TRAINING MANUAL

HEPATITIS B VIRUSHEPATITIS B VIRUS

THIS MANUAL IS MEANT FOR INTENAL CIRCULATION ONLY.

INTRODUCTION Chronic hepatitis B is a major global healthcare problem. Affecting an estimated 350 to 400 million people worldwide, chronic hepatitis B results in approximately one million deaths each year, making it the world's tenth leading cause of death. The progression of chronic hepatitis B is variable, ranging from mild asymptomatic infection to severe chronic liver disease. In about 15% to 25% of patients, ongoing viral replication leads to serious complications including hepatic decompensation, cirrhosis, and hepato-cellular carcinoma (HCC).

EPIDEMIOLOGY The world can be divided into three areas where the prevalence of chronic HBV infection is high (>8%), intermediate (2-8%), and low (<2%). High endemicity areas include Southeast Asia and Pacific Basin (excluding Japan, Australia, and New Zealand), sub-Saharan Africa, the Amazon Basin, parts of Middle East, the central Asian Republics and some countries in Eastern Europe. In these areas, about 70-90% of the population becomes HBV- infected before the age of 40, and 8-20% of people are HBV carriers. In countries such as China, Senegal, Thailand, infection rates are very high in infants, and continue through early childhood. At this stage, the prevalence of HBsAg in serum may exceed 25%. In other countries such as Panama, Papua New Guinea, Solomon Islands, Greenland, and in populations such as Alaskan Indians, infection rates in infants are relatively low and increase rapidly during early childhood. Low endemicity areas include N. America, Western and Northern Europe, Australia, and parts of South America. The rest of the world falls into he intermediate range of HBV prevalence, with 2 to 8% of a given population being HBV carriers.

THE VIRUS Hepatitis B belongs to the hepadnaviridae class of viruses. It is transmitted by direct percutaneous or permucosal exposure to infected blood. The hepatitis B infection occurs in adolescents and adults and can lead to acute hepatitis, sub clinical infection, or the development of chronic infection. The incubation period ranges from 45–160 days, with an average of 75 days, followed by an insidious onset of acute disease HBV is a small, partially double-stranded DNA genome (3.2 kb) encoding four genes—HBsAg (surface envelope glycoprotein), HBcAg (viral capsid protein), HBV Pol/RT (polymerase reverse transcriptase), and X gene (transcriptional activator).

Thus there are four major serologic types (phenotypes) of Hepatitis B virus. But due to the variation in ‘w’ determinant 11 subtypes are formed which are following:-

Ø ayw1 Ø ayw2 Ø ayw3 Ø ayw4 Ø ayr Ø adw2 Ø adw4 Ø adrq+ Ø adyw Ø adrq- Ø ayw30 (F) The clinical significance of the four types remains unclear. Common to all of these subtypes, however, is an immuno-dominant epitope, the “a” determinant, that is the target of a neutralizing antibody in hepatitis B viral infection (anti-HBsAg). Recently, mutations in the “a” determinant have been reported to be associated with recurrence of hepatitis B viremia in serum despite the presence of protective antibodies (anti-HBsAg).

GENOTYPES AND THEIR DISTRIBUTION. The clinical expression of hepatitis B in different parts of the world depends not only on the prevalent genotypes but also on the prevalent modes of transmission. In fact, the clinical expression of hepatitis B is influenced dramatically by host and epidemiologic factors. Below given figure compares areas of low endemicity (such as the US, with a prevalence of 0.1% to 0.2%) versus areas of high endemicity (such as Asia and Sub-Saharan Africa, with prevalences of 10% to 15% or higher). In Asia, for example, the high prevalence of hepatitis B in the general population is reflected by a high prevalence among women of childbearing age. Thus, many infections are transmitted perinatally from mother to offspring. Neonatal infection is associated with a high level of immunological tolerance, which is very difficult to overcome. As a result, the likelihood of chronicity exceeds 90%.

Also in highly endemic areas, the lifetime risk of succumbing to complications has been estimated to be as high as 40%, whereas in low prevalence areas the risk of HCC in those with adult-acquired chronic hepatitis B is very low.

TRANSMISSION Currently, there are four recognized modes of transmission:-

1. From mother to child at birth. 2. By contact with an infected person. 3. By sexual contact. 4. By parental (blood-to- blood) exposure to blood or other

infected fluids. There is considerable variation between areas, countries and continents as to the age at which most transmission takes place. There can be carriers with or without Hepatitis. Faeces are not a source of infection as enzymes of internal mucosa inactivates the virus. Thus food, water, insects or other vector does not transmit the disease. Transmission occurs by percutaneous and permucosal exposure to infective body fluids. Percutaneous exposures that have resulted in HBV transmission include transfusion of unscreened blood or blood products, sharing of unsterilized injection needles for IV drug use, haemodialysis, acupuncture, tattooing and injuries from contaminated sharp instruments sustained by hospital personnel. Sexual and perinatal HBV transmission usually results from mucous membrane exposures to infectious blood and body fluids. HBV is stable on environmental surfaces for atleast 7 days, and indirect inoculation of HBV can occur via inanimate objects like Toothbrush, baby bottles, toys, razors, eating utensils, hospital equipments and other objects, by contact with mucous membranes or open skin brakes.

HBV is about 100 times more infectious than HIV.

REPLICATION

• After the virion enters the host cell, it is uncoated and its genome is delivered to the nucleus.

• In the nucleus, second strand DNA synthesis is completed and the gaps in both the strands are repaired to yield a covalently closed circular (ccc) supercoiled DNA molecule that serves as a template for transcription of 4 VIRAL Rna’s.

• These transcripts are transported to cytoplasm, where they are translated into the viral nucleocapsid polymerase, envelope and transcriptional transactivating proteins.

• The envelope proteins insert themselves in as integral membrane proteins into the lipid membrane of the Endoplasmic Reticulum (ER).

• Pre-genomic RNA (one of the 4 RNA’s formed earlier) is packaged together with HBV polymerase and protein kinase into core particles where it serves as a template for reverse transcription of Negative strand DNA. The RNA to DNA conversion takes place inside the particles.

• The new, mature, viral nucleocapsid can then follow two different intracellular pathways, one of which leads to the formation and secretions of new virions, whereas the other leads to amplification of viral genome inside the cell nucleus.

• In the viral assembly pathway, the nucleocapsid reach the ER, where they associate with the Envelope proteins and bud into the lumen of ER, from which they are selected via the Golgi Apparatus out of the cell.

IMMUNOPATHOGENESIS HBV is not cytotoxic but destroys liver cells indirectly by provoking an immunologic response. Kupffer cells endocytose viral antigens and present them bound to MHC class II molecules to T-helper cells. These CD4+ cells recognize the antigens and release cytokines that direct B-cell and cytolytic T-cell (CTL) activity. Stimulated B cells produce specific antibodies, including neutralizing antibodies. CTLs recognize viral peptides bound to MHC class I molecules on hepatocyte surfaces, leading to destruction of infected hepatocytes. In persons who fail to mount a sufficiently vigorous immune response to HBV during acute

infection, chronic infection develops, and the persistent, ineffective immune response results in progressive liver damage and fibrosis.

SYMPTOMS

Viral hepatitis may develop without clinical signs or symptoms, or with nonspecific symptoms that may appear for a short time with or without jaundice. These symptoms may vary from nonspecific flu-like indications to fatal liver failure. Diagnosis of viral hepatitis often depends on an accumulation of findings considered together.

Early in the disease process, generally called the prodromal phase, some patients experience a serum-type sickness that may include fever, arthralgia, arthritis, rash, and angioneurotic edema. These symptoms usually occur 2-3 weeks before jaundice and generally subside before jaundice develops, although they may be concomitant with its appearance. In the pre-icteric phase, patients may experience respiratory and gastrointestinal tract symptoms, including malaise, fatigue, myalgia, anorexia, nausea, and/or vomiting. They may also experience moderate weight loss, headache, coryza, fever, or pharyngitis and cough. Many patients complain of mid-epigastric pain, right upper quadrant discomfort, or diarrhea. Also characteristic of this phase is the development of dark urine and the lightening of stool color. This duration of this stage of the disease may range from 2-3 days to 2-3 weeks. The icteric phase is signaled by the development of jaundice. General constitutional symptoms may subside. There may be worsening of anorexia, nausea, and vomiting along with scratching and irritated skin lesions related to pruritis.

DIAGNOSIS:

Acute Infection The incubation period for Hepatitis B—from acute exposure to clinical symptoms—may range from 60–180 days. Clinical presentation may vary from asymptomatic infection of cholestatic hepatitis to fulminant liver failure. Presence of HBV DNA or HBeAg in serum implies active viral replication. These tests may remain positive throughout the prodromal phase and early clinical phases of the illness. Chronic Infection Chronic hepatitis B infection is defined as the presence of HBsAg in the serum for more than 6 months. HBsAg is a marker of hepatitis B viral

Figure: - Progression of chronic hepatitis B viral infection. infection, whereas HBeAg and HBV DNA (using a polymerase chain reaction (PCR) assay) are markers of viral replication and infectivity. Chronic infection may lead to serious sequelae, e.g., cirrhosis, liver failure, hepatocellular carcinoma, or death.

In a small number of patients, HBV DNA may be present in serum in the absence of HBeAg. These patients have a “precore” mutant, which prevents completion and excretion of the “e” antigen despite active replication of HBV DNA, and is associated with progressive liver disease. Physical Examination Physical examination of patients with hepatitis B may reveal posterior cervical lymphadenopathy, splenomegaly, and hepatomegaly. Hepatic enlargement may be minimal, with slight tenderness on palpation or percussion. However, in some patients, the examination may be unremarkable. Diagnostic Tests Serum Enzymes Ornithine, carbamyltransferase, sorbitol dehydrogenase, glutamate dehydrogenase, isocitrate dehydrogenase, malate dehydrogenase, and guanase levels are all helpful in the diagnosis of viral hepatitis. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) remain the most sensitive for establishing a diagnosis of acute viral hepatitis. ALT and AST are the first enzymes to reveal abnormalities during the disease process and the last to normalize. They may reach levels 100 times the upper limits of normal, and ALT is usually more abnormal than AST in the early and late stages of the disease. Serum Bilirubin Bilirubin values of 2.5-3.0 mg/dl or greater establish the presence of the icteric phase of hepatitis. Bilirubin levels in excess of 30 mg/dl suggest hemolysis (over production of bilirubin) or renal failure (failure of excretion). Serum bilirubin levels are not always of clinical value. Hematological Changes Leukopenia is commonly observed in the presence of viral hepatitis. Anemia and thrombocytopenia are less frequently observed.

Liver Biopsy Liver biopsy is generally not necessary, but should be

considered if, the diagnosis is uncertain. Liver biopsy should be performed if there is an atypical clinical course, or the clinical course is prolonged. It should also be undertaken if clues of chronic liver disease are present, or if there are complications such as encephalopathy or fluid retention.

SEROLOGY & SEROLOGICAL TESTS

• For clinical diagnostic purposes, we rely on an understanding of serologic events that appear during acute and chronic hepatitis B. Following figure illustrates the serologic course of a typical case of acute, self-limited hepatitis B.

• The first viral protein to appear (within several weeks of infection) is surface antigen, HBsAg, which increases in titer over the next several weeks, even before symptoms begin.

• During clinically apparent illness, levels of HBsAg are high, symptoms occur, and aminotransferase levels are elevated.

• Once symptoms appear, HBsAg begins to decline and is ultimately replaced by anti-HBs (HBsAg seroconversion), The time lag between the disappearance of HBsAg and appearance of anti- HBs is called as Window Period.

• Anti-HBs is recognized to be a neutralizing antibody, coinciding temporally with recovery from infection.

• Within a week or two after the appearance of HBsAg, antibody to the core protein, anti-HBc, can be detected. Early during acute infection, anti-HBc is primarily of the IgM class; however, after approximately 6 months, IgM anti-HBc is replaced by anti-HBc of the IgG class (reported by clinical laboratories as total anti-HBc reactivity with undetectable IgM anti-HBc).

• HBeAg, a non-particulate, circulating protein derived from translation of the core gene, typically appears during the period of peak active virus replication. Because HBeAg is always present during acute HBV infection, testing for it during acute infection is not indicated.

• Ultimately, HBeAg is replaced by anti- HBe (HBeAg seroconversion); when recovery from acute hepatitis B is delayed beyond 3 to 4 months, the persistence of HBeAg may be a harbinger of chronic infection.

• Thus, a person who recovers from hepatitis B generally has all three antibodies-anti-HBs, anti-HBc, and anti-HBe.

• Above figure illustrates the serologic course when acute hepatitis B fails to resolve and, instead, progresses to chronic hepatitis B. In such cases, HBsAg remains reactive indefinitely, and anti-HBc is of the IgG class. Two relative phases of chronic hepatitis B are recognized. A relatively highly replicative phase occurs early during the natural history of chronic

infection and is associated with HBeAg and high levels of HBV DNA (usually >106 copies/mL). About 10% to 15% of patients per year convert spontaneously to a relatively nonreplicative phase-associated with HBeAg seroconversion-in which HBV DNA circulates at levels �103 copies/mL.

HBsAg Anti - HBs

Anti - HBc

…………………….INTERPRETATION…………………………

+ - - Early Acute HBV infection + +/- + Acute or Chronic HBV infection. Differentiate with

IgM- anti- HBc. - + + Indicates previous HBV infection or immunity to

HBV. - - + Possibilities include: Past HBV infection, low level

HBV carrier, time span between disappearance of HBsAg and appearance of anti –HBs; or false positive or non specific reaction. Investigate with IgM anti-HBc.

- - - Another infectious agent, toxic injury to the liver, disorder of immunity, hereditary disease of liver, or disease of the bilary tract.

- + - Vaccine type response.

THUS FROM THE ABOVE EXPLAINATION IT CAN BE INFERED THAT HBsAg IS THE BEST MARKER FOR THE EARLY DETECTION OF HBV INFECTION

JML RANGE OF PRODUCTS FOR THE DIAGNOSIS OF INFECTION WITH

Hepatitis B Virus

HEPACARD HEPALISA

HEPACARD:

SALIENT FEATURES

• One step, rapid, visual and qualitative immunoassay.

• No sample preparation required • Ready to use • Shelf life 15 months at 4 – 80 C. • Unique see through device for maximum user

safety. Specificity: 99% Sensitivity: 0.5 ng /ml EVALUATIONS HEPACARD.

Ø WHO EVALUATION: In this evaluation the Sensitivity obtained was 100% and Specificity of 99.4%

Ø PATH USA: In this the Sensitivity obtained was 100% and Specificity of 100%.

Ø DEPT. OF CLINICAL VIROLOGY, CMC VELLORE: In this evaluation of 90 samples the Sensitivity obtained was 100% and Specificity of 100%.

Ø University of Madras: In this evaluation the Sensitivity obtained was 100% and Specificity of 100%

HEPALISA:

INTENDED USE Hepalisa is designed for in-vitro qualitative detection of Hepatitis B surface antigen (HBsAg) in human serum or Plasma and is used as s screening test for testing of collected blood prior to transfusion. PRINCIPLE Hepalisa is a solid phase enzyme immunosorbent assay (ELISA) based on the “Direct Sandwich” principle. The microwells are coated with Monoclonal antibodies with higher reactivity for HBsAg. The samples added in the wells followed by the addition of enzyme conjugate (polyclonal antibodies linked to Horse Radish Peroxidase). A sandwich complex is formed in the well wherein HBsAg (from serum sample) is “

trapped” or “Sandwich” between the antibody and antibody HRPO conjugate. Unbound conjugate is ten washed off with wash buffer. The amount of bound peroxidase is proportional to the concentration of HBsAg present in the sample. Upon addition of the substrate buffer and chromogen, a blue colour develops. The intensity of developed lue colour is proportional to the concentration of HBsAg in sample. To limit the enzyme – substrate reaction, stop solution is added and a yellow colour develops which is finally read at 450nm spectophotometrically. SALIENT FEATURES

• Detects all the known eleven sub-types of Hepatitis B virus.

• Break away wells for minimal wastage. • Easy interpretation of results • Shelf life 12 months at 2-80 C. • No sample dilution, thus reducing handling time and

errors. • Available in convenient pack sizes: 96, 480 T & 960T. • Specificity: increased power of discrimination between

Negatives and Positives.

Specificity: 99.7 % Sensitivity:0. 2 ng /ml

EVALUATIONS HEPALISA Ø WHO EVALUATION: In this evaluation of 277 samples the

Sensitivity obtained was 99% and Specificity of 100% Ø Armed Force Transfusion Centre, New Delhi: Found the kit

satisfactory for use. Ø Bangalore Medical College: Evaluation claim that the kit is found

to be Satisfactory, with Sensitivity: 100%, Specificity: 100% Ø Dhanvandri Blood Bank & Research Centre, Chennai: The

evaluation has found Hepalisa to be a excellent product, and has high specificity and sensitivity.

Ø K.C.G Hospital Bangalore: Has claimed our kit to be Satisfactory.

PREVENTION AND CONTROL Therapy The treatment goals for chronic hepatitis B infection are:

1) Suppression of viral replication, 2) Seroconversion to an anti-HBs positive, anti-HBe positive, HBV

DNA negative status, 3) Improvement in hepatic necroinflammation, and 4) Reduction in the likelihood of long-term sequelae of HBV

infection, such as cirrhosis and hepatocellular carcinoma. Medical Therapy Interferon alfa-2b is the only FDA-approved interferon product for hepatitis B. The recommended dose is 5 million units sub-cutaneously 5 times per week for 4 months. This has been associated with histological benefit primarily in patients with loss of serum HBeAg. However, most patients do not lose either HBeAg or HBsAg.

Recently, synthetic nucleoside analog, lamivudine (Epivir-HBV™), is used for patients with chronic hepatitis B viral infection associated with evidence of viral replication and active liver inflammation. Lamivudine inhibits HBV polymerase reverse transcriptase activity resulting in chain termination of nascent HBV DNA strands. Preclinical studies demonstrate a satisfactory safety profile with no evidence of mutagenicity, carcinogenicity, or teratogenic potential. Lamivudine (Epivir-HBV™) leads to dose-related reductions in HBV DNA levels after oral absorption, with an equimaximal effect at doses greater than 100 mg/day. Prevention Two agents are currently available for prophylaxis against hepatitis B viral infection. The first is hepatitis B immune globulin (HBIG), which provides temporary protection from HBV. HBIG is prepared from plasma that contains high titers of anti-HBs. The second is the hepatitis B vaccine, which, to date, has exerted its greatest impact on health care workers (a relatively small subgroup in terms of hepatitis B incidence). Those groups most at risk have not, in most cases, been

vaccinated. There are several reasons for this: 1) lack of awareness about hepatitis B and its consequences, 2) lack of public programs, 3) high cost of the vaccine, (4) inability to identify individuals in high risk groups (injection drug users), and (5) disease concentration in people without risk factors. In an effort to eradicate hepatitis B transmission, HBsAg screening of pregnant women is performed and immuno-prophylaxis is given to infants born to potentially infectious mothers to prevent perinatal infection. In addition, hepatitis B vaccination is integrated into current childhood immunization schedules in high-risk populations. This practice provides immunity to teens and adults before they become at risk for hepatitis B infection.

GLOSSARY

Bilirubin is the chief pigment of bile, formed mainly from the breakdown of hemoglobin. After formation it is transported in the plasma to the liver to be then excreted in the bile. Elevation of bile in the blood (>30 mg/l) causes jaundice.

Carrier is a person who has HBV in his or her blood even if all symptoms have disappeared. Because the virus is present in the blood, it can be transmitted to others.

Cirrhosis a chronic disease of the liver characterized by nodular regeneration of hepatocytes and diffuse fibrosis. It is caused by parenchymal necrosis followed by nodular proliferation of the surviving hepatocytes. The regenerating nodules and accompanying fibrosis interfere with blood flow through the liver and result in portal hypertension, hepatic insufficiency, and jaundice. Cirrhosis is a more severe, irreversible process of liver inflammation, necrosis, and regeneration. In hepatitis C, cirrhosis occurs as a late stage sequel of chronic infection, and may take 20-30 years to develop.

ELISA enzyme-linked immunoassorbent assay

Endemic continuously prevalent in some degree in a community or region.

Endoplasmic reticulum a network or system of folded membranes and interconnecting tubules distributed within the cytoplasm of eukaryotic cells. The membranes form enclosed or semi enclosed spaces. The endoplasmic reticulum

functions in storage and transport, and as a point of attachment of ribosomes during protein synthesis.

Epidemic is an outbreak of disease such that for a limited period a significantly greater number of persons in a community or region suffer from it than is normally the case. Thus an epidemic is a temporary increase in incidence. Its extent and duration are determined by the interaction of such variables as the nature and infectivity of the casual agent, its mode of transmission and the degree of preexisting and newly acquired immunity.

Genotype:- the genetic constitution of an individual.

Hepatocytes are liver cells.

Histopathology the study of the structural alterations of cells and tissues caused by disease.

Humoral: pertaining to the humors, or certain fluids, of the body.

Incidence the number of cases of a disease, abnormality, accident, etc., arising in a defined population during a stated period, expressed as x cases per 1000 persons per year.

Interferon a protein produced in organisms infected by viruses, and effective at protecting those organisms from other virus infections. Interferons exert virus-nonspecific but host-specific anti viral activity by inducing the transcription of cellular genes coding for anti viral proteins that selectively inhibits the synthesis of viral DNA and proteins. Interferons also have immuno-regulatory functions. Production of interferon can be stimulated by viral infection, especially by the presence of double stranded RNA, by intracellular parasites, by protozoa, and by bacteria and bacterial products. Interferons have been divided into three distinct types associated with specific producer cells and functions, but all animal cells are capable of producing interferons, and certain producer cells (leukocytes and fibroblasts) produce more than one type (both a and ß).

Jaundice is a yellow discoloration of the skin and mucous membranes due to excess of Bilirubin in the blood, also known as icterus.

Lymphocyte:- a leukocyte of blood, bone marrow and lymphatic tissue. Lymphocytes play a major role in both cellular and humoral immunity, and thus several different functional and morphologic types must be recognized, i.e. the small, large, B-, and T-lymphocytes, with further morphologic distinction being made among the B-lymphocytes.

Prevalence is the number of instances of infections or of persons ill, or of any other event such as accidents, in a specified population, without any distinction between new and old cases.

Prophylaxis is the prevention of disease, or the preventive treatment of a recurrent disorder.

Seroconversion:- the production in a host of specific antibodies as a result of infection or immunization. The antibodies can be detected in the host’s blood serum following, but not preceding, infection or immunization.

Serotype a subgroup within a species, defined by reaction of one or more antigens with the corresponding anti serum.

Serum is the clear, slightly yellow fluid, which separates from blood when it clots. Sera containing antibodies and antitoxins against infections and toxins of various kinds (antisera) have been used extensively in prevention or treatment of various diseases.

Viremia:- the presence of viruses in the blood, usually characterized by malaise, fever, and aching of the back and extremities.

REFERENCES

• Margolis HS, Altrer MJ, Holder SC. Hepatitis B: Evolving epidemiology and implication for control. Sem Liv Dis 1991;112 :84-90.

• Maynord JE. Hepatitis B: Global importance and need for control. Vaccine 1990;8 :s18-s20.

• Chakravarti A, Rawat D, Jain M. A Study on the Perinatal Transmission of the Hepatitis B Virus. Indian J Med Microbiol 2005;23:128-130.

• Centers for Disease Control and Prevention (CDC). Hepatitis B virus. http://www.cdc.gov/ncidod/%20diseases/hepatitis/slideset/hep_b/hep_b.pdf (accessed August 27, 2004).

• Moyer LA, Mast EE. Hepatitis B: virology, epidemiology, disease, and prevention, and an overview of viral hepatitis. Am J Prev Med. 1994;10Suppl:45-55.

• Chu CJ, Keeffe EB, Han SH, et al. Hepatitis B virus genotypes in the United States: results of a nationwide study. Gastroenterology. 2003 Aug;125(2):444-51.

• Chu CJ, Hussain M, Lok AS. Hepatitis B virus genotype B is associated with earlier HBeAg seroconversion compared with hepatitis B virus genotype C.

• RESOURCE CENTRE ON HEPATITIS B VIRUS, Deptt. Of Gatroentrology and Hepatology. JOHNS HOPKINS MEDICAL INSTITUTION.

• Jules L. Dienstag, MD.Massachusetts General Hospital arvard Medical School “ Introduction to Chronic Hepatitis B virus, Diagnosis, Clinical features and Natural History

• Division of Viral Hepatitis Centers for Disease Control and Prevention National Center for HIV/AIDS, STD and TB Prevention.

• Thio CL. Management of chronic hepatitis B in the HIV-infected patient. AIDS Read. 2004 Mar;14(3):122-9, 133, 136-7.

• Soriano V, Puoti M, Bonacini M, et al. Care of patients with chronic hepatitis B and HIV co-infection: recommendations from an HIV-HBV International Panel. AIDS. 2005 Feb 18;19(3):221-40.

• Keeffe E. Clinical Care Options Management Series: Diagnosis, Treatment, and Chronic Care Options for Hepatitis B. Accessed February 7, 2006.

• Centers for Disease Control and Prevention, National Institutes of Health, HIV Medicine Association/Infectious Diseases Society of America. Treating Opportunistic Infections Among HIV-Infected Adults and Adolescents. MMWR Recomm Rep. 2004 Dec 17; 53(RR15);1-112. Available online at aidsinfo.nih.gov/Guidelines/GuidelineDetail.aspx?GuidelineID=14. Accessed May 19, 2006.