Serology

1
Arankalle Serological virological diagnosis

• Serology
• The preferred test is ELISA, though Western blot
  formats were developed earlier
• For surveys, IgG-anti-HEV, for diagnosis,
  IgM-anti-HEV are the markers of choice
• Virological diagnosis
• Detection of HEV RNA in feces or serum by nested
  RT PCR
• The only marker in very acute cases
• Not practical, when serological assay is
  available
• Primers should be able to detect all the known
  genotypes
• Not a preferred method

2
Arankalle Serological virological diagnosis
Immunoassay Developments In the absence of
availability of in-vitro or in-vivo amplified
virus, all the tests use either recombinant
antigen or peptides or both for coating the solid
phase
3
Arankalle Serological virological diagnosis

• Evaluation of ELISA Two types of ELISAs need to
  be considered
• 1. In-house ELISAs developed by several
  investigators
• 2. Commercially available ELISAs
• Comparison of 12 assays (1997)
• No System Sensitivity Specificity
• 1 R, ORF-2, Insect cells 96 98
• 2 R,ORF-2, Insect cells 98 93
• 3 R, ORF-2, Insect cells 98 79
• 4 R, ORF-2 3, E.coli 83 98
• 5 R, ORF-2, E.coli 89 100
• 6 R, ORF-2 3, E.coli 67 100
• 7 R, ORF-2 3, E.coli 91 95
• 8 R, ORF-2 3, E.coli 72 98
• 9 R, ORF-3, Peptides 15 84
• 10 R, ORF-2, Peptides 24 100
• 11 R, ORF-3, Peptides 24 100
• 12 R, ORF-2 3, Peptides 57 98
• Though commercial ELISAs are mainly used for
  diagnosis, no report of comparison of all
  commercially available ELISAs is available

4
Arankalle Serological virological diagnosis

• Nepal experience, 1993
• 67 Hepatitis E patients (Onset of illness lt 14
  days prior to hospitalization) 18 patients
  followed for 27-100 PODs.
• Sensitivity Specificity
• Viremia (93) 85 100
• Fecal shedding (70) 69 ND
• IgM-anti-HEV (79) 74 99
• 14 patients IgM Neg, RNA pos Diagnostic
  Biotechnology, Singapore, 3 rHEV fusion proteins
  of Burma Mexican strains
• Nepal experience, 2002
• 56 kD ORF-2 protein based ELISA
• 197 cases and 449 controls examined
• HEV-IgM detected in gt 95 hepatitis E patients
• IgM/IgG Ab ratio can distinguish primary
  infection from reinfection
• lt5 show possibility of reinfection

5
IgM IgG-anti-HEV positivity and titres Week
post onset
Arankalle Serological virological diagnosis
Indian epidemics, SAR-55-based ELISA (1994)
6
Arankalle Serological virological diagnosis

• Pakistan epidemic IgM-anti-HEV
• Weeks No Pos/No tested
• -5 to -3 2/8 25
• -2 5/7 71
• -1 15/15 100
• 1 to 4 96/101 95
• 5 to 7 18/18 100
• 86 to 92 0/33 -
• SAR-55 (Genotype-I) rORF2 antigen-based ELISA
• Sporadic cases, Spain (n11), 2004
• BioElisa HEV IgG IgM kits, Biokit, Barcelona,
  Spain. Antigens Similar to genelabs
• IgG anti-HEV was detected in all cases
• IgM anti-HEV HEV RNA pos in 3 cases
• The follow up serum samples showed disappearance
  of IgG abs in 5 cases (neg for IgM abs and HEV
  RNA)
• Conclusion Rapid decline of IgG abs

7
Arankalle Serological virological diagnosis

• Comparison with Genelab kit At NIV, we compared
  Genelab (Singapore) Equipar (Italy) ELISAs for
  IgM-anti-HEV detection
• Equipar (Peptide-based) had very poor sensitivity
• IgM-anti-HEV positivity was 187/263 (Gene Lab),
  222/263 (human-ORF-2) and 221/263 (swine-ORF-2).
• Same 35 samples negative by Gene Lab ELISA were
  scored positive by both the human and swine
  HEV-based ELISAs.
• Another seven samples showed discrepant results
  with respect to different ELISAs
• IgG-anti-HEV positivity was 204/243 (Gene Lab),
  222/243 (human-ORF-2) and 223/243 (swine-ORF-2)
• The same 18 samples were positive by NIV ELISAs
  and negative by Gene Lab ELISA
• 9 samples showed discrepant results with respect
  to different ELISAs

8
Arankalle Serological virological diagnosis
9
Arankalle Serological virological diagnosis

• Indian scenario
• AIIMS
• ORF-2 3 proteins expressed in E coli
• Anti-ORF-2 abs were found to be non-specific
• Anti-ORF-3 IgM correlated with disease
• NIV
• ORF-2 proteins from human genotype-I and swine
  genotype IV expressed in baculovirus system
• Excellent correlation of IgM antibodies disease
• IgG antibodies could be detected for several
  years