UK Standards for Microbiology Investigations
Investigation of Hepatitis
Issued by the Standards Unit, Microbiology Services, PHE
Clinical Guidance | G 5 | Issue no: 1.2 | Issue date: 03.03.14 | Page: 1 of 44
© Crown copyright 2014
Investigation of Hepatitis
Acknowledgments
UK Standards for Microbiology Investigations (SMIs) are developed under the
auspices of Public Health England (PHE) working in partnership with the National
Health Service (NHS), Public Health Wales and with the professional organisations
whose logos are displayed below and listed on the website
http://www.hpa.org.uk/SMI/Partnerships. SMIs are developed, reviewed and revised
by various working groups which are overseen by a steering committee (see
http://www.hpa.org.uk/SMI/WorkingGroups).
The contributions of many individuals in clinical, specialist and reference laboratories
who have provided information and comments during the development of this
document are acknowledged. We are grateful to the Medical Editors for editing the
medical content.
For further information please contact us at:
Standards Unit
Microbiology Services
Public Health England
61 Colindale Avenue
London NW9 5EQ
E-mail: standards@phe.gov.uk
Website: http://www.hpa.org.uk/SMI
UK Standards for Microbiology Investigations are produced in association with:
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UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Investigation of Hepatitis
Contents
ACKNOWLEDGMENTS .......................................................................................................... 2
CONTENTS ............................................................................................................................. 3
AMENDMENT TABLE ............................................................................................................. 4
UK STANDARDS FOR MICROBIOLOGY INVESTIGATIONS: SCOPE AND PURPOSE ....... 5
SCOPE OF DOCUMENT ......................................................................................................... 8
INTRODUCTION ..................................................................................................................... 8
1
HEPATITIS A ............................................................................................................. 12
2
HEPATITIS B ............................................................................................................. 14
3
HEPATITIS C ............................................................................................................. 20
4
HEPATITIS D ............................................................................................................. 23
5
HEPATITIS E ............................................................................................................. 25
6
HEPATITIS G ............................................................................................................. 29
7
CYTOMEGALOVIRUS (CMV) .................................................................................... 29
8
EPSTEIN BARR VIRUS (EBV) .................................................................................. 30
9
YELLOW FEVER ....................................................................................................... 31
10
Q FEVER.................................................................................................................... 33
REFERENCES ...................................................................................................................... 35
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Investigation of Hepatitis
Amendment Table
Each SMI method has an individual record of amendments. The current
amendments are listed on this page. The amendment history is available from
standards@phe.gov.uk.
New or revised documents should be controlled within the laboratory in accordance
with the local quality management system.
Amendment No/Date.
2/03.03.14
Issue no. discarded.
1.1
Insert Issue no.
1.2
Section(s) involved
Amendment
Document has been transferred to a new template
to reflect the Health Protection Agency’s transition
to Public Health England.
Front page has been redesigned.
Whole document.
Status page has been renamed as Scope and
Purpose and updated as appropriate.
Professional body logos have been reviewed and
updated.
Scientific content remains unchanged.
Amendment No/Date.
1/11.11.11
Issue no. discarded.
1
Insert Issue no.
1.1
Section(s) involved
Amendment
Whole document.
Formerly QSOP 54 now G 5.
Document presented in a new format.
Relevant NSM and Glossary of
terms sections.
Removed.
References.
Some references updated.
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Investigation of Hepatitis
UK Standards for Microbiology Investigations:
Scope and Purpose
Users of SMIs

SMIs are primarily intended as a general resource for practising professionals
operating in the field of laboratory medicine and infection specialties in the
UK.

SMIs provide clinicians with information about the available test repertoire and
the standard of laboratory services they should expect for the investigation of
infection in their patients, as well as providing information that aids the
electronic ordering of appropriate tests.

SMIs provide commissioners of healthcare services with the appropriateness
and standard of microbiology investigations they should be seeking as part of
the clinical and public health care package for their population.
Background to SMIs
SMIs comprise a collection of recommended algorithms and procedures covering all
stages of the investigative process in microbiology from the pre-analytical (clinical
syndrome) stage to the analytical (laboratory testing) and post analytical (result
interpretation and reporting) stages.
Syndromic algorithms are supported by more detailed documents containing advice
on the investigation of specific diseases and infections. Guidance notes cover the
clinical background, differential diagnosis, and appropriate investigation of particular
clinical conditions. Quality guidance notes describe laboratory processes which
underpin quality, for example assay validation.
Standardisation of the diagnostic process through the application of SMIs helps to
assure the equivalence of investigation strategies in different laboratories across the
UK and is essential for public health surveillance, research and development
activities.
Equal Partnership Working
SMIs are developed in equal partnership with PHE, NHS, Royal College of
Pathologists and professional societies.
The list of participating societies may be found at
http://www.hpa.org.uk/SMI/Partnerships. Inclusion of a logo in an SMI indicates
participation of the society in equal partnership and support for the objectives and
process of preparing SMIs. Nominees of professional societies are members of the
Steering Committee and Working Groups which develop SMIs. The views of
nominees cannot be rigorously representative of the members of their nominating
organisations nor the corporate views of their organisations. Nominees act as a
conduit for two way reporting and dialogue. Representative views are sought through
the consultation process.

Microbiology is used as a generic term to include the two GMC-recognised specialties of Medical Microbiology (which
includes Bacteriology, Mycology and Parasitology) and Medical Virology.
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SMIs are developed, reviewed and updated through a wide consultation process.
Quality Assurance
NICE has accredited the process used by the SMI Working Groups to produce SMIs.
The accreditation is applicable to all guidance produced since October 2009. The
process for the development of SMIs is certified to ISO 9001:2008.
SMIs represent a good standard of practice to which all clinical and public health
microbiology laboratories in the UK are expected to work. SMIs are NICE accredited
and represent neither minimum standards of practice nor the highest level of
complex laboratory investigation possible. In using SMIs, laboratories should take
account of local requirements and undertake additional investigations where
appropriate. SMIs help laboratories to meet accreditation requirements by promoting
high quality practices which are auditable. SMIs also provide a reference point for
method development.
The performance of SMIs depends on competent staff and appropriate quality
reagents and equipment. Laboratories should ensure that all commercial and inhouse tests have been validated and shown to be fit for purpose. Laboratories
should participate in external quality assessment schemes and undertake relevant
internal quality control procedures.
Patient and Public Involvement
The SMI Working Groups are committed to patient and public involvement in the
development of SMIs. By involving the public, health professionals, scientists and
voluntary organisations the resulting SMI will be robust and meet the needs of the
user. An opportunity is given to members of the public to contribute to consultations
through our open access website.
Information Governance and Equality
PHE is a Caldicott compliant organisation. It seeks to take every possible precaution
to prevent unauthorised disclosure of patient details and to ensure that patientrelated records are kept under secure conditions.
The development of SMIs are subject to PHE Equality objectives
http://www.hpa.org.uk/webc/HPAwebFile/HPAweb_C/1317133470313. The SMI
Working Groups are committed to achieving the equality objectives by effective
consultation with members of the public, partners, stakeholders and specialist
interest groups.
Legal Statement
Whilst every care has been taken in the preparation of SMIs, PHE and any
supporting organisation, shall, to the greatest extent possible under any applicable
law, exclude liability for all losses, costs, claims, damages or expenses arising out of
or connected with the use of an SMI or any information contained therein. If
alterations are made to an SMI, it must be made clear where and by whom such
changes have been made.
The evidence base and microbial taxonomy for the SMI is as complete as possible at
the time of issue. Any omissions and new material will be considered at the next
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review. These standards can only be superseded by revisions of the standard,
legislative action, or by NICE accredited guidance.
SMIs are Crown copyright which should be acknowledged where appropriate.
Suggested Citation for this Document
Public Health England. (2014). Investigation of Hepatitis. UK Standards for
Microbiology Investigations. G 5 Issue 1.2. http://www.hpa.org.uk/SMI/pdf.
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Investigation of Hepatitis
Scope of Document
Type of Specimen
N/A
Scope
This SMI describes the examination of samples for Hepatitis.
This SMI should be used in conjunction with other SMIs.
Introduction
Background
The term "hepatitis" means inflammation of the liver. Hepatitis may be caused by
viruses, bacteria, drugs, toxins, or excess alcohol intake. Viral hepatitis was first
recognized as a distinct clinical entity during the late 18th and early 19th centuries. At
that time, reports of scattered outbreaks of this syndrome, referred to as infectious
hepatitis, epidemic hepatitis, or catarrhal jaundice, began to appear in the medical
literature. The viruses associated with the main clinical feature of hepatitis or
jaundice are known as the hepatitis viruses, of which there are currently five
described - hepatitis A, B, C, E and D viruses, although other viruses can cause
hepatitis as part of a wider illness, for example, CMV and EBV. There are likely to be
others, as yet uncharacterised, hepatitis viruses.
Viral hepatitis is the most common of the serious contagious diseases, with
significant morbidity and mortality resulting from acute illness (hepatitis A, B, C, D, E)
and cirrhosis and liver cancer associated with chronic infection (hepatitis B, C, D) in
addition to adverse economic effects.
Viral hepatitis can be transmitted enterically and parenterally.
Enterically Transmitted Hepatitis
This occurs when a sufficient amount of the virus enters the mouth to cause
infection. This can occur through direct contact with an infected person’s faeces or
indirect faecal contamination of food, water supply, shellfish or utensils. This is the
main route of transmission for hepatitis A and E.
Parenterally Transmitted Hepatitis
This is when the disease is transmitted from one person to another by infectious
blood and body fluids. The people most likely to become infected include:

The family of infected persons (including newborn infants)

Injecting drug users (current or previous)

Recipients of transfusions, blood or blood products, or transplanted organs
prior to 1990 when blood banks began testing for hepatitis C

Persons with needle-stick injury

Institutionalised persons

Persons with multiple sex partners
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
Persons with tattoos

Healthcare professionals – rare in the UK
Hepatitis B, C and D are transmitted parenterally, hence including them in the bloodborne viruses.
The Liver
The liver weighs approximately 1.5 kilograms in the normal adult and it is located in
the right side of the abdomen, immediately under the diaphragm.
Liver cells perform many diverse chemical activities including:

Production of bile, which aids digestion through emulsification of lipids

Storage of vital substances such as glycogen, iron, copper, and certain
vitamins especially vitamins A, B12 and D

Disposal of amino acid wastes, producing urea from ammonia

Production of energy through production of glucose from amino acids and
breakdown of glycogen, and breakdown of fats

Production of protein substances including coagulation factors that
regulate blood clotting and plasma proteins notably albumin

Filtration of toxic substances that could damage the body if allowed to
accumulate
All the above aspects of normal liver cell function may be affected by hepatitis,
resulting in general systemic symptoms as well as more specific infection and
hepatic damage related symptoms and signs (see below).
However, the liver is one of the most remarkable and versatile organs in the human
body. In fact, it is the only organ able to regenerate itself – up to 80% to 85% of the
liver can be destroyed and the remaining tissue may recover.
Symptoms of Hepatitis
Early symptoms of infective hepatitis are similar to common flu symptoms—general
fatigue, joint and muscle pain, and loss of appetite. Nausea, vomiting, and diarrhoea
or constipation may follow with a low-grade fever (up to 39°C). As the disease
progresses, the liver may enlarge and become tender. Chills, weight loss, and
distaste for food and, curiously, cigarettes may occur. Occasionally, the urine and
faeces will change colour. There is a spectrum of clinical features found in all types
of viral hepatitis, ranging from asymptomatic or subclinical to classical jaundice, and
through to acute liver failure and death. Any combination of fatigue, fever, loss of
appetite, nausea, vomiting, diarrhoea, or abdominal discomfort may occur.
A proportion of patients notice dark urine and light-coloured stools, followed by
jaundice, in which the skin and whites of the eyes appear yellow. Itching of the skin
may be present. With the onset of jaundice, other symptoms tend to subside.
Jaundice is due to accumulation of bilirubin in the blood. It is formed primarily from
the breakdown of “haem” in red blood cells and is a waste product. In a healthy
individual, there is only a small amount of bilirubin circulating in the blood. However,
increased levels of serum bilirubin are found in patients that are suffering from
conditions that increase the destruction of red blood cells, and conditions such as
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liver disease, where there is a decreased removal of the substance from the blood
stream. Bilirubin is a yellow pigment and when accumulated in the blood in
abnormally high levels, will make the skin and the whites of the eyes appear yellow.
Jaundice (or icterus) is the clinical term for this condition.
In some cases the cause of acute hepatitis can be suggested by clinical features and
the patient’s history. However, specific laboratory tests must be used to establish a
diagnosis.
Biochemical Tests
“Liver function tests” (LFTs) is a commonly used term to describe a group of blood
tests that assess the general state of the liver and biliary system. Routine blood tests
can be divided into those tests that are true LFTs, such as for serum albumin,
prothrombin time, or serum bilirubin, and those tests that are simply markers of liver
or biliary tract disease, such as for the various liver enzymes. Serum bilirubin is
generally considered a true test of liver function, since it reflects the liver’s ability to
take up, process, and secrete bilirubin into the bile. It is relatively non-specific,
however, because many forms of liver or biliary tract disease can result in elevated
levels of serum bilirubin. In addition to the usual liver tests obtained on routine
automated chemistry panels, physicians may order more specific tests such as viral
serologic tests, or tests for autoimmunity, to determine the specific cause of a liver
disease.
Liver Enzymes
There are two general categories of “liver enzymes”. The first group includes alanine
aminotransferase (ALT) and aspartate aminotransferase (AST), formerly referred to
as SGPT and SGOT. Presence of high levels of these enzymes is an indicator of
liver cell damage. The other frequently used liver enzyme tests are for alkaline
phosphatase and gamma-glutamyltranspeptidase (GGT). High levels of the latter
enzymes indicate obstruction to the biliary system, either within the liver or in the
large bile channels outside the liver.
ALT and AST enzymes are found in liver cells (hepatocytes). When liver cells are
injured, these enzymes are released into the bloodstream. In general, the level of
ALT is thought to be a more specific indicator of liver inflammation, since AST may
be elevated in diseases of other organs such as the heart. In acute liver injury, such
as acute viral hepatitis, ALT and AST are often dramatically elevated; in chronic
hepatitis or cirrhosis, the elevation of these enzymes may be minimal, ie 2-3 times
normal, or moderate. Mild or moderate elevations of ALT may be caused by a wide
range of liver diseases. Determination of ALT and AST level is often used to monitor
the course of chronic hepatitis and the response to treatments such as interferon and
other antivirals.
Alkaline phosphatase and GGT are elevated in a large number of disorders that
affect the drainage of bile. Because alkaline phosphatase is also found in other
areas, such as bone, placenta, and intestine, an elevated level is not specific for liver
disease; measurement of GGT is utilized as a supplementary test to be sure that the
elevation of alkaline phosphatase is indeed coming from the liver or the biliary tract,
since GGT is not elevated in diseases of bone, placenta, or intestine. Mild or
moderate elevation of GGT in the presence of a normal level of alkaline phosphatase
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is difficult to interpret and is often caused by changes in the liver cell enzymes
induced by alcohol or medications but not associated with injury to the liver.
Albumin and Prothrombin Time
Two other commonly used indicators of liver function are the serum albumin level
and prothrombin time. Albumin is a major protein formed by the liver, and chronic
liver disease causes a decrease in the amount of albumin produced. Therefore, in
more advanced liver disease, the level of the serum albumin is reduced. The
prothrombin time or PT is a test that is used to assess blood clotting. Blood clotting
factors are proteins made by the liver; when the liver is significantly injured, these
proteins are not normally produced. The prothrombin time is a useful test of liver
function, since there is good correlation between abnormalities in coagulation
measured by the prothrombin time and the degree of liver dysfunction. Prothrombin
time is usually expressed in seconds and is compared to a normal control patient’s
PT.
Other Biochemical Tests
There are many other specialised biochemical tests that may be used to diagnose
the cause of liver disease. In addition, there are also specific blood tests used to
diagnose the cause of viral hepatitis.
Histological Assessment
Liver biopsy to remove samples of liver cells (hepatocytes) is generally used to
assess liver cell damage. Assessment of these biopsies is highly subjective and
qualitative. In an attempt to qualify comparisons of liver histology, scoring systems to
quantify all aspects of the necroinflammatory-fibrotic process of chronic hepatitis are
used such as the Ishak, Knodell and Metavir scores. Serologic and molecular testing
has made liver biopsy less valuable as a diagnostic tool, but it remains important to
exclude other liver diseases and to assess the extent of injury. Other, non-invasive,
methods of assessing liver fibrosis have recently been introduced, such as Fibroscan
(a measure of liver elasticity) and Hepascore based on blood parameters.
Notification of Hepatitis
Suspected acute infectious hepatitis must be notified by a registered medical
practitioner to the proper officer of the local authority in which the patient was seen,
as stated in the Department of Health. Health Protection Legislation (England)
Guidance 2010; this notification does not need to await laboratory confirmation1.
Laboratories are to notify the PHE of cases of hepatitis A, and both acute and
chronic cases of hepatitis B and C.
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1
Hepatitis A
1.1
Basic Virology and Taxonomy
The enterically transmitted disease known as ‘infectious hepatitis’ was known for
many years before its causal agent was identified in 1973 by demonstration of a
27nm icosahedral virus in patient faeces using immune electron microscopy2.
Biophysical and biochemical studies later established Hepatitis A (HAV) as a
member of the family Picornaviridae. In 1992 HAV was assigned to a separate
genus Hepatovirus, on the basis of a number of differences from the enteroviruses,
including stability of the virion at 60°C, lack of reactivity with an enterovirus groupspecific monoclonal antibody, low percentage nucleotide homology with the genome
of the enteroviruses and certain differences in the replication cycle. The genome is
single stranded positive sense RNA in an unenveloped icosahedral particle3. The
capsid comprises 60 copies of each of the four structural proteins VP1-4. A single
copy of the genome-linked protein, VPg, is attached covalently to the 5’ end of the
RNA.
All isolates of HAV are of a single serotype with seven genotypes recognised on the
basis of less than 85% nucleotide identity in selected regions of the genome.
1.2
Laboratory Diagnosis
HAV is present in stools before the onset of clinical symptoms and can be
demonstrated by electron microscopy. Isolation in cell cultures is difficult but when
necessary for research purposes virus can be recovered from faeces in primary or
continuous lines of primate cells, derived from monkey kidney or from human
fibroblasts or hepatoma. Detection of nucleic acid with PCR can be useful as an
epidemiological tool and in environmental studies.
Antibodies can be demonstrated from the onset of the clinical symptoms. Diagnosis
of acute infection requires demonstration of anti-HAV IgM antibodies or
seroconversion. IgM antibodies disappear about 3-6 months after the onset of
disease. IgG antibodies persist for life and indicate immunity against reinfection.
Antibodies are demonstrated by means of EIA. Hepatitis A IgM is also seen after
vaccination, and false positive results are not uncommon, especially in older age
groups.
1.3
Clinical Symptoms
The incubation period is usually 4 weeks (2-6 weeks). The site of primary infection is
in the alimentary tract, although the sequence of events that eventually results in
hepatitis is not well understood. The short prodromal phase, varying from 2 to 7
days, usually precedes the onset of jaundice. The most prominent symptoms of this
phase are fever, headache, muscular and abdominal pain, anorexia, nausea,
vomiting and sometimes arthralgia. Hepatomegaly and leukopenia are often present
during this period followed by bilirubinuria, then pale faeces and jaundice about 10
days later.
The liver is usually enlarged and liver function tests are abnormal with elevated
levels of serum alanine aminotransaminase (ALT) and serum aspartate amino
transaminase (AST). Jaundice is sometimes accompanied by itching and by
urticarial or papular rashes4.
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Worldwide, most infections occur in young children and are subclinical. The disease
is usually mild in older children and young adults with severity increasing with age 5.
Symptoms are likely to disappear after 10-14 days except in adults where they may
last up to 4-5 weeks. Liver function tests rapidly return to normal when clinical
symptoms disappear, but in some cases may persist for several months. Prolonged
excretion of virus in faeces has been observed in neonates nosocomially infected in
an intensive care unit6.
There are no long term sequelae and no chronic carrier state. The infection may
produce a relapsing course, and less often an illness with cholestasis as the principal
feature4. Acute hepatitis A is a major cause of fulminant hepatitis and has been
reported to account for 10% of liver transplants in children4,7.
1.4
Transmission
Transmission can be via various routes:

The most important means of transmission is via the faecal-oral route from
person to person. Transmission is generally limited to close contact especially
within families

In many countries an important mechanism of transmission is consumption of
raw or partially cooked shell fish, for example oysters harvested from waters
that have been contaminated with human sewage8. Other foods have been
implicated in outbreaks of HAV including milk, strawberries or salad 9. Various
waterborne outbreaks have also been noted

Several large outbreaks have occurred in injecting drug users (IDUs) and
among men who have sex with men10,11
1.5
Epidemiology
Hepatitis A is present in a worldwide distribution3. Epidemiological patterns of
hepatitis A infection vary in different parts of the world although the differences are
linked more to socio-economic conditions than to geographic regions. In developing
countries where sanitation and hygiene are poor, subclinical childhood infection is
common and 90% of the adult population is immune. In industrialised countries, most
people are susceptible to infection and unimmunised travellers to developing
countries thus risk contracting the infection.
1.6
Prevention and Control
As hepatitis A is transmitted via the faecal-oral route, high standards of public and
personal hygiene are still the cornerstone of control. Clean water supplies and
efficient modern methods of collection, treatment and disposal of sewage are
important. Those employed in the food industry should be required to observe high
standards of hygiene.
Passive immunisation against hepatitis A is a well established practice.
Administration of 0.02-0.06mL/kg normal immunoglobulin before exposure gives 8090% protection for a period of 4-6 months and has been used for travellers to
endemic areas.
Currently, four inactivated vaccines against HAV are internationally available. All four
vaccines are safe and effective, with long-lasting protection. All four vaccines are
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similar in terms of efficacy and side-effect profile. The vaccines are given
parenterally, as a dose plus booster series, 6-18 months apart. The dose of vaccine,
vaccination schedule, ages for which the vaccine is licensed, and whether there are
paediatric and adult formulations varies from manufacturer to manufacturer. No
vaccine is licensed for children younger than one year of age. Hepatitis A vaccines
are all highly immunogenic. Nearly 100% of adults develop protective levels of
antibody within one month after a single dose of vaccine. Although one dose of
vaccine provides at least short-term protection, the manufacturers currently
recommend two doses to ensure long-term protection. Contraindications to hepatitis
A vaccination include a known allergy to any of the vaccine components. Combined
hepatitis A/hepatitis B vaccines are also available.
Recommendations for hepatitis A vaccination in outbreak situations depend on the
epidemiology of hepatitis A in the community, and the feasibility of rapidly
implementing a widespread vaccination programme. The use of hepatitis A vaccine
to control community-wide outbreaks has been most successful in small, selfcontained communities, when vaccination is started early in the course of the
outbreak, and when high coverage of multiple-age cohorts is achieved. Vaccination
efforts should be supplemented by health education and improved sanitation12,13.
Human normal immunoglobulin should be given as well as vaccine to those over 50
years of age and to individuals with chronic liver disease14.
At present no specific antiviral therapy is available for hepatitis A.
2
Hepatitis B
2.1
Basic Virology and Taxonomy
The hepatitis B virus (HBV) is the human representative of the Hepadnaviridae
family, a group of small DNA viruses which infect a range of animal species. In many
of these animals eg the Eastern woodchuck, the Beechey ground squirrel, and the
Pekin duck, the viruses are hepatotropic15. The hepatitis B virus is an enveloped
virus with a partially double-stranded circular DNA genome of 3.2 kb; the plus strand
is incomplete prior to replication. Eight genotypes of HBV, designated A-H, are
described by sequence analysis, based on nucleotide variation of over 8% across
the genome16,17.
The genome of HBV contains four overlapping open reading frames: S, C, P, X. The
S ORF codes for surface (envelope) proteins, which can be translated from three
initiation points to give three proteins of varying size – L (large) includes all three
regions of the S ORF ie pre-S1, pre-S2, S, M (major) includes pre-S2 and S, and S
(small) includes the product of the S region of the S ORF. The surface proteins
together are designated hepatitis B surface antigen HBsAg. Four principal subtypes
of virus can be recognised by HBsAg heterogeneity in response to anti-HBs binding,
subtypes adw, ayw, adr, ayr15,18. The C gene similarly has two initiation points, the
longer comprising the products of pre-C and C (this long translational product is
cleaved to produce the soluble core component HBeAg) and the shorter the
nucleocapsid C (core) protein HBcAg. The P ORF codes for viral polymerase which
includes a reverse transcriptase function. The X gene has multiple functions but its
role is not well characterized; it may have a role in oncogenicity of HBV.
The replication cycle begins with closure of the gap in the + DNA strand and the
DNA moves to the cell nucleus in a covalently closed circular form (ccc DNA). The
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negative strand of ccc DNA is transcribed by cellular RNA polymerase II to
pregenome RNA (longer than genome length) and shorter subgenomic transcripts,
which serve as mRNA producing P and C proteins and the pre-S and S envelope
proteins respectively. The polymerase P has reverse transcriptase activity and
transcribes DNA from the RNA template in the new virions. In the nucleus ccc DNA
copies accumulate and remain19,20.
2.2
Laboratory Diagnosis
Testing for hepatitis B infection is based on detection of HBsAg in the first instance.
HBsAg will be detectable in the blood if there is current hepatitis B infection. For
many years very sensitive immunoassays have been available for this purpose
including radioimmunoassay (RIA) and a range of enzyme immunoassays (EIA).
Many tests now are done using EIA tests on automated analysers, for example
where HBsAg bound by anti-HBs antibody on microparticles is detected by a
chemiluminescent reaction21. Mutations in the ‘S’ gene can lead to reduced
sensitivity or failure to detect HBsAg, especially if monoclonal anti-HBs is used for
both capture and probe in the immunoassay22. Although the published specification
for the minimum level of sensitivity for HBsAg detection in the UK National Blood
Service is at present 0.2 IU/mL there is general acceptance that assays should
detect 0.05 IU/mL HBsAg or less23. Assays should be CE marked. Because of the
important implications of a positive finding the initial HBsAg reactivity should be
confirmed by a neutralisation test or by repeat HBsAg reactivity in a second assay,
and a second sample tested as well. HBsAg appears about 2-4 weeks before the
ALT rises. However, HBV DNA can be detected about 3-4 weeks earlier24.
Detection of HBsAg by immunoassay at a single time point does not give information
on the duration of infection. Its presence in samples 6 months or more apart defines
‘chronic’ hepatitis B infection. Presence of detectable IgM antibody to hepatitis B
core antigen (anti-HBc IgM) is used to help determine whether the HBsAg is
associated with an acute or a chronic infection. In acute hepatitis B anti-HBc IgM
appears in high levels (>50 Paul Ehrlich units/mL) in both symptomatic and
asymptomatic individuals and is commonly said to be detectable for about 3-6
months. However, this antibody however is a marker of HBV activity and its
appearance and disappearance are quite variable. In a study using RIA anti-HBc IgM
appeared in most within a week of symptoms, but was delayed to 2 weeks in about
8%25. Median duration of anti-HBc IgM was 32 weeks, with a range from two weeks
to over two years; 14% had detectable IgM for over one year25. Thus anti-HBc IgM
results need to be interpreted with caution and with the clinical and biochemical
information. Because it can be found in chronic hepatitis B with active viral
replication, quantitation of anti-HBc IgM is also important in differentiating acute and
chronic hepatitis B with high levels of anti-HBc IgM correlating with acute infection
rather than chronic infection with exacerbation of symptoms26,27. HBsAg levels tend
to be higher in acute infection, and in early infection determination of anti-HBc avidity
might also be a useful test27.
Antibody to hepatitis B core antigen (anti-HBc) was recognised as persisting after
hepatitis B infection by counterimmunoelectrophoresisand, as it persists generally for
lifeis used as a marker of infection with HBV at some time28,29. Most commercial
tests are total antibody tests detecting both IgG and IgM, most are competitive
assays. It is a useful test for validating a positive HBsAg result, and is found together
with anti-HBs antibody in past resolved infections. Isolated anti-HBc found in the
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absence of HBsAg or any other serological markers of hepatitis B infection is difficult
to interpret.
Detection of circulating HBeAg, a soluble derivative of the core ORF product, occurs
when virus is actively replicating in the liver, so it is associated with high levels of
HBV DNA in the blood and high potential infectivity. Its association with progressive
liver disease varies with the stage in the natural history, so both HBeAg and antiHBe antibody are monitored to follow the stage of infection and response to
treatment. Both HBeAg and anti-HBe antibody are detected using enzyme
immunoasays, with anti-HBe tests usually in a competitive format24. Both HBeAg and
anti-HBe may coexist30.
Anti-HBs assays use HBsAg bound to solid phase to capture the antibody.
Automated assays usually use recombinant antigen as capture antigen and for the
labelled probe24. Anti-HBs is used to monitor post-vaccination immunity, where an
initial level of 10 mIU/mL is recognised as conferring protection against HBV31. It is
also used as a marker of resolution of infection (absent HBsAg, positive anti-HBc)
but HBsAg and anti-HBs can coexist, particularly where the subtype specificities are
different, so the presence of anti-HBs cannot exclude chronic hepatitis B
infection30,32.
Hepatitis B DNA is measured by PCR (both in house and commercial assays are
available) or by other amplification assays such as branched chain DNA. Assays
may not be directly comparable and vary in performance and dynamic range.
Detection of HBV DNA is useful in early diagnosis in at risk individuals before HBsAg
appears, and for monitoring viral load during therapy; the best endpoint for
management is to reach a level of HBV DNA which is undetectable by current
methods with a sensitivity of 10-15 IU/mL33,34. HBV DNA is also a significant
prognostic marker for cirrhosis. In the UK, health care workers (HCW) who are
HBsAg positive and HBeAg negative must be tested for HBV DNA level before doing
blood exposure prone procedures. A level below 103 genome equivalents/mL is
acceptable but must be rechecked yearly35. If viral load is between 103 to 105
geq/mL, the HCW may work while taking antiviral therapy provided the HBV DNA
level is less than 103 geq/mL on treatment and HBV DNA monitoring is done at 3monthly intervals36.
Assays for detecting ccc DNA in blood have been developed but as yet their clinical
relevance is uncertain37.
2.3
Transmission
HBV is transmitted by blood and blood products and by other secretions. Blood
represents a particular risk, as there may be over 108 virions/mL, which can be
directly visualised by electron microscopy38. In the past, blood transfusion was an
important route of transmission, with 6% of multiply transfused individuals acquiring
hepatitis B infection prior to the reduction in risk resulting from the introduction of
screening for HBV in the early 1970s39. Screening for HBsAg alone does not entirely
eliminate HBV transmission since there are occasional blood donations where the
donor has early hepatitis B before appearance of HBsAg. In an experimental animal
model, blood taken early in infection prior to appearance of anti-HBc is significantly
more infectious than that taken later in the acute phase 40. Infectivity persists
throughout the course of active infection. Transmission from a surgeon with chronic
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HBV to a patient in the UK has been associated with levels of circulating HBV DNA
as low as 4 x 104 copies/mL40.
Those with chronic hepatitis B with undetectable HBsAg, so called ‘occult’ hepatitis
B, are generally positive for anti-HBc antibody and sometimes anti-HBs, but with low
levels of HBV DNA consistent with ongoing viral replication40. Measures to exclude
such individuals from the blood donor pool require use of anti-HBc testing and HBV
DNA testing39.
Vertical transmission from mother to baby of HBV is one of the most important routes
of HBV transmission worldwide. Most transmission occurs in the intrapartum period,
with exposure of the baby to maternal blood and genital secretions. Less than 3% of
cases of neonatal infection arose from transmission in utero in a Taiwanese study39.
Although possible micro-leakage of blood across the placenta in threatened abortion
or premature labour might be a factorthis was not noted in other studies 41,42. The
principal risk of vertical transmission is from HBeAg positive mothers from whom
transmission to the neonate occurs in about 85% of cases, compared to 31% from
HBeAg negative women43.
There is an increased risk of transmission to the neonate in acute hepatitis B during
pregnancy, particularly with hepatitis B acquired later in pregnancy44.
Breast milk contains HBV in some 70% of mothers with circulating HBsAgand can
presumably transmit infection to the neonate45. In general the risk from this route is
relatively unimportant as the risk to the baby during the birth process is greater, and
prophylaxis is given to the at risk baby.
In the developed countries, hepatitis B is principally associated with sexual
intercourse, both heterosexual and between men, and injecting drug use (IDU).
Incidence of acute hepatitis B in IDU may have fallen in parts of the UK where active
vaccination programmes have been pursued but overall in the UK this is not the
case, as vaccination uptake among IDU varies widely46. In 2003, drug use was the
commonest risk factor and an increase in seroprevalence to anti-HBc from 3.4% in
1997 to 10% in 2006 suggests that this is a continuing risk47. In the North West of
England, for example, the most likely transmission route was not known in two thirds
of cases; of clear risk factors homosexual sex was the most common (17%),
followed by heterosexual exposure (9%)46. The majority of cases are in males,
predominantly in the 25 to 45 year age group46,48.
2.4
Clinical Features
The incubation period for hepatitis B infection is between 2 and 6 months, with
higher viral inocula associated with shorter incubation periods.
Symptoms of infection vary with the age at which the individual is infected. Acute
infection is usually asymptomatic in the infected neonate and young child, but even
in infections acquired in adult life, asymptomatic infection is common (around 50%).
If symptoms occur, the patient may have jaundice, feel nauseated and lethargic, and
may have right upper quadrant discomfort or pain. Jaundice is seldom marked but
there may be lightening of faeces colour and darkening of urine, and sometimes
pruritus towards the end of the symptomatic illness.
Acute hepatitis B is preceded by a serum sickness-like prodrome with fever,
arthralgia or arthritis, and rash, in up to 10% of cases. These symptoms occur about
2 weeks prior to the onset of jaundice, and usually subside soon after jaundice
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appears. Another characteristic rash, papular acrodermatitis (Gianotti-Crosti
syndrome), is seen in childhood in association with hepatitis B; maculopapular
erythematous nonpruritic lesions occur on the face and extremities, and persist for
15-20 days19.
Also included among the extrahepatic manifestations of hepatitis B is membranous
glomerulonephritis49. Although more common in children it is more likely (30-60%) to
spontaneously resolve in childhood, whereas one third of cases in adults progress to
renal failure. Resolution is generally associated with HBeAg clearance 49.
Fulminant hepatitis occurs in less than 0.5% of acute cases but is a severe lifethreatening condition. The proportion of cases of acute liver failure due to hepatitis B
has been declining in recent years and it now causes less than 10% of cases50. It is
important to note that HBsAg may be weak or undetectable in some cases of
fulminant infection, but that anti-HBc IgM and HBeAg are generally found51. This is
usually due to an enhanced immune response, but a proportion may be due to
failure of HBsAg assays to detect ‘S’ mutant virus52.
Chronic hepatitis B is defined (arbitrarily) as persistence of detectable HBsAg for
over 6 months. This generally correlates with the persistence of raised liver
transaminases for over 6 months, consistent with ongoing viral replication and liver
damage. The course of chronic infection can be divided into several phases,
reflecting the dynamics of virus replication and host immune response. These are
not necessarily sequential. The phases are: 1. immune tolerance; 2. immune
elimination (immune reactive or immune clearance); 3. low replication (inactive); 4.
HBeAg-negative chronic hepatitis B (reactivation)33,53. In the phase of immune
tolerance, high levels of HBV DNA and HBeAg are seen in the blood but there is little
or no liver damage. The ALT remains normal and there is little or no inflammation
seen on liver biopsy. This phase is characteristic of hepatitis B acquired in the
perinatal period, generally by vertical transmission, and it may last 10-30 years54.
After some time immune tolerance is lost and an immune response occurs against
hepatitis B infected hepatocytes. This immune elimination (immune clearance or
immune reactive) phase, which can last weeks or years, is characterised by falling
HBV DNA levels, raised or fluctuating ALT, and histological evidence of
necroinflammation with progression of fibrosis54. There is increased loss of HBeAg
with seroconversion to anti-HBe antibody positivity; such seroconversion is often
accompanied by a symptomatic exacerbation of hepatitis.
The low replication inactive carrier state may follow immune clearance and
seroconversion to anti-HBe positivity. Liver enzymes – ALT and AST – are normal
and HBV DNA level is low or undetectable. Prognosis is favourable with low rate of
progression to cirrhosis and HCC. Loss of HBsAg occurs in about 1-3% of chronic
carriers per year.
HBeAg negative chronic hepatitis B occurs in patients in whom mutations appear in
the pre-C or basal core promoter regions giving rise to virus that cannot express
HBeAg. The most common mutation is the A1896G pre-C mutation55. This cannot
occur in genotype A viruses, although basal core promoter mutations (notably the
double mutation A1762T plus G1764A) are seen as in other genotypes 56. As these
mutations are selected by anti-HBe antibody, the typical serological pattern is HBsAg
positive, anti-HBc positive, HBeAg negative, anti-HBe positive. During this phase
reactivation can occur, giving fluctuating liver enzyme levels and HBV DNA levels, so
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monitoring regularly throughout this period is essential in differentiating individuals
with active disease with risk of progression to cirrhosis and those with inactive antiHBe positive disease.
In what is termed by some a fifth phase, the state after loss of HBsAg, prognosis is
generally good. However, low level HBV replication can persist in the liver despite
undetectable HBV DNA in the blood. These individuals are HBsAg negative, are
positive for anti-HBc and may or may not have anti-HBs antibody. Reactivation of
infection may occur if the patient is immunosuppressed, particularly if monoclonal
antibodies targeting the immune system eg rituximab are given57,58.
Cirrhosis is the result of progressive liver damage, a liver with abnormal architecture
and loss of function, characterised by proliferation of fibrous tissue and scarring, with
nodular regeneration of hepatocytes. Principal causes include hepatitis B, hepatitis
C, and alcohol. Some 15-40% of patients with chronic hepatitis B develop liver
damage16.
A sustained high HBV DNA viral load seems to be a major predictor of progression
to cirrhosis and hepatocellular carcinoma59-61.
Hepatocellular carcinoma (HCC) may arise after many years of chronic infection with
hepatitis B. In a large Taiwanese study HCC developed at a rate of 1169 per 100000
person years for those who were HBeAg positive and at 324 cases for those with
HBeAg negative HBV. The pathogenesis of HCC is not entirely clear, but the long
period required for its development suggests that several low probability events must
occur. Continued proliferation of hepatocytes in response to loss of infected cells
may be one factor, and it is likely that the X protein of HBV has oncogenic
properties19,62. Risk of HCC increases with higher HBV viral load, especially for viral
load above 20000 IU/mL60. Risk may vary among hepatitis B genotypes. Cofactors
are also important in development of HCC in chronic hepatitis B. These include
aflatoxins, alcohol and coinfection with hepatitis C63.
2.5
Epidemiology
The prevalence of hepatitis B varies markedly around the world, reflecting to a large
extent differing modes of transmission. Rates of seroprevalence for anti-HBc
antibody, a marker of infection at any time, vary for instance from 1-2% in the UK,
0.5-1% in the Netherlands, to 96% in China and South Korea, while Ag positive rates
can reach over 5-10% in some regions, particularly in China and South East Asia,
Africa and Oceania64. In the Far East, transmission occurs in the neonatal or
perinatal period, whilst in African there is a more complex pattern involving perinatal
transmission, and horizontal transmission in early childhood through close contact
with abraded skin and during tribal rituals65. Infection around the time of birth is
highly efficient (around 90% transmission rate from an HBeAg positive mother to the
infant and is likely to result in chronic infection in the child66. Transmission in utero is
relatively uncommon (less than 5% of mother/infant transmissions)66,67.
Genotype distribution varies geographically; genotype A predominates in Northern
Europe and North America, B in the Far East, C in West and Central Africa, D
especially in North Africa, the Mediterranean littoral, the Middle East, and Asia, F in
indigenous peoples of North and South America, G in France and the USA (rarely
found and then usually in association with genotype A virus), H in Central America17.
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2.6
Prevention and Control
Hepatitis B is notifiable in the UK; both acute cases and chronic cases should be
notified, so that risk factors can be determined and screening and prophylaxis may
be offered where appropriate14.
2.6.1 Vaccination
Spread of hepatitis B infection can be controlled by vaccination. Given the global
importance of hepatitis B infection, most countries have opted for universal
vaccination against this virus68,69. Countries with very low overall prevalence such as
the UK have preferred targeted vaccination of high risk groups.
A number of commercial vaccine preparations are available, generally containing a
recombinant yeast-derived HBs protein segment which includes the major
immunodominant ‘a’ epitope. The failure of currently licensed vaccines to include
pre-S may be one factor in suboptimal responses but other important factors include
increasing age, injection into fat, smoking, alcohol use, renal impairment and HIV
infection70-72.
Hepatitis B Immunoglobulin (HBIG) provides passive immunity for post exposure
prophylaxis for known high risk exposures. It is used as an adjunct to vaccination for
example in non immune adults, or for individuals at risk who have failed to mount a
satisfactory immune response after a vaccine course. HBIG should ideally be given
intramuscularly within 48 hours of exposure but this can be extended to a week
particularly after sexual exposure73.
For babies born to mothers who are HBeAg positive, HBeAg negative and anti-HBe
negative, or anti-HBe positive with HBV DNA >106 IU/mL HBIG is offered together
with vaccine in the UK; in this situation HBIG should be given within 24 hours of
delivery at a site different from that used for the first dose of vaccine73,74. HBIG is
also used for prevention of recurrence of hepatitis B after liver transplantation for
chronic hepatitis B infection, together with antiviral agents such as lamivudine or
adefovir33.
2.6.2 Treatment
Treatment is considered for severe acute hepatitis B and for chronic infection. A
detailed description of treatment regimens and monitoring response is outside the
remit of this document. There are a number of current published guidelines which
should be consulted on this subject. They include recommendations on specific
antivirals from the National Institute for Health and Clinical Excellence (NICE), and
comprehensive guidelines on management from bodies including the European
Association for the Study of the Liver, the Asian-Pacific Association for the Study of
the Liver, and the American Association for the Study of Liver Diseases 33,65,75.
3
Hepatitis C
3.1
Basic Virology and Taxonomy
Hepatitis C virus (HCV) is a blood-borne virus of the Flaviviridae family and the only
member of the hepacivirus genus76,77. It is a single-stranded, positive sense
enveloped RNA virus with a genome of approximately 9600 bases76. The virus was
discovered in 1989 and subsequently the genome was patented by the Chiron
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Corporation, the first virus to have ever been patented. Prior to this discovery, a
class of blood-borne hepatitis transmissible by blood known to be negative for
hepatitis A and B and known as non-A, non-B hepatitis (NANBH), was recognised to
cause hepatitis after blood transfusion and use of blood products78.
Only a small percentage of HCV early infections cause the patient to seek medical
intervention79. It is estimated that in 50-85% of all cases the virus is not cleared,
leading to chronic infection, even though there is evidence of seroconversion 78,79. In
the case of HCV, the relationship between neutralising antibodies and the control of
viraemia is more complex due to the enormous genetic variability of the virus (much
greater than the variability seen with HIV), particularly in the E2 envelope
glycoprotein region where many antibodies are targeted79. This variability leads to
HCV being regarded as existing in an individual as a ‘quasispecies’, as essentially
HCV is likened to a horde of closely-related variants circulating in the one infected
host76. The window between detection of HCV RNA and antibodies to HCV can be
months, with an average of 60 days80. There are six genotypes of HCV (1-6) with
multiple subtypes, although a new genotype has been proposed as genotype 7 81,82.
Transmission of hepatitis C virus is through contact with blood or blood derived
products. Therefore, the main risk factors are contaminated blood supply, injecting
drug use, unsafe therapeutic needle use, or medical procedures83. There is a clear
divide between the modes of transmission depending on whether in the developed or
developing world83. In the developed world, the primary mode of transmission is
through injecting drug use, particularly since blood and blood derived products are
so thoroughly screened for HCV. In developing countries, where supplies of sterile
syringes are short or non-existent, unsafe therapeutic needle use is much higher
than in the developed world, where a person may receive multiple injections adding
a cumulative risk of acquiring HCV83. An example of this is seen in countries such as
Egypt, where prevalence of HCV is extremely high in the older population, probably
due to shared syringes during nationwide schistosomiasis treatment policies that
date back to the 1920’s84. Blood transfusion related HCV incidence in Egypt has
become almost non-existent since the introduction of all-volunteer donor systems,
screening of blood using highly sensitive and specific assays for anti-HCV and HCV
RNA, and detailed screening of donors for HIV risk factors83. In fact, the safety of
donated blood in the developed world is at a level where methods to measure risk
are not sensitive enough to be useful in the estimation of transfusion-related
transmission of HCV85. Most countries in the developing world, however, do not
screen blood donors for HCV83. Other sources are occupational, perinatal and sexual
exposures, but these occur very infrequently. Prevalence of HCV infection is higher
in persons having received blood transfusions, tissue/organ transplants or blood
products such as haemophiliacs, prior to 1992 before sensitive screening tests were
available86.
3.2
Laboratory Diagnosis
Laboratory diagnosis is based upon the detection of antibodies for the virus using
serological methods followed by the detection of HCV RNA using molecular methods
to determine viraemia76,86. Antibodies to HCV are detected using EIA. Assays have
progressed in development to second generation (core proteins and non-structural
proteins 3 and 4) and third generation (inclusion of antigen non-structural protein
5)81. Assays are now available that will detect free HCV core antigen in
serum/plasma and display similar sensitivity to some molecular methods 87.
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The presence of HCV in blood is a good marker of replicating virus80. Detection is
possible 1-3 weeks after infection with the use of molecular methods to detect HCV
RNA. Molecular methods can also be used to distinguish between the genotypes
and subtypes, of particular importance as some genotypes are more prevalent than
others in different global regions81. Knowledge of the genotype is crucial in the
treatment selection process81.
Real-time PCR provides an excellent quantitative range which allows determination
of viral load up to 7-8 Log10 IU/mL and as low as 10-15 IU/mL to assess treatment
efficacy80. Qualitative PCR is not often used even though sensitivity is often
considered superior to quantitative real-time PCR; there may be a role for a
qualitative assay in cases where extremely low levels of viraemia are suspected 86.
The presence of RNA and antibodies to HCV do not give an indicator as to whether it
is acute or chronic infection80. However, antibodies do take a long time to develop
and are therefore only detectable in 50-70% of symptomatic acute infections88.
3.3
Clinical Features
HCV is often asymptomatic (85-90% of cases) and therefore is rarely diagnosed
during the acute phase of infection81,88. Symptoms may include jaundice, nausea
and malaise; however, although fulminant hepatitis is extremely rare, it has been
described during the first 8 weeks of infection81. A majority of acute cases go on to
become chronic and, without treatment, the majority of chronic cases do not clear
the virus81. HCV RNA detectable for longer than 6 months is considered chronic
infection with sequelae including end-stage cirrhosis and hepatocellular carcinoma
(HCC), although the length of time this may take can vary from less than 20 years to
greater than 30 years81. Co-infection with HIV displays accelerated disease
progression as HCV behaves as an opportunistic infection83. Co-infection with
hepatitis B virus displays more severe liver disease progression than either single
infection83. Increased alcohol intake also accelerates the progression of chronic HCV
towards end-stage cirrhosis and HCC83. Transplantation is often necessary due to
HCV infection related liver failure, leading to further complications with progression
towards cirrhosis in over 25% of patients within 5 years of transplantation89.
3.4
Epidemiology
HCV is a leading cause of chronic liver disease and a serious public health problem
with high global prevalence estimates at around 2-3%, or around 120-180 million
people90,91. Approximately 1.5 million people die of viral hepatitis related chronic liver
disease80. This includes end-stage cirrhosis and HCC.
In the UK, data shows that HCV remains common in the injecting drug use
population, with 10% and 70% antibody positivity prevalence for those injecting for 2
years and 15 years respectively92.
There is an estimated 10 million people worldwide believed to be co-infected with
HIV, possibly linked to high-risk traumatic sexual practices and drug use in men who
have sex with men (MSM)93.
3.5
Prevention and Control
Knowledge of the genotype can be critical in the treatment schedule chosen, as well
as the rate of success of treatment81. Treatment involves use of interferon usually in
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combination of ribavirin88. Of the six major genotypes described genotypes 2 and 3
respond more favourably to treatment with interferon and ribavirin than genotype 1.
The recommended length of treatment in the UK is 24 weeks for genotypes 2 and 3,
and 48 weeks treatment for genotype 1 and remaining genotypes. Pegylated
interferon is often used to increase the time it takes to clear the drug and therefore
reduce the number of times interferon needs to be taken to once per week78.
Ribavirin is required to be taken daily88. Infection is considered eradicated when
there is no HCV RNA detected for more than 6 months after cessation of therapy
(also known as a sustained virological response)86,91,94. All the major genotypes have
multiple subtypes but there is little evidence of differences in response among these.
Other antivirals specific for hepatitis C are being trialled. The protease inhibitors
telaprevir and boceprevir (which inhibit the NS3/4A protease) appear to be very
active. As resistance develops readily with monotherapy, telaprevir must be
combined with ribavirin and interferon95. Vaccines that induce protection via the
production of neutralising antibodies have two hurdles to overcome – heterogeneity
(the various genotypes) and mutability (the rapid generation of antigenically diverse
E2 envelope glycoproteins) of HCV79. There are a host of vaccine development
initiatives that aim to prevent initial infection, viral persistence or clear the virus
entirely.
4
Hepatitis D96,97
4.1
Basic Virology and Taxonomy
HDV is closely associated with HBV because it is a defective virus that requires a
helper function provided by HBV. It is unique among animal viruses and has
similarities with both viroids and virusoids of plants in its structure and mode of
replication. It is classified in the floating genus Deltavirus. The virus is an enveloped,
spherical particle with an average diameter of 36 nm. The outer capsid is HBsAg,
and the interior nucleocapsid is the genome complexed with hepatitis delta antigen
(HDAg) which is the product of the sole structural gene. The HBsAg is responsible
for attachment to the host cell and the HDAg for nuclear localisation. The genome is
single stranded, circular negative-sense RNA of 1679 nucleotides. The genome has
extensive secondary structure due to intramolecular base pairing. The HDV genome
can function as a ribozyme, capable of self-cleavage. This is a necessary part of the
complex rolling circle mechanism of replication. The virus exists as various
genotypes, originally three major genotypes and recently seven genotypes are
described. Within an infected patient the virus is a mixture of different related species
known as quasispecies.
4.2
Laboratory Diagnosis
The most sensitive method of laboratory confirmation is detection of HDV RNA by
PCR using primers from the most conserved sequences. HDAg and HDV antibodies
(IgG or IgM) in the serum can be detected by EIA. High titres of HDV IgG are found
during chronic infection, and IgM may also persist; but EIA is less sensitive than
PCR in the acute phase of infection. Quantitation of the viral load has been used in
studies of antiviral therapy. Simultaneous infection with HBV and HDV can be
differentiated from super-infection of a chronically HBV infected patient by the
appearance of anti-HBV core IgM with anti-HDV IgM, or by the appearance of HDV
RNA without detectable HDV antibody. Immunohistochemical staining for HDAg is
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still used, and this is the technique that was responsible for the discovery of hepatitis
D in the 1970s.
4.3
Clinical Symptoms
HDV may be transmitted with HBV to a susceptible person, this is known as coinfection. Super-infection is infection of an individual already chronically infected with
HBV. Co-infection may result in an acute HBV / HDV infection that is mild to severe.
Most cases are clinically indistinguishable from acute B virus infection. However,
acute HDV / HBV co-infections have a much greater risk of evolving into acute liver
failure than acute HBV infection.
After super-infection, chronic co-infection causes a rapidly progressive chronic liver
disease in 90% of cases with spontaneous resolution being rare. Thus superinfection has a poorer prognosis than chronic HBV infection alone, with higher rates
of cirrhosis, hepatocellular carcinoma and death. It may present as an exacerbation
of an already diagnosed HBV disease or lead to the presentation and diagnosis of a
previously asymptomatic and undetected HBV infection. The latter situation may be
initially considered a case of acute hepatitis B until negative anti-HBc IgM test results
and positive HDV test results indicate the true picture. Most patients with chronic
HDV infection have antibodies to HBeAg and low levels of HBV DNA. Different from
HDV negative chronic hepatitis B, there is no correlation between serum HBV DNA
and ALT levels in patients with chronic hepatitis D, suggesting that the liver damage
in these patients is mainly caused by HDV.
High doses of interferon have been reported in a randomised clinical trial to improve
survival, reduce fibrosis and reduce viral replication98. Nucleoside analogues given
without interferon have so far been ineffective. HDV genotype I has been
independently associated with a poorer prognosis99.
4.4
Epidemiology
It is reported than 15 to 23 million of the estimated 460 million people chronically
infected with HBV are also infected chronically with HDV, that is up to 5% 100. The
prevalence of HDV varies widely geographically, and does not always mirror the
prevalence of HBV. The literature suggests a declining trend at least in southern
Europe. Increasing use of universal hepatitis B vaccination will be expected to
reduce the prevalence of HDV also. HDV can be spread by blood contact in injecting
drug users, though blood product screening for HBV has reduced the iatrogenic
route. Molecular epidemiology has confirmed sexual transmission and an inapparent
parenteral route is suggested to be important in families in endemic areas. Foci of
infection are identified in southern Europe (including Italy and Albania), the Middle
East, Saudi Arabia, northern India, Russia, West Africa, Japan, Taiwan, some South
Pacific islands and South America.
4.5
Prevention and Control
There is currently no vaccine against HDV itself, but vaccination against HBV also
protects against HDV.
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5
Hepatitis E
Hepatitis E infection is increasingly recognised in the UK, including a high proportion
of cases without the traditional risk factor of travel abroad to a high incidence area. It
is important to consider hepatitis E as a potential cause of viral hepatitis early on in
the assessment of the patient.
5.1
Basic Virology and Taxonomy
Following the development of sensitive and specific tests for acute hepatitis A and B
infections, it became clear from clinical, epidemiological, and laboratory data that
another enterically transmitted viral hepatitis agent existed101. This agent was
descriptively called epidemic non-A non-B hepatitis virus. Characterisation of this
new virus began in 1983 when Dr Balayan infected himself using stool associated
with non-A non-B hepatitis from an outbreak in the former Soviet Union 102. He
subsequently developed hepatitis and analysed his own samples to visualise the
virus by immune electron microscopy, and to reproduce hepatitis in Macaca
fasicularis. Cloning and sequencing of the virus was reported in 1990 and
1991respectively, and it was subsequently named hepatitis E virus103,104.
Hepatitis E virus is classified as the sole member in the family Hepeviridae, genus
Hepevirus105. A typical virion is non-enveloped, 32-34nm diameter containing a
positive sense single stranded RNA genome of around 7.2Kb. The genome has
three open reading frames encoding non-structural proteins (ORF1), the capsid
(ORF2), and a small protein of unclear function (ORF3).
There are four main genotypes (G1-4), G1 and 2 are likely to be exclusive human
pathogens, whereas G3 and 4 are probably zoonotic with swine as the main host. An
avian HEV is proposed as a fifth genotype106. G1-4 show geographical variation in
prevalence.
5.2
Laboratory Diagnosis
The clinical presentation of acute symptomatic hepatitis E is difficult to distinguish
from that of any other viral hepatitis. Whilst epidemiological features may suggest
HEV infection, laboratory tests should be performed to confirm any clinical diagnosis.
Virus can be detected in stool by immune electron microscopy, and viral nucleic acid
can be detected in stool and blood by RT-PCR. Some cell lines support HEV
replication but virus isolation is not used as a diagnostic technique. Non-human
primates are susceptible to HEV and have been used in research only; no animal
model shows the spectrum of HEV infection seen in humans. A number of antibody
detection formats have been developed but the commercially available systems are
solid phase EIA (IgG and IgM). These assays are usually based upon antigens
derived from G1 and 2 viruses and although there is probably a single HEV serotype
for human infection, they may be suboptimal for G3 virus endemic in the UK.
In common with many of the other viral hepatitides, the maximal liver injury appears
to coincide with development of the immune response, leading to a typical picture of
the relationship between the timings of virus detection, liver injury, and antibody
detection. Available data is potentially complicated by variable assay performances,
in general and via genotype specific differences.
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HEV is detectable in the stool from around 1 week before, and up to three weeks
after, symptoms appear. There are reports of more prolonged faecal shedding of
virus107,108. Viraemia detectable by RT-PCR probably mirrors the period of faecal
shedding, but may persist for much longer107,108.
IgM antibody becomes detectable just prior to the maximal liver injury, potentially
coinciding with the onset of symptoms, and remains at detectable levels for several
months. Some patients do not mount a detectable IgM response. There is a
spectrum of IgM persistence but over 50% of patients will be negative six months
after onset.
IgG antibody appears shortly after IgM (when present), persisting for several years in
the majority.
Laboratory diagnostic criteria can be drawn up to account for the variability in natural
immune responses and assay performance, particularly in the setting of low
endemicity. An acute case (symptomatic presentation) is best defined by being HEV
RNA positive or by showing IgG seroconversion. Other combinations of IgG and IgM
results may be best interpreted according to antibody titre/ reactivity levels. IgG
avidity may be useful in inconclusive cases109.
The histopathological appearance of acute infection occurring in hyperendemic
regions is of focal hepatic necrosis, ballooned hepatocytes and a lymphocytic
inflammatory infiltrate110. A cholestatic picture may occur but it is uncommon. The
histopathological appearance of acute infection acquired in the UK may be different.
5.3
Clinical Features
There is a spectrum of outcomes after HEV infection, ranging from asymptomaticand
also implied by seroprevalence data to acute liver failure111. Although it is not
possible to predict the course of the infection using genotype data or host features, a
striking feature of hepatitis E is the enhanced severity in late pregnancy.
HEV infection in the immunocompetent is believed to be self-limiting, without chronic
infection. Several reports have been published describing likely chronic hepatitis E in
immunocompromised organ transplant recipients112-114. The extent and clinical
relevance of this condition are yet to be fully defined.
Data from volunteer studies, case reports, and outbreaks have defined the typical
features of symptomatic HEV infection occurring in hyperendemic areas115,116.
Although these features can be applied to hepatitis E indigenous to developed
countries, there may not be complete concordance.
Incubation period: 15 to 60 days
The spectrum of illness is wide. Typically a self-limiting viral hepatitis occurs with
recovery after 2-3 weeks. Asymptomatic and fulminant hepatitis are both described.
Rarely there is a prolonged illness lasting a few months, or a cholestatic picture. A
prodrome or pre-icteric phase may occur lasting 3-4 days on average (range 1-10)
characterised by epigastric pain, nausea, malaise, mild pyrexia (<38°C). The icteric
phase starts abruptly with jaundice, pale stools and dark urine, often accompanied
by fever and arthralgia.
The overall mortality rate for HEV is 1-3% but in pregnancy this increases to
5–25%. Pregnant women appear to be more frequently affected by hepatitis E during
an outbreak, and the disease is often more severe, especially in late
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pregnancy117,118. Vertical transmission may be relatively common and associated
with poor fetal outcome117,119. The reasons for this excess mortality have not been
clearly defined, complicated by the lack of an appropriate animal model.
Co-infections with hepatitis A and superinfection on a background of chronic viral
hepatitis (HBV and HCV) are likely to be common in areas of endemicity and they
may not run a typical course or may lead to hepatic decompensation 120. However, it
is unclear whether the severity is always increased.
The clinical management of hepatitis E is based upon assessment of severity of liver
damage and associated effects, influencing the degree of supportive measures
needed. No specific antiviral therapy is available. Liver transplant is an option for
acute liver failure. Acute hepatitis E is a notifiable disease14.
5.4
Epidemiology
HEV causes epidemics and sporadic cases, and is responsible for the majority of
acute clinically apparent cases of hepatitis in central and south-east Asia.
Transmission is faecal-oral, often by contaminated water, and potentially via food.
Epidemics associated with poor water quality have occurred in many areas of the
world (Pakistan, India, South-East Asia, Nepal, Africa). The largest epidemic
described occurred in China, with over 119,000 cases of jaundice121. No outbreaks
of hepatitis E have been described in the USA, Canada, Europe, or developed areas
of Asia. Sporadic cases of infection occur worldwide, often as the result of travel to
an endemic area but increasingly recognised as indigenous. Some countries, such
as Egypt appear to have a high number of sporadic cases yet have never reported
an epidemic122. Estimates implicate HEV as the cause of around 2 million cases of
hepatitis a year in India, where it is a common cause of jaundice123. Blood-borne
transmission is described, reflecting the viraemic phase of the illness, however, this
is thought to be an uncommon event124. Seroprevalence studies investigating the
potential for bloodborne transmission involving injecting drug users, haemodialysis
patients, and patients infected with HBV or HCV have been inconclusive. There is no
convincing evidence for sexual transmission.
In broad terms, genotype 1 is the predominant type found throughout Asia and North
Africa. Genotype 2 is represented by the prototype strain from an outbreak in
Mexico, with variants detected in parts of Africa. Genotype 3 has been recovered
from humans in North and South America, Japan and many countries in Europe.
Genotype 4 has been recovered from humans in China, Japan and Taiwan. The
prevalent genotype impacts on the epidemiology (age at infection), influenced by the
likely source of infection (human or zoonotic)125. Genotypes 3 and 4 have been
recovered from swine throughout the world.
Waterborne epidemics in developing countries due to genotypes 1 and 2 exhibit an
attack rate of 1-15%, with a typical age related bias for clinically apparent hepatitis
towards older children and young adults (15-40 years). Sporadic cases caused by
genotypes 3 and 4 in developed countries affect an older age group on average.
Given that the person to person transmission rate is low, rarely above 1-2%, it is
likely that these G3 and G4 cases are acquired from an animal reservoir126. Indeed,
there is increasing evidence for HEV as a zoonosis in developed parts of the
world127. HEV is considered to be enzootic in swine, and anti-HEV can be found in
many other species, including chickens, cats, dogs, goats, sheep, and rodents.
Experimental transmission studies have shown G1 and G2 can infect some nonClinical Guidance | G 5 | Issue no: 1.2 | Issue date: 03.03.14 | Page: 27 of 44
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human primates but not other animals, whereas G3 viruses from humans or swine
can infect non-human primates and other animals128,129.
Some studies show a significantly higher anti-HEV prevalence amongst pig handlers
than in age matched controls from the same areas130. Nucleotide sequencing of
isolates from humans and swine from Taiwan, Japan and the USA show a high
degree of similarity108,131,132. HEV appears to be endemic in other animals that may
act as a reservoir (rats, cows, sheep, goats, monkeys, dogs), but there is as yet no
clear evidence implicating them as a zoonotic source. Recent reports suggest food
as a vector for transmission in some countries: shellfish, pig liver,venison, boar133-137.
The reasons for these geographically linked differences are unclear, potentially
reflecting variable viral virulence, host susceptibility to infection or propensity to
develop disease.
HEV IgG seroprevalence studies have shown the different clinico-epidemiological
patterns mentioned previously, and suggest exposure to HEV may be more
widespread than implied by clinical disease rates. There appears to be a background
prevalence of seropositivity for HEV IgG worldwide, although the patterns in
epidemic regions do not fit those expected for a waterborne agent, with rates in India
of around 10% in children, reaching a maximum of around 40% in adulthood 138. The
background rate of anti-HEV IgG in European blood donors is often reported to be
between 1 and 9%, although there are reports of rates reaching around 16%139-141. In
the USA, rates of between 2% and 26% have been found129. Interpretation of such
seroprevalence data should be made taking into account variable assay
performance, the use or not of confirmatory assays, the validity of low antibody
indices for indicating true past infection, and the potential for pockets of high
prevalence within countries.
Data from the UK has been limited but does indicate a pattern of HEV disease
consistent with that of the USA and most of Europe, with HEV endemic in the pig
population. Reports of HEV hepatitis acquired in the UK have been available for
many years142. The epidemiological profile of HEV acquired in England and Wales
has been recently summarised143. This work showed that the typical patient was
caucasian male and over 55 years of age, but a common source of infection could
not be identified. Further work is required to define whether those patients presenting
with a clinical illness subsequently identified as hepatitis E have been more at risk of
infection or are simply more likely to be symptomatic.
5.5
Prevention and Control
In regions where outbreaks occur, prevention strategies should focus upon
improving the quality of the water supply and sewage disposal, identification of
infectious sources, and vaccine development. In countries such as the UK where no
common source of infection is known, improved case ascertainment and targeted
vaccination may be more appropriate.
Travellers to hyperendemic regions should be advised to take precautions against
enteric infections, including HEV. Boiling drinking water is likely to inactivate HEV.
Immune serum globulin has not been shown to protect against infection in endemic
areas, possibly due to the relatively low titre of HEV IgG in such preparations.
The results from trials of active immunisation are promising. A recombinant vaccine
comprising a truncated capsid protein expressed in insect cells was evaluated by a
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randomised controlled trial including 1794 subjects based in Nepal144. The vaccine
was found to be safe and efficacious, with an intention to treat efficacy of 88.5%.
Further studies are required to define the value of such a vaccine in alternative
settings.
6
Hepatitis G
Hepatitis G virus is a blood-borne human virus of the Flaviviridae family. The virus
was first discovered in 1995, the same year GB virus C was discovered 145. Both
GBV-C and hepatitis G virus were showed to be the same virus and the two have
since been known as GBV-C. There is no supporting evidence that GBV-C causes
chronic hepatitis possibly due to poor replication in hepatocytes (replication appears
to occur in lymphocytes). There is, however, strong evidence to suggest that
persistent GBV-C co-infection with HIV reduces disease progression of HIV disease
thus reducing the hazard for mortality due to HIV infection145.
7
Cytomegalovirus (CMV)
7.1
Basic Virology and Taxonomy146
Cytomegalovirus (CMV) was first thought of solely as a human salivary gland virus
until 1956, when it was isolated from the adenoids of children undergoing
tonsillectomies and adenoidectomies. The virus itself belongs to the family
Herpesviridae and is a β herpes virus. As with all herpes viruses, it has the ability to
establish latency in the host after recovery. The CMV viral particle is about 102nm in
diameter and has a lipid envelope that is studded with glycoproteins, surrounding a
matrix of phosphoproteins, an icosahedral nucleocapsid and a DNA genome with a
molecular mass of 155kDa146.
7.2
Laboratory Diagnosis
The diagnosis of CMV hepatitis is made from a combination of excluding other viral
causes of hepatitis, such as HBV, HAV and EBV and the demonstration of IgM
antibody to CMV or sero-conversion for CMV. The presence of IgM however may be
due not only to primary infection but also to reinfection or reactivation of latent virus,
as well as the possibility of heterotypic responses and false IgM reactivity. A new
CMV infection will give rise to a four fold increase in complement fixing antibodies or
a single high titre; importantly in the three months after a primary infection the CMV
IgG will have low avidity147. The role of a molecular assay is unclear at the present
time for diagnosis, but the presence of CMV DNA in blood is consistent with an
active CMV infection, which may need to be monitored especially in the
immunocompromised.
7.3
Clinical Features
Symptomatic primary CMV infection is uncommon; it can be associated with hepatitis
in immunocompetent adults, usually as part of a heterophile antibody-negative
mononucleosis syndrome. This is usually accompanied by hepatosplenomegaly,
minor elevations of serum aminotransferases and aytypical lymphocytes. It may
rarely be associated with a pure hepatitis. CMV hepatitis in this context is self limiting
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and treatment is supportive. There is no role for antiviral agents. CMV hepatitis is a
feature of symptomatic congenital infection.
7.4
Epidemiology
Human CMV is endemic and infects humans of all ages, the peak of acquisition
occurring in childhood. The sero-prevalence of CMV in young adult populations
depends upon socio-economic status ranging from 40% to 80% (high to low).
The virus is well adapted and in patients who are immunocompetent primary
infection is often asymptomatic (90-95%). The virus causes more significant disease
in an immunocompromised host, such as patients that have undergone transplant
operations and those that are infected with HIV. It is also an important cause of
congenital infection when it may be either symptomatic or asymptomatic.
7.5
Prevention and Control
In patients that are immunocompromised, both primary CMV infection and
reactivation, can give rise to CMV disease. As part of CMV disease, there may be
liver involvement. There is a role for antiviral therapy in preventing and treating CMV
in this context. Antiviral agents that can be used to treat CMV disease include
ganciclovir, foscarnet and cidofovir.
8
Epstein Barr Virus (EBV)
8.1
Basic Microbiology and Taxonomy148
The Epstein-Barr virus was first discovered in 1964 in cells that had been cultured
from tumour specimens from patients in Kampala, Uganda. EBV belongs to the
Herpesviridae family and has morphologic features that are characteristic of this
family. It has a hexagonal nucleocapsid that is surrounded by a complex envelope
and is approximately 180 – 200nm in diameter.
8.2
Laboratory Diagnosis
This section should be read in conjunction with V 26 - Epstein-Barr Virus Serology.
Heterophile antibodies are used to diagnose infectious mononucleosis (IM) and
specific EBV serology to diagnose EBV hepatitis and IM with negative heterophile
antibody such as in children:
8.2.1 Primary EBV infection
Antibodies to viral capsid antigen (VCA):

IgG, IgM and IgA are all typically detectable in the serum of patients with EBV
infection by the time of onset of symptoms

IgA and IgM decline to undetectable levels during convalescence

IgM detection by indirect immunofluorescence (IF) is a highly sensitive test for
EBV infection
Complexes of IgG and IgM class rheumatoid factor can cause false positive IgM
results. EBV IgG can cause false negative IgM results by competing with EBV IgM.
Serum samples should be pre-treated to remove IgG before the IF test. Non-specific
anti-cell fluorescence may be seen but should be readily differentiated from specific
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fluorescence by an experienced observer. ELISA tests for EBV IgM are less
sensitive, but are more widely used. Weak cross-reactions may cause false positive
EBV VCA IgM results, particularly during primary CMV.
Antibodies to EBNA-1 IgG is not usually detectable until the convalescent period. Its
detection excludes recent or current IM. Commercial EBNA-1 IgG ELISAs are
available and are widely used.
EBV DNA tests (quantitative PCR assays) are used in the monitoring of patients who
are immunocompromised and at risk of EBV associated lymphoproliferative disease,
where very high levels may be observed. Lower levels are seen in uncomplicated IM.
A role for EBV DNA quantitation in the diagnosis and monitoring of EBV infection
and its complications has been suggested. Levels are higher in IM than in healthy
carriers and higher still in complicated IM.
8.3
Clinical Features
IM, or glandular fever, is one of the commonest causes of prolonged illness in
adolescents and young adults in developed countries. Signs of IM are typified by
fever, pharyngitis, lymphadenopathy, splenomegaly, and hepatocellular dysfunction.
Jaundice occurs in 5% of cases, although hepatocellular enzymes are raised in 80 to
90% of cases149. Most cases resolve spontaneously over a 2 to 3 week period.
Malaise may occur for weeks or months in some patients.
Rarely primary EBV in a person who is immunocompetent may be complicated by
fulminant IM associated with haemophagocytosis caused by activation of the
monophagocytic system in multiple organs150. Liver failure is the cause of death in
about half of patients with fatal infectious mononucleosis, the remaining deaths
resulting from opportunistic infections. Chronic active EBV (CAEBV) infection is a
rare condition, distinct from chronic fatigue, that is typified by severe, chronic, or
recurrent IM-like symptoms after a well documented primary EBV infection. There is
a marked increase of EBV load in peripheral blood, often with infection of T and/or
natural killer cells, and evidence of major organ involvement, eg hepatitis. CAEBV
has a high morbidity and mortality from hepatic failure, lymphoma, sepsis, or
haemophagocytic syndrome149. Some cases of autoimmuneor granulomatous
hepatitishave also been associated with EBV infection151,152. Clinically significant
EBV liver damage is mostly observed in the immunocompromised states, including
HIV infection, post-transplant and X-linked lymphoproliferative disorders.
8.4
Epidemiology
Transmission occurs predominantly sub-clinically during childhood, often by spread
via salivary contact. In developed countries, primary infection often occurs during
adolescence, presenting as infectious mononucleosis (IM). More than 90% of adults
worldwide have been infected with EBV and carry the virus as a life-long persistent
infection, with latent infection of B lymphocytes.
9
Yellow Fever
9.1
Basic Virology and Taxonomy
Yellow fever virus, isolated in West Africa in 1927, is the prototype member of the
family Flaviviridae, and is an enveloped virus with a single positive sense RNA
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genome, containing approximately 11,000 nucleotides153. The family Flaviviridae is
divided into the genera Flavivirus, Pestivirus and Hepacivirus, and the genus
Flavivirus is further classified antigenically into 8 complexes (yellow fever has not,
however, been assigned to an antigenic complex)154. Flavivirus particles are 40-50
nm in diameter and have a spherical nucleocapsid surrounded by a lipid bilayer
envelope. Analysis of the yellow fever virus Pre-Membrane/Membrane and a portion
of the E (envelope) gene sequence and 3’-non-coding region has distinguished at
least 7 genotypes, with homology >90% across the genotypes. These include two
South American genotypes, two West African genotypes (I and II), a single
Central/South African genotype (Angola), and two East African genotypes (East and
East/Central)155,156.
9.2
Laboratory Diagnosis
In the UK, testing is performed at the Special Pathogens Reference Unit (SPRU) at
CEPR, Porton Down. The information they require in all cases is the onset date of
illness, the countries and dates of visit, the date of return to the UK, and whether
yellow fever vaccination has been given.
In the first 7-10 days post onset of symptoms, an EDTA sample can be tested for
yellow fever RNA detection. Serologic testing on serum is also performed to
determine virus-specific IgM and IgG antibodies (ELISA, Immunofluorescence). The
IgM response is very specific but may be short-lived. The IgG response can show
cross-reactivity between the flavivirus complexes/strains (JE, WN, Dengue, TBE,
YF), thus other serologic assays (eg neutralisation tests) need then be utilised to
determine the strain involved. There is also the facility to culture the virus from tissue
samples or to perform PCR on tissue samples.
9.3
Clinical Features and Transmission
Yellow fever is spread by the bite of an infected mosquito. Humans and monkeys are
the main virus reservoir, with the principle mosquito vector either Aedes Aegypti or
Haemagogus species.
There are 3 main transmission cycles, recently reviewed157:
1. The sylvatic (jungle or forest) transmission cycle causes sporadic cases in
South America and Africa. The cycle occurs between monkeys and
mosquitoes, with humans infected when they live or work in the cycle areas
2. An intermediate sylvatic cycle occurs in the moist African savannah where
semi-domestic mosquitoes infect animals and humans to cause small
epidemics in rural villages
3. Urban transmission can occur between humans via domestic (ie breed around
human habitation) Aedes aegypti and can lead to large epidemics if the
population is unvaccinated
The Aedes mosquito is active during the day and once infected with virus remains
infectious for life (2-3 months). The virus can survive over the dry season in
mosquito eggs.
The incubation period is 3-6 days and there can be three distinct phases to the
illness. Initial symptoms are fever, prostration, extremity pain (including knee joint),
headache, photophobia, lumbosacral pain, anorexia, epigastic pain and vomiting.
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Laboratory abnormalities include leukopenia from the onset and elevation of serum
transaminases on days 2-3. In many patients gradual recovery occurs after 3-4 days.
However, in approximately 15-25% patients, the second phase is an abatement of
fever for 2-24 hrs, followed by a more serious illness in the third phase. Hepatic
coagulopathy produces haemorrhagic symptoms, including haematemesis, epistaxis,
bleeding, petechial and purpuric haemorrhage. There is a marked thrombocytopenia,
a deepening jaundice and proteinuria. Patients may develop shock, metabolic
acidosis, acute tubular necrosis, myocardial dysfunction, cardiac arrhythmias,
confusion, seizures and coma. Death rates of 20-50% occur and there is often
secondary bacterial infection and renal failure158. Survival gives lifelong immunity.
9.4
Epidemiology
The disease is endemic in 10 South American countries and over 30 countries in
sub-Saharan Africa. WHO estimates approximately 200,000 cases of yellow fever
annually with 30,000 deaths (although there is under-reporting of disease). Yellow
fever can reappear with outbreaks after long periods of apparent quiescence 157.
9.5
Prevention and Control
There is no specific antiviral available, thus good supportive management is
essential. Prevention entails avoidance of mosquito bites, control of mosquito
populations and immunisation.
A live attenuated vaccine has been available for more than 50 years and is
recommended for laboratory workers handling infective material and for everyone
visiting countries with a risk of yellow fever transmission. With the recent recognition
of rare severe adverse events related to yellow fever vaccine, it is essential to make
a careful risk assessment prior to administering vaccine159. Currently yellow fever is
the only disease for which an ICVP (international certificate of vaccine or
prophylaxis) may be required for entry. There is only one licensed yellow fever
vaccine in the UK which contains an attenuated strain of the 17D strain of yellow
fever virus that protects against all yellow fever genotypes (Stamaril, manufactured
by Sanofi Pasteur MSD). A single dose of vaccine, correctly administered, confers
immunity in 95-100% of recipients and this immunity persists for at least 10 years160162. Vaccination should be performed at least ten days prior to travel to an endemic
area to allow protective immunity to develop and for the ICVP to become valid.
International Heath Regulations (2005) require re-vaccination at 10 year intervals to
retain a valid ICVP.
10 Q Fever
10.1 Basic Microbiology and Taxonomy
Coxiella burnetii, the causative agent of Q fever, a zoonotic infection, is a Gram
negative, pleomorphic coccobacillus. It is an obligate intracellular gamma
proteobacterial organism.
10.2 Laboratory Diagnosis
Q fever diagnosis is commonly based upon serological methods. Both the
complement fixation test (CFT) and the indirect immunofluorescence assay (IFA)
may be utilized. In acute illness, antibodies to phase II antigen are higher than those
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of phase I, whereas, in chronic Q fever, antibodies to phase I predominate163.
C. burnetii PCR offers a sensitive direct detection approach to the diagnosis of acute
Q fever164.
10.3 Clinical Features
Acute Q fever is mostly a self-limiting febrile illness and may present with a wide
variety of symptoms. The fever normally lasts for 1 to 2 weeks and many patients will
develop pneumonia. In addition a majority of patients will have abnormal liver
function tests and some will develop hepatitis163. Q fever hepatitis usually manifests
with a moderate rise in liver enzyme levels rising to around two to three times the
upper limit of normal163. The hepatitis may occur in the absence of pulmonary
involvement165.
10.4 Transmission
Transmission of Q fever is primarily via the aerosol route by inhalation of
contaminated material. The most infectious source is parturient fluids from animals,
usually infected sheep, cattle or goats, but other means of infection include animal
blood, milk, urine and faeces. The organism is very robust and may survive in the
environment for many weeks163.
10.5 Epidemiology
Q fever is found worldwide, but is relatively rare in the UK3. The infection is
associated with rural living and is most common in those regions associated with
agriculture. Q fever is capable of causing outbreaks and should be considered in
certain circumstances166.
10.6 Prevention and Control
Prevention and control measures are directed at those handling possibly infected
animals with emphasis on appropriate disposal of animal birth products. Acute Q
fever usually resolves without treatment in about two weeks, although treatment of Q
fever with tetracycline or doxycycline or a fluoroquinolone is normally effective in
reducing the duration of illness167,168. At present there is no vaccine against Q fever
available in the UK.
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