Performance of serologic assays specific to IgM antibodies
against hepatitis E virus: pan-genotypic evaluation
Jan Drobeniuc1, Jihong Meng1,2, Gabor Reuter3, Tracy Greene1, Natasha Khudyakov1, Thao Le1, Zoya Dimitrova1,
Saleem Kamili1 and Chong-Gee Teo1
1Centers
for Disease Control and Prevention, Atlanta, GA, USA; 2SE University School of Medicine, Nanjing, China; 3ANTSZ Regional Institute of State Public Health Service, Pecs, Hungary
53.038
Background
Classical hepatitis E, which manifests as water-borne
outbreaks of jaundice, is associated with transmission of
hepatitis E virus (HEV) belonging to genotypes 1 and 2.
Non-imported hepatitis E encountered in industrialized
countries is associated with autochthonous transmission of
HEV strains belonging to genotypes 3 and 4. In the U.S.,
clinically manifest hepatitis E is seldom reported although
HEV infection appears widespread , a disparity that may be
due to low-dose exposure to HEV, asymptomatic infection
by relatively avirulent HEV strains, or missed or underdiagnosis of hepatitis E. Assays have been developed for
anti-HEV IgM detection, but evaluations of their
performances were based on analyzing serum samples
obtained from case-patients infected by ≤ 2 HEV
genotypes. It would be desirable, in clinical practice and for
disease surveillance, to rely on assays that identify incident
HEV infection regardless of the genotypic background of
the infecting strain.
Results
Final constitution of study panels. 53 samples in the specificity panel were reactive in at least one
of the assays. One sample in the acute hepatitis C group tested positive for HEV RNA (arrows, Fig.
1A) and characterized to carry genotype 3. It was considered a constituent of the sensitivity panel.
This panel therefore comprised 51 serum samples, and the specificity panel comprised 228 samples.
Analytical sensitivity. Analytical response curves generated by Assays III-VI after application to
dilutions of Pool 6 are shown in Fig. 1B.
Figure 1. Geographic distribution
of HEV infection
http://wwwnc.cdc.gov/travel/yellowbook/2010/chapter-5/hepatitis-e.aspx
Objective
To evaluate serologic assays specific for IgM antibodies against
HEV originating from all 4 genotypes.
Methods
Two in-house assays and 4 commercial assays were evaluated.
• Assay I used a recombinant protein expressed in baculovirus from ORF2 amino-acid positions 112606 of the Sar-55 HEV strain (genotype 1);
• Assay II used proteins expressed in E. coli from ORF2 amino-acid positions 452-617 originating
from 4 genotypes: HEV-Morocco strain (genotype 1), the Mexico-14 strain (genotype 2), the US-1
strain (genotype 3) and the China-9829 strain (genotype 4);
• Assay III was purchased from International Immuno-Diagnostics (Foster City, CA), Assay IV from
MP Biomedicals (Singapore), Assay V from Diagnostics Systems (Nizhniy Novgorod, Russia) and
Assay VI from Mikrogen GmbH (Neuried, Germany).
Figure 1A. Distribution of signal to cut-off ratios (SCRs) generated
in serum samples that initially constituted the acute hepatitis E
panel (red symbols, each symbol denoting HEV genotypes), and
the non-acute hepatitis E panel (circles in colors other than red).
Arrows indicate single serum in the acute hepatitis C group,
subsequently characterized to carry HEV genotype 3. For each
assay, the inter-panel difference in the mean SCRs was highly
significant (P <0.0001; Mann-Whitney test), which remained after
exclusion of convalescent samples from the specificity panel.
Diagnostic sensitivity and specificity, and inter-assay agreement. The mean sensitivity and
specificity values were 90.2% and 91.4%, respectively. Individual assay sensitivities and specificities
are presented in Table 1. Inter-assay agreement “κ” coefficients ranged from 0.46-0.8 (mean: 0.53),
with the highest value attained between Assays II and V, and between Assays V and VI.
Table 1. Anti-HEV-IgM reactivities among 6 immunoassays in the finally constituted acute hepatitis E
panel and non-acute hepatitis E panel, and corresponding diagnostic sensitivity and specificity values.
Assay
Evaluation of diagnostic sensitivity. Fifty sera that fulfilled the following criteria were included:
•
origin from acutely jaundiced patients in whom acute hepatitis A, B and C, and chronic hepatitis
B and C were serologically excluded;
•
presence of HEV RNA;
•
assignment of the HEV genotype after nucleotide sequencing
The HEV genotype, provenance, year of collection and sample size were as follows:
•
Genotype 1- Turkmenistan (1985 and 1994, n=5); Chad (2004, n=10);
•
Genotype 2- Mexico (1986, n=4);
•
Genotype 3- U.S. (2004-2008, n=4); Mexico (1986, n=1); Hungary (2001-2006, n=9);
•
Genotype 4- China (2005-2006, n=17).
Evaluation of diagnostic specificity. 229 serum samples originating from:
•
anti-HEV-IgG-seropositive household contacts of hepatitis E cases in Chad and Mexico, not
jaundiced at the time of interview but reported being jaundiced > 6 mo before (n=31);
•
U.S. patients with acute hepatitis A (n=35), acute hepatitis B (n=35) and acute hepatitis C
(n=28) identified from 1996-2006 in CDC’s Sentinel Counties Study of Acute Viral Hepatitis;
•
U.S. blood donors (n=100, Vital Products Inc., Boynton Beach, FL).
Figure 1B. Analytical response curves generated from the
application of Assays III, IV, V and VI to indicated dilutions of Pool
6. Approximation was done with polynomials of degree 4 (R 2 =
0.989, 0.966, 0.995 and 0.964, respectively). Dotted horizontal
line denotes SCR value of 1.. The detection limits of Assays III, V
and VI were estimated as 137, 9 and 49 WR units/ml, respectively.
Pool 7 was reactive in all 4 assays. The sensitivity limit of Assay IV
therefore lay between the lowest dilution of Pool 6 and the upper
limit of anti-HEV IgM estimated in Pool 7.
Acute Hepatitis E panel
Genotype (gt) and number of samples
reactive
Non-acute hepatitis E
panel
Sensitivity
(%)
Number
of
samples
reactive
Specificity
(%)
gt 1
(n=15)
gt 2
(n=4)
gt 3
(n=15)
gt 4
(n=17)
Total
(n=51)
I
15
4
14
17
50
98
49
78.5
II
15
3
15
17
50
98
15
93.4
III
15
2
13
12
42
82.4
19
91.7
IV
13
1
10
13
37
72.5
16
93
V
15
4
14
17
50
98
10
95.6
VI
15
3
13
16
47
92.2
9
96.1
Conclusions
Samples found reactive in any one assay were tested for HEV RNA by RT-PCR.
• Appreciable variability in sensitivity, specificity and inter-assay agreement among
Evaluation of analytical sensitivity. An anti-HEV IgM quantitation standard, Pool 6* (860 Walter
Reed (WR) units/ml) and an positive control, Pool 7* (195-304 WR units/ml) were tested in
Assays III-VI.
anti-HEV-IgM immunoassays was revealed.
• A testing strategy adopting commercially available standardized assays with the
highest sensitivity and specificity is recommended.
*Seriwatana J, et al. Clin Diagn Lab Immunol 2002; 9:1072-8
Acknowledgements: Authors are grateful to K Foytich and D Chen for assistance with panel
assembly and assay performance, and R Purcell, R Jarman and K Myint for providing reagents.
Potential conflict of interest: none reported.
The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.