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CHAPTER 1 - VIRUSES CAUSING HEPATITIS

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ENUMERATE THE VIRUSES CAUSING HEPATITIS

• Cytomegalovirus (CMV)
• Epstein-Barr virus (EBV)
• Herpes simplex virus (HSV)
• Adenoviruses
• Rubella virus
• Hantaviruses
• Mumps virus
• Enteroviruses (Coxsackieviruses)
• Yellow fever virus - an arbovirus (Flaviviridae) causing hemorrhagic fever with hepatitis

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HEPATITIS B VIRUS (HBV) - COMPREHENSIVE

1. MORPHOLOGY (Structure)

• Size: 42 nm in diameter
• Enveloped virus
• Contains:
  • Outer envelope: carries HBsAg (Hepatitis B surface antigen) in three forms: L (large/gp42), M (medium/gp36), S (small/gp27)
  • Inner icosahedral nucleocapsid/core: composed of HBcAg (Hepatitis B core antigen)
  • Genome: Partially double-stranded circular DNA, ~3200 base pairs (unique among DNA viruses - replicates via RNA intermediate using reverse transcriptase)
  • DNA polymerase with reverse transcriptase and RNase H activity
  • Protein kinase
  • HBeAg: non-structural protein encoded by precore region; secreted into serum

1. Spherical particles: 22 nm diameter, composed mainly of S-form HBsAg
2. Tubular/Filamentous particles: 22 nm wide, 100-700 nm long, composed of S, M, and L forms

• S gene: encodes HBsAg (S, M, L forms = surface antigen)
• C gene / Precore: encodes HBcAg (core) and HBeAg (precore)
• P gene: encodes DNA polymerase (reverse transcriptase)
• X gene: encodes HBxAg - a transactivating factor implicated in hepatocellular carcinoma (HCC)

• HBV divided into 4 major serotypes (adr, adw, ayr, ayw) based on antigenic epitopes on HBsAg envelope protein
• 8-10 major genotypes (A through H/J) based on DNA sequence; immunity is NOT serotype-specific
• Genotype C: associated with higher risk of cirrhosis and HCC; low response to interferon treatment

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2. MODES OF TRANSMISSION

1. Sexual transmission - most common route in developed countries (particularly homosexual males at higher risk)
2. Parenteral / Blood-borne transmission:
   • Blood transfusions (residual risk ~1 in 600,000 with screening)
   • Sharing IV drug use needles
   • Needle-stick injuries (health care workers)
   • Hemodialysis
   • Tattooing, acupuncture with unsterilized equipment
3. Vertical (Perinatal) transmission - from HBsAg-positive mother to neonate (usually at or around delivery, not in utero). Carrier rate if infected perinatally: ~90%; if infected as adults: ~5-10%
4. Horizontal transmission in childhood - skin abrasions, contact with infected secretions (common in endemic areas)
5. HBV is NOT transmitted by the fecal-oral route, breast milk (generally), casual contact (hugging, kissing), contaminated food, or water

Key risk factor for chronicity: Age at infection inversely correlates with chronicity - perinatal infection → 90% chronic; early childhood → 30%; adults → 5-10%

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3. PATHOGENESIS

1. Entry: HBV attaches to hepatocytes via HBsAg glycoproteins binding to the sodium/bile acid cotransporter NTCP (sodium taurocholate cotransporting polypeptide)
2. After entry, the nucleocapsid delivers the partially ds-DNA genome to the nucleus where it is completed into a covalently closed circular DNA (cccDNA) - a viral minichromosome that persists in infected hepatocytes
3. The genome is transcribed, producing 4 types of mRNA including a 3500-base pregenomic RNA (pgRNA)
4. Replication: pgRNA is packaged into core particles; reverse transcriptase synthesizes negative-sense DNA from the RNA template; RNA is degraded; positive-sense DNA is partially synthesized → new virions released by exocytosis

• Hepatocytes carrying viral antigens are recognized by CD8+ cytotoxic T cells and NK cells - these kill infected hepatocytes → liver damage
• In adults with intact immunity: robust immune response → clearance (~95%)
• In neonates/infants: immature immune response → virus tolerated → carrier state (immune tolerance phase)

• Chronic active hepatitis → cirrhosis → hepatocellular carcinoma (HCC)
• HBV X gene product (HBxAg) transactivates cellular growth genes, promotes carcinogenesis
• Integration of HBV DNA into host genome triggers chromosomal rearrangements
• PHC (primary hepatocellular carcinoma) - HBV one of the three most common causes of cancer mortality worldwide

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4. LABORATORY DIAGNOSIS OF HBV - SEROLOGICAL MARKERS AND SIGNIFICANCE

A. Hepatitis B Surface Antigen/Antibody (HBsAg / Anti-HBs)

• First detectable marker; appears 1-10 weeks after exposure (before symptoms)
• Indicates active infection (acute or chronic)
• Presence for >6 months = chronic infection
• Detected by: ELISA, chemiluminescence, ICT

• Appears after clearance of HBsAg (after resolution)
• Indicates: recovery + immunity OR successful vaccination
• Only marker positive in vaccinated individuals
• HBsAg and anti-HBs cannot be detected simultaneously (immune complex obscures both) → "window period"

B. Hepatitis B Core Antigen/Antibody (HBcAg / Anti-HBc)

• Hidden antigen; surrounds the viral core
• NOT detectable in serum (non-secretory, enclosed within HBsAg coat)
• Detectable only in hepatocytes by immunofluorescence

• First antibody to appear; detected during the "window period" (after HBsAg disappears but before anti-HBs appears)
• IgM anti-HBc = marker of acute HBV infection (diagnostic during window period)
• IgG anti-HBc = marker of past infection; persists for decades
• Anti-HBc is present in both acute AND chronic infection; absent only in vaccinated persons

Window Period: The HBsAg window is the diagnostic gap when HBsAg has cleared but anti-HBs has not yet appeared. During this time, IgM anti-HBc is the only detectable marker - it is the diagnostic marker of acute HBV.

C. Hepatitis Be Antigen/Antibody (HBeAg / Anti-HBe)

• Encoded by precore region; a soluble, secreted protein
• Present in serum when active viral replication is occurring
• Best correlate of viral infectivity - its detection indicates high viral load and high infectiousness
• Its persistence beyond 3 months indicates progression toward chronic hepatitis
• NOT detectable in "precore mutant" strains of HBV

• Appears after clearance of HBeAg
• Signals reduction in infectivity and viral replication
• In chronic HBV, transition from HBeAg+ to anti-HBe+ (seroconversion) = good prognostic sign

Serological Marker Interpretation Table

Additional Tests:

• HBV DNA (Viral Load): Detected by PCR; quantified by real-time PCR; most sensitive indicator of viral replication; used for monitoring treatment response and guiding antiviral therapy
• Liver enzymes (ALT, AST): Elevated in hepatitis; not specific but indicate hepatocyte injury
• Liver biopsy: "Gold standard" for staging HBV infection (degree of fibrosis); Fibroscan (elastography) is non-invasive alternative
• HBcAg: Detected in liver biopsy specimens by immunofluorescence

Markers of ACUTE HBV infection (summary):

1. HBsAg - earliest marker
2. HBeAg - active replication marker
3. IgM anti-HBc - the most specific marker of ACUTE infection (present even during window period)
4. Elevated serum ALT/AST

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5. PREVENTION OF HBV

A. Passive Immunization (Post-exposure prophylaxis)

• Hepatitis B Immunoglobulin (HBIG): Contains high-titer anti-HBs
  • Given within 7 days of exposure (needle-stick) or within 24 hours of birth to neonate of HBsAg+ mother
  • Provides immediate but temporary protection

B. Active Immunization - Hepatitis B Vaccines

• Plasma-derived vaccine: HBsAg purified from plasma of chronic carriers
• Safe but concerns about blood-borne pathogens

• Recombinant DNA (yeast-derived) vaccine - most widely used globally
• HBsAg (S-protein) expressed in Saccharomyces cerevisiae by recombinant DNA technology
• Trade names: Recombivax HB, Engerix-B
• Highly immunogenic, safe (no blood products)

• Contains pre-S1 and pre-S2 antigens in addition to S protein
• Better immune response in non-responders

• 3 doses: 0, 1, and 6 months (IM in deltoid)
• Infants of HBsAg+ mothers: HBIG + vaccine within 24 hours of birth
• For newborns: 0, 1-2, and 6-18 months

• Healthy adults: ~95% respond
• Non-responders (~5-10%): do not develop anti-HBs even after 6 doses; HLA-linked non-response
• Immunocompromised, obese, smokers, elderly: lower response rates

• Twinrix = HAV + HBV vaccine
• Pentavalent vaccine (includes HBV, DTP, Hib)

• Screening blood donations for HBsAg and anti-HBc
• Universal precautions for healthcare workers
• Needle exchange programs
• Safe sex (condoms)
• No sharing of razors, toothbrushes, needles

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HEPATITIS A - LABORATORY DIAGNOSIS

1. Anti-HAV IgM (Method of choice for acute diagnosis):
   • Detected by ELISA
   • Appears at onset of symptoms; persists 3-6 months
   • Positive = acute HAV infection
2. Anti-HAV IgG:
   • Appears 1 week after IgM
   • Persists for decades (lifelong)
   • Detection in absence of IgM = past infection or vaccination (immunity)
   • Used in seroprevalence studies
3. HAV Antigen detection (rarely used clinically):
   • ELISA or immunofluorescence on stool samples
   • HAV shed in feces 2 weeks before to 2 weeks after onset of jaundice
4. PCR (RT-PCR for HAV RNA):
   • Detects HAV RNA in blood and stool during viremic phase
   • Used for genotyping and outbreak investigation
5. Liver function tests (ALT/AST): Elevated, non-specific

Key point: HAV does NOT cause chronic infection. No carrier state. ELISA is the method of choice; many rapid test formats available.

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HEPATITIS C - LABORATORY DIAGNOSIS

1. Anti-HCV (ELISA / CIA):
   • Screening test of choice
   • First and second generation ELISA: now obsolete (less sensitive)
   • Third-generation ELISA: uses antigens from NS5 region in addition to core, NS3, NS4 regions; sensitivity/specificity >99%; becomes positive ~5 weeks post-infection
   • Limitation: does not distinguish acute from chronic or resolved infection
2. HCV RNA (RT-PCR/NAAT):
   • Most sensitive test; detects viremia within 1-2 weeks of infection
   • Qualitative PCR: for diagnosis and confirmation of anti-HCV positive results
   • Quantitative RT-PCR (viral load): for monitoring antiviral treatment
   • HCV RNA prevalence in general population is >0.1%
3. Recombinant Immunoblot Assay (RIBA) - formerly used as confirmatory test after ELISA; now obsolete as PCR is preferred
4. HCV Core Antigen:
   • ELISA-based
   • Detectable earlier than anti-HCV; correlates with viral load
   • Useful when NAT unavailable
5. HCV Genotyping (PCR-based): Required before initiating antiviral therapy (direct-acting antivirals)
6. Liver biopsy / Fibroscan: Staging of liver fibrosis

HCV does NOT spread through breast milk, food, water, hugging, kissing, or casual contact. Vertical transmission rate is ~4% (much lower than HBV ~20%). Sexual transmission is rare.

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YELLOW FEVER

• Jungle (sylvatic) cycle: Between forest mosquitoes and monkeys
• Urban cycle: Aedes aegypti → human → Aedes aegypti

• Mosquito inoculates virus into skin
• Virus multiplies in local lymph nodes → spreads to liver, spleen, kidney, bone marrow, heart
• Viremia during early phase (source of infection for mosquitoes)
• Liver: midzone necrosis of hepatocytes, Councilman bodies (eosinophilic inclusion bodies - necrotic hepatocytes), fatty change
• Kidneys: fatty degeneration and necrosis of tubular epithelium

• Incubation: 3-6 days
• Three phases:
  1. Acute phase (infection): fever, chills, headache, myalgia, backache, nausea, vomiting, bradycardia
  2. Remission: brief improvement
  3. Toxic phase (~15% of cases): jaundice, renal failure, hemorrhage (hematemesis - "black vomit"), myocarditis; mortality 20-50%

1. Virus isolation: From blood (first 4 days); intracerebral inoculation of mice; cell culture
2. Antigen detection: ELISA antigen capture, immunohistochemistry in tissue
3. PCR (RT-PCR): Detects viral RNA in blood/tissue
4. Serology:
   • Anti-YF IgM (ELISA): Appears after viremia; diagnostic for recent infection
   • Plaque Reduction Neutralization Test (PRNT): Most specific; confirms diagnosis
   • IgG antibodies persist for years
5. Histopathology (postmortem): Midzone necrosis, Councilman bodies, fatty change in liver

• Live attenuated vaccine 17D strain: Highly effective, single dose provides lifelong immunity; required for travel to endemic areas
• Vector control: eradication of Aedes aegypti breeding sites

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CHAPTER 2 - PARASITIC INFECTIONS OF HEPATOBILIARY SYSTEM

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CESTODES (TAPEWORMS)

Classification of Cestodes

• Diphyllobothrium latum

General Features of Cestodes:

1. No alimentary canal - absorb nutrients through tegument
2. Hermaphroditic - both male and female reproductive organs in each segment
3. Scolex (head): attachment organ with suckers (and hooks in some)
4. Neck: zone of proliferation
5. Strobila: chain of proglottids
6. Proglottids: segments - immature, mature, gravid (contain eggs)
7. Life cycle requires two hosts (definitive + intermediate), except H. nana

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ECHINOCOCCUS GRANULOSUS (HYDATID DISEASE / CYSTIC ECHINOCOCCOSIS)

Morphology

• 3-6 mm long; smallest tapeworm of medical importance
• Scolex: with 4 suckers + double row of hooklets (rostellum)
• Neck: short
• Strobila: only 3-4 proglottids: 1 immature + 1 mature + 1-2 gravid
• Gravid proglottid: contains 500-800 eggs

• Spherical, thick-walled, 30-40 μm
• Contain oncosphere (hexacanth embryo) with 6 hooklets
• Indistinguishable from Taenia eggs

• Unilocular fluid-filled cyst
• Three layers:
  1. Pericyst (Host layer) - outermost; fibrous layer derived from host tissue (compressed liver parenchyma)
  2. Ectocyst (Laminated/Outer layer) - middle; white, laminated, acellular, non-nucleated membrane; produced by the parasite
  3. Endocyst (Germinal/Inner layer) - innermost; single layer of nucleated germinal epithelium; gives rise to all internal structures

• Brood capsules: daughter cysts budding inward from the germinal layer; each contains protoscoleces (immature tapeworm heads with hooklets)
• Daughter cysts: smaller cysts within the main cyst
• Hydatid fluid: clear, colorless, under pressure; contains oncospheres and protoscoleces; antigenic and anaphylactogenic
• Hydatid sand: sediment at bottom of cyst fluid; contains free protoscoleces + hooklets + brood capsule fragments

Neat Labeled Diagram of Hydatid Cyst

                 ┌──────────────────────────────┐
                 │  PERICYST (host fibrous wall) │
                 ├──────────────────────────────┤
                 │ ECTOCYST (laminated membrane) │
                 ├──────────────────────────────┤
                 │  GERMINAL LAYER (endocyst)    │◄── produces all structures
                 │    ┌──────────────┐           │
                 │    │ Brood Capsule│           │
                 │    │ ┌──────────┐ │           │
                 │    │ │Protoscoleces│           │
                 │    │ │(with hooks)│ │           │
                 │    │ └──────────┘ │           │
                 │    └──────────────┘           │
                 │   [ Daughter Cysts ]           │
                 │   [ Hydatid Fluid  ]           │
                 │   [ Hydatid Sand   ]           │
                 └──────────────────────────────┘

Life Cycle of E. granulosus

Dog (Definitive Host) ←────────────────────────────┐
    ↓ Adult worm in intestine                       │
    ↓ Gravid proglottids shed eggs in feces         │
    ↓                                               │
Eggs ingested by intermediate host                  │
(sheep, cattle, pig, or HUMAN - accidental host)    │
    ↓ Oncosphere hatches in small intestine         │
    ↓ Penetrates intestinal wall                    │
    ↓ Travels via portal circulation → liver        │
    ↓ (or lung, brain, bone, spleen rarely)         │
    ↓ Forms HYDATID CYST (grows slowly over years)  │
    ↓                                               │
Dog ingests raw viscera of infected sheep/cattle ───┘
(containing protoscoleces which develop into adult worms in dog intestine)

Pathogenesis

1. Eggs ingested by human → oncosphere hatches in duodenum → penetrates gut wall → enters portal circulation
2. Arrested in liver first (most common site, 60-70%)
3. If passes liver → lungs (20-30%); rarely brain, bone, spleen
4. Develops into hydatid cyst over months to years (slow-growing)
5. Clinical manifestations depend on site, size, complications:
   • Liver cyst: hepatomegaly, abdominal pain, obstructive jaundice (bile duct compression)
   • Lung cyst: cough, chest pain, hemoptysis
   • Cyst rupture (most dangerous):
     • Spillage of antigenic cyst fluid → anaphylactic shock (life-threatening)
     • Secondary seeding: daughter cysts implant and grow in peritoneum/other organs
     • Hydatid fluid is extremely antigenic

Laboratory Diagnosis

• Ultrasound (USG): First-line; shows cystic lesion with daughter cysts, calcification, hydatid sand; WHO classification of cysts (CL, CE1-CE5)
• CT scan / MRI: Better anatomical detail, shows daughter cysts within mother cyst
• X-ray: May show calcified cyst (eggshell calcification)

• ELISA: Most sensitive; detects anti-Echinococcus antibodies (IgG); screening test
• Indirect Hemagglutination (IHA): Positive in ~80% of liver cysts
• Latex Agglutination Test (LAT)
• Casoni's Intradermal Test (Casoni's test): Injection of sterile hydatid cyst fluid intradermally
  • Positive: wheal >5 mm at 20 minutes = hypersensitivity to hydatid antigens
  • Sensitivity: 80%; many false positives and negatives; now largely replaced by serology
  • Historically important but no longer widely used

• Puncture, Aspiration, Injection, Re-aspiration - guided by ultrasound
• Aspirated fluid examined microscopically for protoscoleces and hooklets (diagnostic)
• Hydatid sand: scolex + hooks + brood capsule debris seen under microscope
• Risk of spillage limits its use to specialist centers

• Blood count: Eosinophilia (present in ~25% of patients; higher if cyst has leaked)
• Elevated ALP/GGT if biliary obstruction

• 0.2 mL sterile hydatid cyst fluid injected intradermally
• Read at 15-30 minutes: Immediate reaction (wheal + flare >5mm = positive)
• Read at 24-48 hours: Delayed reaction (induration = positive for cellular immunity)
• Sensitivity ~70-80% but lacks specificity (cross-reacts with other cestode infections)

1. ELISA (most sensitive serological test)
2. Casoni's intradermal test (classic, though now obsolete clinically)

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INVASIVE (EXTRAINTESTINAL) AMOEBIASIS

Extraintestinal Manifestations of Amoebiasis

1. Amoebic Liver Abscess (ALA) - most common (accounts for ~50% of liver abscess cases)
   • Route: portal bloodstream from large intestine → right lobe of liver preferentially
   • "Anchovy sauce" pus (chocolate-brown fluid = liquefied hepatic tissue)
2. Pleuropulmonary amoebiasis: direct extension from liver abscess through diaphragm
3. Amoebic brain abscess: rare, hematogenous spread
4. Cutaneous amoebiasis: perianal skin; rare
5. Amoebic pericarditis: extension from left lobe liver abscess (rare, life-threatening)

Laboratory Diagnosis of Extraintestinal Amoebiasis / Amoebic Liver Abscess

• Aspiration of liver abscess fluid: Examine for trophozoites at the wall of the abscess (not in the necrotic pus itself)
• Trophozoites with ingested red blood cells (erythrophagocytosis) seen on wet prep
• Yield is low (~10-40%); better from the last aspirated material

• ELISA / IFA (Immunofluorescence Assay): Most sensitive (~90-99%); detects anti-amoeba IgG antibodies
• Countercurrent Immunoelectrophoresis (CIE): Rapid, highly specific
• Indirect Hemagglutination Assay (IHA): Positive in >90% of ALA
• Latex Agglutination: Rapid, useful in field settings
• Note: Serology may be negative early in illness; repeat if initially negative

• ELISA for Gal/GalNAc lectin antigen in serum (highly specific for E. histolytica)
• Fecal antigen ELISA: for intestinal amoebiasis; not useful for ALA

• PCR (stool, biopsy): Differentiates E. histolytica from non-pathogenic E. dispar and E. moshkovskii (morphologically identical)
• Most specific available test

• Ultrasound: First-line; ALA appears as hypoechoic lesion in right lobe of liver
• CT/MRI: Better characterization; round/oval lesion with peripheral enhancement ("ring sign")

• Direct wet mount: Trophozoites with ingested RBCs (diagnostic), or cysts
• Iodine preparation: Better visualization of cysts (4 nuclei in E. histolytica)
• Culture (Jones' medium / Robinson's medium): rarely done clinically
• Biopsy of colonic ulcer: shows "flask-shaped" ulcers with trophozoites

• Leukocytosis (neutrophilia)
• Elevated ESR, CRP
• Elevated serum ALP (liver involvement)

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LIVER FLUKES - ENUMERATION

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LARVA MIGRANS

1. Cutaneous Larva Migrans (CLM) - "Creeping Eruption"

• Larvae from cat/dog hookworms penetrate human skin
• Unable to complete life cycle in humans → migrate in dermis creating serpiginous (snake-like), intensely itchy tracks
• Diagnosis: Clinical; skin biopsy; serology
• Treatment: Albendazole or ivermectin

2. Visceral Larva Migrans (VLM)

• Systemic VLM: Hepatomegaly (most common), fever, cough, wheeze (pulmonary infiltrates), urticaria
• Ocular Larva Migrans (OLM): Larvae in eye → endophthalmitis, granuloma, retinal damage, can mimic retinoblastoma
• Covert/Common toxocariasis: Mild symptoms in older children; abdominal pain, neurological symptoms

• Eosinophilia (prominent, often >30%)
• Serology: ELISA for anti-Toxocara antibodies (larval excretory-secretory [ES] antigens; highly sensitive and specific)
• Imaging: Liver USG/CT - multiple small hypodense lesions
• Ophthalmic examination (for OLM)
• Liver biopsy (rarely needed): eosinophilic granulomas with larvae

3. Neural Larva Migrans

• Baylisascaris procyonis (raccoon roundworm): causes fatal eosinophilic meningoencephalitis

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SOURCES

• Medical Microbiology 9e (Murray, Rosenthal & Pfaller)
• Tietz Textbook of Laboratory Medicine, 7th Edition
• Jawetz, Melnick & Adelberg's Medical Microbiology, 28th Edition
• Robbins & Kumar Basic Pathology