus (HCV)-infected patient and from a nurse who appeared to become infected by this patient. The hypervariable region 1 (HVR1) and 5'noncoding region (5'NCR) of the HCV genome were amplified from each serum sample by polymerase chain reaction (PCR) and cloned. In the first serum specimen from the patient and the first two serum specimens from the nurse, most of the 20 clones from each serum sample had one common sequence in the HVR1 gene. All later serum samples contained a heterogeneous mixture of HCV quasispecies. The uniformity of the HVR1 sequence in the early samples and the emergence of greater diversity in later serum samples is consistent with the apparent transmission of HCV between the patient and nurse and the eventual emergence of other quasispecies as the virus replicated in the new host. In addition, the immune globulin given to the nurse may have been responsible for some of the HCV quasispecies changes observed in her serum.

Latent-class analysis was used to evaluate the usefulness of markers of hepatitis C virus (HCV) infection in characterizing the true, underlying infection in a community-based Japanese population. Antibodies to HCV were detected in 24%, HCV RNA in 22%, and HCV core protein in 19% of stored serum samples from 372 adults. A 2-class model suggested that positive results for any 2 virus markers defined the current HCV infection class, with an estimated prevalence of 22% (95% confidence interval, 18%‐26%). The sensitivity for detection of current HCV infection was highest for anti-HCV (97%) and was more moderate for HCV RNA (91%) and HCV core protein (85%). The specificity for each marker was 96%. In general, the association between demographic factors and current HCV infection status was strengthened by use of latent-class analysis that combined data for markers of HCV infection, when compared with results of logistic regression analysis for each marker separately. Hepatitis C virus (HCV) is an etiologic factor for both chronic hepatitis and hepatocellular carcinoma [1]. HCV infection is distributed worldwide, and population seroprevalences, as measured by the detection of antibodies to HCV, are within the range 0.5%–2.0% [1]. Since anti-HCV does not discriminate between current infection and resolved infection, methods to detect the virus can assist the classification of HCV infection status into current, resolved, or never infected. Detection of either viral RNA or core protein is indicative of current infection. Elevation of alanine aminotransferase (ALT) is a marker for hepatocyte damage or death that may be attributed to HCV infection. Many serologic surveys either measure anti-HCV alone or include methods to detect viral RNA or core protein only in anti-HCV–positive serum samples. The rationale for this approach may be due to the special handling required for the blood specimens, the high cost of the assays, or the difficulty in performing the assays. Moreover, measure

During the past 50 years, most US-licensed plasma derivatives have maintained an impressive record of not transmitting HBV, HCV, or HIV Albumin (50-year history) has never transmitted these viruses. PPF (40-year history) transmitted HBV on only one occasion, which was associated with a design flaw in one manufacturing plant. IGIM has never transmitted any of these viruses since the requirement of sensitive serologic screening tests for HBV (24 years). IGIV (17-year history) transmitted HCV in only one outbreak involving the product of one manufacturer. Even AHF and FIX have not transmitted these viruses since effective virus-inactivation processes in manufacturing were developed. In summary, there has been no transmission of HBV, HCV, or HIV by US-licensed plasma derivatives since the introduction of effective virus-inactivation procedures. This means, essentially, that there has been no transmission of these viruses since the end of 1987; the sole exception is IGIV, by which there has been no transmission since 1994.

In the present study, genetic alterations of the precore and proximal core regions (codons 1–50) were determined in HBV isolated from the serum of 58 patients with HCC and hepatitis B surface antigen (HBsAg) in their serum to identify any role of such genetic changes in HBV genome for hepatocarcinogenesis. DNA extracted from the serum of 16 patients with chronic hepatitis B but no HCC were used as controls. In HCC patients, mutations of T 1846 , C 1858 , A 1896 , and A 1899 were identified in 48, 5, 86 and 36%, respectively. A 1896 mutation is associated with T 1846 and A 1899 mutations more frequently in HCC patients than in chronic hepatitis B patients. Fourteen mutations of the proximal core region in HBV genomes from HCC patients were observed in codons 5, 13, 21, 22, 26, 27, 31, 35 and 41. The median number of mutations in the proximal core gene was 7 from HBeAg-negative HCCs, 5 in HBeAg-positive HCCs, and 4 in patients with chronic hepatitis B without HCC. These results suggest that mutations of the precore and proximal core gene sequences may preferentially occur in specific nucleotides and the continuous replication of such mutant HBV might play a role in HBV-related hepatocarcinogenesis.

BACKGROUND Hepatocellular carcinoma (HCC) is generally considered a hypervascular tumor when visualized by angiography. However, small HCCs are not always found to be hypervascular. METHODS To evaluate this, 50 HCCs < or =3 cm in diameter were studied. The 50 tumors consisted of 16 well-differentiated HCCs, 25 moderately differentiated HCCs, and 9 that were each a mixture of well- and moderately differentiated HCC. RESULTS The mean number of portal tracts in the well-differentiated HCCs was 34% of the number in the surrounding nontumorous liver, and few intratumoral arterioles were seen. In contrast, the mean number of portal tracts in the moderately differentiated HCCs was 0.6% of the number in the surrounding nontumorous liver, and abundant intratumoral arterioles were seen. For HCCs that contained both well-differentiated and moderately differentiated tumor, the distribution of portal tracts and intratumoral arterioles in each portion was similar to that seen in well-differentiated or moderately differentiated HCC alone, respectively. HCCs that were larger than 1.5 cm in diameter had fewer portal tracts and more intratumoral arterioles than HCCs whose diameters were < or =1.5 cm. CONCLUSIONS As small HCCs increase in size and become increasingly dedifferentiated, the number of portal tracts apparently decreases and intratumoral arterioles develop. These findings may reflect changes in the hemodynamics as the HCC develops.

Two patients with hepatocellular carcinoma (HCC) were identified who had substantial deletions within the hepatitis B virus (HBV) X gene from HCC tissues. In one patient, the deletion was found at nt. 382-389 (codons 128-130) of the X gene, followed by two nucleotide substitutions, a frame shift, and formation of a new stop codon. In the second patient, the deletion was found at nt. 389-396 (codons 130-132) of the X gene, followed by one nucleotide substitution, a frame shift, and formation of a new stop codon. The resulting X proteins in both cases would be truncated at the 3′ end and would be 20 amino acids shorter than the full length X protein. These patients had been identified during a study of 25 HCC patients from Qidong, China in whom a 228-base region of the X gene was sequenced. No deletions were found within this X gene sequence in HCC tissues from the other 23 patients or in the 20 adjacent noncancerous liver samples available from these patients. However, the fact that these deletions encompassed codons 130 and 131, two adjacent codons where point mutations were found in 21 of the remaining 23 patients, suggests that this region may play an important role in hepatocarcinogenesis.

In order to study the mechanisms by which p53 function is regulated, human wild-type p53 cDNA was cloned into a vaccinia virus vector and the expressed p53 protein was used to investigate binding of the p53 by cellular proteins from a cDNA expression library from human liver. One protein that bound wild-type p53 had >99% homology with DNA topoisomerase IIb. p53 protein was coimmunoprecipitated from topoisomerase II-rich cell lysates (but not from topoisomerase II-deficient cell lysates) by an antibody to topoisomerase IIa and IIb. This binding was shown to occur without a dsDNA intermediary. Hepatocellular carcinomas (HCCs) and adjacent nontumorous liver tissues from ten patients were studied to determine the level of expression of topoisomerase II and p53. Overexpressed topoisomerase II proteins were detected by western blot in six of ten HCCs (60%), including several in which presumed wild-type p53 was detected by immunohistochemistry. No topoisomerase II expression was detectable in the ten nontumorous liver tissues from the same patients or in a sample of normal human liver.

OBJECTIVES Hepatitis B virus (HBV) with a stop mutation at precore codon 28 (TGG-->TAG, tryptophan-->stop) was investigated to clarify if such a mutant virus might play a role in hepatocarcinogenesis. METHODS A total of 73 patients with HBV-related hepatocellular carcinoma were included in this study. Polymerase chain reaction (PCR) was performed in DNA samples extracted from 73 sera to amplify a HBV-DNA segment involving the precore and proximal core regions, and sequences of PCR products were analyzed to see the presence of the mutations at precore codon 28 by a direct sequencing method. RESULTS HBV-DNA was detectable in 64 (88%) patients by PCR. The stop mutation at precore codon 28 was identified in 50 of 58 PCR products (86%), in which direct sequencing was performed. Among patients with this mutant HBV, 21/50 (42%) patients were co-infected with wild-type HBV. The mutant virus was found in 23/28 (82%) patients with hepatitis B e antigen (HBeAg) and 27/30 (90%) patients without HBeAg. The mutant HBV alone was found in 10/28 (36%) patients with HBeAg and 19/30 (63%) without HBeAg. Among those patients on whom laparoscopy was performed, 22/24 (92%) with the precore codon 28 stop mutant alone had cirrhosis, compared to 12/19 (63%) co-infected by both the mutant and the wild-type (p < 0.05). The association of this mutant virus with both the presence and absence of HBeAg, and its association with cirrhosis when there is no co-infection with wild-type HBV, suggests an evolving pattern of liver pathology. CONCLUSION The high prevalence of a stop mutation at precore codon 28 in these patients with hepatocellular carcinoma suggests that HBV with this mutation may contribute to the development of hepatocellular carcinoma.

Mutation of the p53 gene in hepatocellular carcinoma has been recognized as one of the most important genetic alterations to occur during hepatocarcinogenesis. This study was performed to analyze the frequency and nature of p53 mutations in advanced hepatocellular carcinomas from Korea. Tissue samples were obtained by laparoscopic biopsy from 35 patients; adjacent nontumorous liver tissue was also obtained from 24 of them. Mutations of the p53 gene were identified in 11/35 (31%) of hepatocellular carcinomas. These included 7 missense mutations and 4 deletion mutations. Only one mutation was detected at codon 249, a “hot spot” at which mutations have been found frequently in hepatocellular carcinomas from some geographic areas; however, this was an A-to-T transversion at the first nucleotide, thus differing from commonly reported G-to-T transversion at the third nucleotide of codon 249 in hepatocellular carcinomas. Patients whose serum alkaline phosphatase levels were higher than the mean value were more likely to have p53 mutations, compared to patients whose alkaline phosphatase levels were lower than the mean value [55% (6/11) v s. 21% (5/24)] (p

Expression of transforming growth factor-alpha (TGF-alpha) and its receptor, the epidermal growth factor receptor (EGFR), in hepatocellular carcinomas (HCCs) and adjacent nontumorous livers from 25 Japanese patients were examined using immunoperoxidase staining of paraffin-embedded sections. TGF-alpha was detected in 24 of 25 (96%) HCCs and 23 of 24 (96%) available adjacent nontumorous livers. EGFR was detected in 16 of 25 (64%) HCCs and 17 of 24 (71%) adjacent nontumorous livers. TGF-alpha and EGFR were not detected by immunohistochemical staining in normal livers. Fifteen of 25 HCCs contained an apparent area of a second tumor (two of the 15 also contained a third tumor) that had a less-differentiated histological grade developing within or adjacent to the first tumor. In those cases, staining in the less-differentiated area of tumor was usually less intense than in the more highly differentiated area (80% of cases for TGF-alpha; 91% for EGFR). These data confirm that increased expression of TGF-alpha and EGFR occur frequently in human HCC. Furthermore, the detection of greater staining in more highly differentiated portions of the tumors suggests that increased expression of TGF-alpha and EGFR may be events of the early stages of human hepatocarcinogenesis.

The effect of interferon alfa (IFN-α) therapy on the expression of transforming growth factor alpha (TGF-α) in the liver during chronic hepatitis B was investigated. Serial liver biopsy specimens were evaluated from 35 patients who had participated in a randomized, controlled trial of recombinant human IFN-α for the treatment of chronic hepatitis B. Percutaneous liver biopsy specimens obtained before and 1 year after entry in the trial were sectioned and stained with a monoclonal antibody to TGF-α in an avidin-biotin-peroxidase-complex system. The expression of TGF-α in each section was evaluated blindly (with respect to treatment group and order of biopsies) and was numerically scored. There was no significant difference in TGF-α expression before or after therapy between 13 patients receiving daily IFN-α, 13 receiving alternate-day IFN-α, and 9 receiving no therapy. Sustained clearance of HBV-DNA and DNA polymerase activity occurred in 8 of 26 treated patients (“responders”); the 18 other patients were “nonresponders”. Expression of TGF-α before IFN-α therapy was significantly higher in responders than in nonresponders; after IFN-α therapy, TGF-α expression decreased significantly among responders compared with nonresponders and untreated controls. Thus, the level of expression of TGF-α in the liver, which was correlated with the severity of inflammation in the liver in this study, appeared to be predictive of the response to IFN-α therapy in chronic hepatitis B, with a higher level of expression indicating a greater likelihood that the patient would respond.

A recombinant vaccinia virus was constructed using the wild-type human p53 gene as an insert. The p 53 protein produced by this recombinant virus was used to investigate p53 binding proteins in seventeen cell lines, including 10 derived from human hepatocellular carcinoma, four from other human cancers, and three from non-human primate tissues. In all 17 cell lines tested, two proteins of 40 kD and 50 kD were identified that bound to wild-type p53 and that may be cellular regulators of p53 function.

Recent studies have identified epithelial cell populations in human livers that are similar to the "oval cells" and "transitional cells" seen in rat livers during the early stages of chemical carcinogenesis. It has been suggested that these cells might be precursors of hepatocytes and theoretically could be involved in hepatocarcinogenesis. The hepatitis B virus (HBV) also is believed to play a role in the etiology of hepatocellular carcinoma (HCC). Therefore, a study was conducted in nontumorous livers adjacent to HCCs obtained from 26 patients from China to determine whether HBV antigens could be identified in oval cells and transitional cells using an immunohistochemical technique. Hepatitis B surface antigen (HBsAg) was detected in the nontumorous livers of 22/26 (85%) patients. HBsAg was detected in oval cells in 18/26 (69%), in transitional cells in 21/26 (81%), and in mature hepatocytes in 22/26 (85%), but not in bile duct or ductule cells. Transforming growth factor-alpha (TGF-alpha) was expressed in oval cells, transitional cells, and bile duct cells in 24/26 (92%) patients, an in mature hepatocytes in 25/26 (96%). Coexpression of HBsAg and TGF-alpha was identified in the same cells in populations of oval cells and transitional cells of selected patients. Because of the possibility that oval cells could be a source of evolving HCC, these findings suggest that expression of TGF-alpha associated with HBV infection of oval cells could be a mechanism of human hepatocarcinogenesis. Thus, oval cells could be a site (or one of the sites) where HBV participates in the development of HCC.

One hundred and one of 255 recipients of a plasma-derived hepatitis B vaccine were evaluated in 1990, 9 years after the first vaccine dose in a study in Zambia to evaluate the efficacy of one, two, or three doses. In 1983, 2 years after the first vaccine dose, antibody to the hepatitis B surface antigen (anti-HBs) had been detectable in 90 of these 101 participants (89%). In 1990, anti-HBs was still detectable in 72 of 101 (71%), and was present at a protective level ( ≥ 10 mlU ml) in 68 of 101 (67%). Although the original vaccine study elicited a protective level of antibody in a greater percentage of children and adolescents than in adults, there were no significant differences among the three groups at 9 years. (In 1990, anti-HBs was still detectable in 52 of 70 [74%] who had had no serologic markers of the hepatitis B virus in 1981, and a protective level was detected in 47 of 70 [67%].) A protective level of anti-HBs was detected in 1990 in 26 of 36 (72%) recipients of three doses and in 23 of 31 (74%) recipients of two doses; the slightly lower prevalence among recipients of one dose (19 of 34 [56%]) was not statistically significant. However, between the years 1983–1990, hepatitis B virus infections had occurred in one of 36 (3%) of those who had been vaccinated with three doses, one of 31 (3%) vaccinated with two doses, and eight of 34 (24%) of those vaccinated with one dose (P < .02 for either two or three doses compared with one dose). These data support the long-term immunogenicity and protective efficacy of a two- or three-dose regimen of the hepatitis B vaccine in a rural African setting. © 1993 Wiley-Liss, Inc.

Background Mutations of the p53 tumor suppressor gene have been reported in 50% of patients with hepatocellular carcinoma (HCC) from China and South Africa. These reports suggested an association of p53 mutations with high levels of aflatoxin in the diet. Most studies of p53 and HCC, however, have not fully evaluated the possible role of the hepatitis B virus (HBV). Aflatoxin is a substance produced by food mold that is known to cause HCC in experimental animals. Purpose The purpose of this study was to evaluate the relationship of p53 gene mutation to high or low levels of aflatoxin in the diet and to HBV infection. Methods p53 protein and hepatitis B surface antigen (HBsAg) were evaluated by immunohistochemistry using the avidin-biotin-peroxidase system in paraffin-embedded specimens of HCC and of adjacent nontumorous liver tissue from 43 patients. Tissue specimens from three normal human livers were also evaluated. HCCs and adjacent nontumorous liver tissues were obtained from 23 patients from Qidong, China, where aflatoxin levels in the diet are high, and from 20 patients from two regions in the United States (patients from the National Institutes of Health, Bethesda, Md., and Kuakini Medical Center, Honolulu, Hawaii), where aflatoxin levels in the diet are low. Results Mutant p53 protein was detected in the nuclei of HCCs from 14 (61%) of 23 patients from China and from three (30%) of 10 patients and six (60%) of 10 patients, respectively, from the two regions of the United States. A statistically significant association between detection of mutant p53 protein in HCC cells and the detection of HBsAg in hepatocytes of the adjacent nontumorous liver tissue was observed in patients from China and the United States considered together. Conclusion Mutations of the tumor suppressor gene p53 in hepatocellular carcinomas are not limited to patients from geographic regions where the ingestion of aflatoxin is high. In many patients, these mutations may be associated with HBV infection. Implications The possible interaction of chronic HBV infection and p53 gene mutation, suggested by these data, indicates a mechanism by which HBV infection beginning early in life could contribute to the subsequent development of HCC.

BACKGROUND Hepatitis B virus (HBV) infection is closely associated with the development of hepatocellular carcinoma (HCC) in many patients, but the mechanisms by which HBV contributes to HCC are not known. Transforming growth factor-alpha (TGF-alpha), a regulator of growth and regeneration in rat liver that can be found in high levels in some human cancers, theoretically could play such an intermediate role in the development of HCC. METHODS The expression of TGF-alpha and its relation to the HBV antigens were evaluated in human HCC and adjacent nontumorous livers from 33 patients from the United States and China using immunoperoxidase staining of paraffin-embedded sections. RESULTS TGF-alpha was detected in HCC from 27 of 33 (82%) patients; the frequencies were similar in patients from the United States and China. TGF-alpha was detected in HCC more frequently in patients whose adjacent nontumorous livers had detectable hepatitis B surface antigen (HBsAg) and/or hepatitis B core antigen (HBcAg) than in those whose adjacent livers lacked HBsAg and HBcAg. Detection of TGF-alpha was not affected by tumor size, histologic type, or grade. TGF-alpha was detected in adjacent nontumorous livers from 31 of 33 patients (94%). Coexpression at a high intensity of TGF-alpha and HBsAg in the same hepatocytes could be demonstrated by specific staining of consecutively cut sections for 17 of 33 patients (52%). CONCLUSIONS TGF-alpha is expressed at a high level in 82% of human HCC. Localization of HBsAg within the same hepatocytes as TGF-alpha suggests a possible interaction between HBV and TGF-alpha during hepatocarcinogenesis in humans. Stimulation of TGF-alpha expression could be part of a chain of events by which HBV contributes to the development of HCC in some patients.

A field trial of a plasma-derived hepatitis B vaccine in five rural villages in Zambia was analyzed to determine if adults in a rural African setting respond to this vaccine as well as adults in Western countries and to determine the immunogenicity of fewer than the recommended three doses; 255 residents, including 171 who were susceptible to hepatitis B, were vaccinated. Among those who received three vaccine doses, protective levels of antibody to hepatitis B surface antigen (anti-HBs) developed in 67% of adults (ages 21 to 70 years), 87% of adolescents (ages 12 to 19 years), and 100% of children (ages 0 to 11 years). The 67% of vaccinated adults who developed anti-HBs at the protective level was lower than the 96% reported among adults receiving the same vaccine at the same dose and dosage schedule in studies in Western countries. No difference was seen in the response of those receiving two doses compared with those receiving three doses among adults and adolescents, suggesting that a two-dose regimen may be acceptable in these age groups in developing countries to reduce costs and improve compliance. Use of hepatitis B vaccine in a region where prevaccination hepatitis B serologic screening was not available did not appear to increase the number of severity of adverse reactions.

Hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) remains the most common form of HCC in large areas of Asia and Africa. It remains common even in some countries where hepatitis C virus (HCV)-associated HCC has become the predominant form, such as Japan. Integration of HBV in HCC DNA is found at random sites in the host genome in nearly all patients with HBV-associated HCC. It is not clear how often this integration results in insertional mutagenesis, but previously unknown growth regulating genes and cancer-associated genes have been found frequently near HBV integration sites in HCC in recent studies. In addition, HBV encodes a transactivating protein, the X protein, which could enable the randomly integrated HBV to alter the function of host genes that are not near the integration site. Mutations at two adjacent codons in HBV (1762(T)/1764(A) mutations) within the X gene are frequently found in HCC patients, and may play a role in the mutagenic or transactivational role of HBV in HCC. The presence of cirrhosis in most patients with HBV-associated HCC, and the presence of mutations in tumor suppressor genes in many, suggests that these are also factors in hepatocarcinogenesis. Few studies have examined the mutations of more than one gene in the same HCC patients. Fewstudies have evaluated the interactions between HBV mutations, host gene mutations, cirrhosis, and other potentially mutagenic stresses at the cellular level, with progression to HCC, and few studies have been conducted to determine whether these changes must accumulate in succession to lead to HCC. The recent availability of rapid sequencing methods and DNA microarray technologies has permitted expression profiling and permutation analysis of an array of genes to explore the pattern and succession of molecular changes leading to HBV-associated HCC. To date, these methods have been used to show patterns of molecular changes that differ in HBV-associated HCC (compared to HCV-associated HCC or to HCC in patients lacking either virus) and patterns that can predict survival (and hence may directly indicate different mechanisms of disease), and may soon make possible a universally accepted clinical classification scheme for HCC.

Hepatitis B virus (HBV) mutants have usually been studied in patients in Asia because of the wider use of HBV immunization there and the resultant emergence of viral mutants. Nevertheless, HBV surface antigen (S) gene mutants also are found in Europe and North America. In Europe and North America, HBV with mutations in the portion of the S gene coding the "a" determinant of the hepatitis B surface antigen (HBsAg) have been documented in small numbers of infants born to HBV-infected mothers following post-natal HBV vaccine and hepatitis B immune globulin (HBIG) prophylaxis and in many liver transplant recipients who develop HBV re-infection despite HBIG prophylaxis. In some cases, these mutations have included a glycine to arginine substitution at position 145 (G145R), which results in a conformational change and different reactivity to monoclonal antibody reagents than that of the wild-type virus. Mutations in the a determinant (but not G145R) also have been reported in European patients with chronic HBV infection who have not received HBV vaccine or HBIG. However, it appears that such mutations are only responsible for a small proportion of "occult" or "silent" HBV infections, which are characterized by the presence of HBV DNA in serum in the absence of detectable HBsAg. However, some of these mutant forms of HBV in cases of occult HBV may theoretically escape detection and could present a risk to blood safety.

Publisher Summary This chapter discusses viruses that are emerging or that threaten to emerge among human populations in the 21st century. Some viruses that emerged in the late 20th century are discussed in an historical context; many of the experiences faced in the 20th century with viruses such as human immunodeficiency virus (HIV) and human T-lymphotropic virus provide models for developing programs to monitor and counteract emerging viruses in the 21st century. Diseases caused by emerging viruses are a permanent part of the human condition. There is a current but controversial theory that all DNA, including that in the cells of humans, originated when RNA viruses underwent adaptive changes to avoid host defenses. The genes of viruses are prone to changes that allow the viruses to adapt easily to new hosts. The relatively simple structure of viruses allows mutations to occur easily; the explosive replication of viruses magnifies the mutations. RNA viruses are particularly prone to these modifications because they lack molecular “proofreading” mechanisms to correct mutations and errors in replication.

Summary. This study was conducted to evaluate reports that hepatitis B virus (HBV) DNA sequences can be found in the serum and/or tumour tissue from some hepatocellular carcinoma (HCC) patients who have no detectable hepatitis B surface antigen (HBsAg) in their sera. Such HBV infections would be highly atypical, because prospective studies have shown a clear succession of specific serologic markers during and after most HBV infections. As most HBsAg-negative HCC patients in Japan have hepatitis C virus (HCV) infections, the present study was conducted to determine whether some of these patients actually have unrecognized HBV infections. Thirty newly diagnosed HCC patients from Kurume, Japan, with antibody to the hepatitis C virus (anti-HCV) were studied. None of the 30 had HBsAg detectable in their serum. Of 22 for whom test results for antibodies to the hepatitis B core antigen (anti-HBc) and antibodies to HBsAg (anti-HBs) were available, 14 (64%) had anti-HBc and anti-HBs, four (18%) had anti-HBc alone, and four (18%) had no HBV markers. Nested polymerase chain reaction was used to detect the HBV surface (S), core (C), polymerase (P) and core promoter gene sequences in the HCC tissues and in the adjacent nontumorous liver tissues. HBV DNA was detected in HCC and/or adjacent nontumorous liver in 22 of 30 (73%) patients [detected in both HCC and nontumorous liver in 19/30 patients (63%)]. Among the 22 patients with detectable HBV DNA, more than one HBV gene was detected in 10 (46%). Among the four patients whose sera were negative for all HBV markers, three had HBV DNA in either HCC and nontumorous liver (two cases) or only in the nontumorous liver (one case); HBV DNA could not be detected in tissues from the fourth patient. In 18 of 21 (86%) patients with detectable HBV core promoter sequences, mutations at both nucleotides 1762 (A–GT) and 1764 (G–A) in the core promoter region were found. No deletions were detected in the core promoter gene region of the type reported to be associated with some cases of HBsAg-negative HBV infection. Thus, HBV DNA was detectable in 22 (73%) HBsAg-negative, anti-HCV-positive HCCs, including three (10%) who were also negative for anti-HBc and anti-HBs. HBV mutations at both nucleotides 1762 (A–GT) and 1764 (G–A) in the core promoter region were found in the majority of cases, mutations that have previously been reported in HBV that is integrated in HCC DNA. In serologic surveys to determine etiologic associations of HCC, patients such as those in this study would have been incorrectly designated as having ‘HCV-associated HCC,’ whereas the data in this study suggest that HBV could have played a role in the development of their HCCs.

Studies have suggested that a truncated form of the hepatitis C virus (HCV) core protein can enter hepatocyte nuclei and might play a role in HCV-associated hepato-carcinogenesis. In the present study, the HCV core gene from hepatocellular carcinomas (HCC) and/or adjacent non-tumorous liver tissues from eight patients was amplified by nested RT-PCR and sequenced. Mutations in the HCV core gene that would encode a truncated core protein were found in 4 of the 8 patients. Since truncated core proteins have been shown to be capable of entry into the hepatocyte nucleus (unlike HCV itself, which is an exclusively cytoplasmic virus), the detection of mutated sequences encoding them in these four HCC patients suggests that these mutations may have played a role in the development of these HCCs.

Integration of hepatitis B virus is thought to be an essential step in hepatitis B virus associated hepatocarcinogenesis. Mutations at nucleotides 1762 and 1764 in the hepatitis B virus, within a sequence encoding both the core promoter gene and the X gene, have been found frequently in patients with hepatocellular carcinoma. However, integration of these mutant sequences has not been reported to date. Methods. A 228-base pair segment of the hepatitis B virus core promoter gene was amplified from hepatocellular carcinomas and adjacent non-tumourous liver tissue by nested PCR and sequenced. Integration of hepatitis B virus into human genomic DNA was investigated using the ‘genome walking’ method. Results. Point mutations were found in both hepatitis B virus nucleotides 1762 and 1764 in 8 of 14 hepatocellular carcinoma tissues (57%) and in 11 of 14 adjacent non-tumourous liver tissues (79%). Three patients were evaluated using the ‘genome walking’ method; all were found to have hepatitis B virus DNA integrated in their hepatocellular carcinoma (two patients) and/or in their non-tumourous liver tissue (three patients). Integration occurred in all tissues near host genomic sites that are prone to integration. Hepatitis B virus was integrated at or near the hepatitis B virus DR1 site in all samples, and all contained truncated X gene sequences that have been reported to be capable of producing fusion transcripts with transactivation potential. Conclusions. Integrated hepatitis B virus DNA containing core promoter mutations at nucleotides 1762 and 1764 was found in hepatocellular carcinoma and/or adjacent non-tumourous liver tissue of three patients. These findings leave open the possibility that insertional mutagenesis or transactivation by fusion transcripts resulting from hepatitis B virus integration could play a role in hepatocarcinogenesis in some patients.

BACKGROUND: A study was designed to estimate relative analytic sensitivity and window-period (WP) closure and to project incremental yield of newer HBsAg tests, pooled-sample NAT, and single-sample NAT, compared to currently licensed HBsAg tests. STUDY DESIGN AND METHODS: HBV DNA and HBsAg test results for 23 HBV seroconversion (SC) panels were first analyzed to construct a model of primary HBV viremia. One-hundred representative samples were then selected from 10 panels and coded with 28 analytical controls. All 128 samples were tested by seven HBsAg tests and by four pooled-sample and three single-sample NAT assay formats. Results were analyzed to obtain differential times to HBV detection and combined with HBV incidence rates to project comparative yields. RESULTS: HBV doubling time during the ramp-up phase was estimated at 2.56 days. HBsAg concentrations at cutoff for new tests ranged from 0.07 to 0.12 ng per mL, compared with 0.13 to 0.62 ng per mL for licensed tests. Estimated viral load at cutoff ranged from 102 to 267 IU per mL for new tests and from 363 to 1069 IU per mL for licensed tests. HBsAg tests detected 31 to 63 percent of early ramp-up phase samples in the 100-member seroconversion panel study, while pooled-sample NAT detected 55 to 71 percent and single-sample NAT, 82 to 99 percent. Compared with currently licensed HBsAg assays, newer HBsAg assays would reduce the WP by 2 to 9 days; pooled-sample NAT would reduce the WP by 9 to 11 days; and single-sample NAT would reduce the WP by 25 to 36 days. CONCLUSION: Newer HBsAg tests would be expected to detect an additional 15 to 21 infected units per 107 donations, compared to licensed HBsAg tests. Sensitivity, WP closure, and yield projections for newer HBsAg assays and pooled-sample NAT are comparable. Single-sample NAT would increase yield by 13 to 15 units per 107 donations over pooled-sample NAT and newer HBsAg assays and by 35 to 50 units per 107 donations over currently licensed HBsAg assays.

Hepatitis C virus (HCV) is an important cause of liver disease throughout the world. However, the natural history and pathogenesis of this infection is still not completely understood. The aim of this study was to characterize the evolution of incident, asymptomatic HCV infection in a community-based population in Japan. The Miyazaki Cohort Study is a prospective study of adult residents in two villages, one of which has a very high prevalence of HCV. Nine hundred and seventy-three people from this village were enrolled in the cohort between 1984 and 1995, with antibodies to HCV (anti-HCV) found in 23%. During subsequent visits to annual health screens, new HCV seroconverters were identified among susceptible individuals, and their sequential samples were tested for anti-HCV, HCV-RNA, and HCV core antigen. Fourteen participants (six males, eight females) acquired anti-HCV during the first 11 years of study follow-up, at an incidence rate of 362 per 100 000 person-years. Detectable HCV-RNA and high anti-HCV titres (> 1:2048) were observed for more than 5 years following seroconversion in 80% (8/10) of seroconverters with sufficient information, indicating the development of persistent infection in these subjects. Three (37.5%) of the eight sero converters with persistent infection had fairly consistent, albeit mild, alanine aminotransferase elevations (30-130 IU/L) during the study. Anti-HCV seroconversions occurred at a very high rate in this community-based population in Japan, in which this infection is endemic. Persistence also developed at a high frequency among the cases of newly acquired infection, although the associated liver enzyme abnormalities were mild.