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ABSTRACT |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Most HEV cases are diagnosed serologically using enzyme immunoassays (EIAs) for IgM and IgG antibodies to HEV or a reverse transcriptase–polymerase chain reaction (RT-PCR). IgM antibody to HEV appears early during acute clinical illness but wanes over a few months. IgG antibody to HEV appears a few days later but persists for at least a few years.15 The detection of HEV-specific IgM or an increasing titer of IgG antibodies to HEV is diagnostic of acute infection. Two commercial kits are currently marketed for detecting IgM antibodies to HEV in blood. Different methods to detect viral-specific nucleic acid by RT-PCR have been developed and evaluated.16 A rapid RT-PCR procedure using universal oligonucleotide primers was also developed for detection of HEV-RNA in serum.17 However, the EIA can be performed only by relatively skilled personnel in well-equipped laboratories and the RT-PCR is time-consuming, expensive, and difficult to conduct. The tests routinely use for the laboratory diagnosis of hepatitis E are too elaborate for widespread application under field or outbreak conditions.
There is an urgent need for a rapid, reliable, and user-friendly assay to diagnose acute HEV infection in disease-endemic areas. A kit based on immunochromatography should be a reliable and rapid method for clinical diagnosis, even in the hands of inexperienced personnel, and easy to perform under harsh field conditions. The aim of this study was to conduct an evaluation of the performance of the rapid immunochromatographic assay based on the antibody capture format for IgM antibodies to HEV with laboratory-confirmed acute-phase sera from patients with hepatitis E, as well as with control sera from hepatitis E–free individuals.
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MATERIALS AND METHODS |
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INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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Serum samples. The following panel selected from existing collections was used: 1) sera (n = 200) obtained from patients with acute HEV infection in Indonesia and Nepal; 2) sera from patients with other confirmed types of hepatitis: acute hepatitis A (n = 80), acute hepatitis B (n = 45), and acute hepatitis C (n = 50) from Thailand, Nepal, and Indonesia; 3) healthy blood donors from Thailand (n = 100); 4) sera from patients with acute Epstein-Barr virus (EBV) infections (n = 20); 5) rheumatoid factor (RF)–positive blood (n = 26); and 6) convalescent-phase HEV sera collected 10 days or more after acute onset sera were negative for HEV by an RT-PCR (n = 70). All HEV specimens were collected under previous protocols. Written, informed consent was obtained from study participants. These studies were reviewed and approved by the U.S. Army Medical Research and Material Command and its associated institutional review board(s), as well as the institutional review boards of the host countries. Some specimens were submitted from outbreak investigations or routine patient care. These specimens were devoid of any patient identifiers. All control sera including EBV- and RF-positive sera did not contain IgM antibodies to HEV. All samples were stored at –70°C, except for sera from healthy blood donors and RF- and EBV- positive blood, which were stored at –20°C. The assay was not evaluated with whole blood specimens in this study.
Quantitative EIA for IgM to recombinant HEV (rHEV) capsid. Serum analysis for the presence of IgM antibodies to rHEV antigen was performed using a WRAIR EIA,18 which can also be used to quantitate antibody to HEV. In this assay, IgM antibody to rHEV was detected by a sandwich EIA in 96-well plates. The antigen is the putative capsid protein of HEV, Sargodha 1987 strain, expressed in Spodoptera frugiperda (SF9) cells by a recombinant baculovirus. More than 100 U/mL of WRAIR IgM to HEV (with an acute hepatitis illness) was considered evidence of an acute HEV infection.
Detection of HEV by RT-PCR. An RT-PCR was used to detect the presence of HEV virus RNA.18,19
ASSURETM HEV IgM rapid test.
The immunochromatographic test device intended for the rapid detection of IgM antibodies to HEV in human serum, plasma, or whole blood was provided by Genelabs Diagnostics (Singapore). The test is an IgM capture solid-phase immunochromatographic assay. IgM, when present in the test sample, can be captured by anti-human IgM monoclonal antibodies immobilized on the membrane. A colloidal gold-labeled antibody to HEV is pre-incubated with HEV antigen prior to being embedded on the membrane. In addition, immobilized rabbit IgG antibodies, which can be recognized by colloidal gold-labeled anti-rabbit IgG antibodies, were used as a procedural control. When present, these rabbit IgG antibodies confirm the validity of the assay by formation of a visible red line. In the test procedure, 25 µL of serum (as used in this study) or plasma or 35 µL of whole blood was placed on the sample application well, and the result was read in 15 minutes. The test was performed according to the manufacturer’s instructions and was read blinded to prevent reader bias. The test result was considered positive if both the internal control and the test band were stained, irrespective of the intensity of the staining (Figure 1
). The test result was considered negative if only the internal control was visible. The test result was considered invalid if the internal control line was not visible.
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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100 U/mL) and 200 acute-phase sera were confirmed as true positive samples (RT-PCR positive and an HEV IgM titer > 100 U/mL). Additionally 70 follow-up samples from convalescent patients following an HEV infection, with negative RT-PCR result for HEV but with IgM titers between 11.3 and 11,885.1 U/mL (mean ± SD = 1,617.5 ± 2,453.1), were analyzed.
In samples collected from acutely ill HEV patients, 186 of 200 tested positive by the rapid test. Table 1 shows the sensitivity and specificity of the rapid test using the HEV IgM seropositive and seronegative samples. The test had a sensitivity of 93% (95% CI = 88.5–96.1%). The specificity was 99.7% (95% CI = 98.3–100%) with HEV-negative samples from healthy donors, as well as other patient samples, including those infected with EBV or hepatitis A, B, or C.
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Tests were read blinded to eliminate reader bias and the staining intensity of the color band was graded from – to 4+ (– = nonreactive, ± = faintly positive; + = clearly positive; ++ = distinctly positive; +++ = moderately positive; ++++ = strongly positive) (Figure 1
). The faintly positive bands were read and confirmed by an independent observer. The test was generally simple to interpret with the majority (65.4%) of positive test results (n = 237) showing line intensities between 2+ (distinct) and 4+ (strong). The line intensity of the positive rapid test results corresponded well with the IgM levels measured by an enzyme-linked immunosorbent assay (ELISA) (R = 0.347, P < 0.001). The median IgM level was 656.7 IU/mL in faintly positive test results, 1,616.6 IU/mL in clearly positive test results, 1,641.1 IU/mL in distinctly positive test results, 1,826.6 IU/mL in moderately positive test results, and 2,417.5 IU/mL in strongly positive test results (Figure 2).
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To simplify the diagnosis of acute HEV infection, a new rapid test for the detection of IgM antibodies to HEV based on immunochromatography has been developed and evaluated with well-characterized sera from HEV-infected patients. Our evaluation showed that the rapid assay has excellent specificity and sensitivity (> 90%) compared with our in-house reference assays to diagnose acute HEV infection (Table 1). The sensitivity was related to individual IgM levels because most of the false-negative samples (85.7%, 95% CI = 57.2–98.2%) had IgM levels consistently below the average value (mean for false-negative samples = 679.1 U/mL compared with the overall mean of 1,617.5 IU/mL). The practicability and reliability of this rapid detection test was investigated with sera collected from three countries in southeast Asia. The rapid assay has a high sensitivity and specificity to detect IgM antibodies to HEV because no cross-reactivity was observed with other hepatitis viruses. Also, most of the acute-phase samples showed a line intensity reading 
2+ that was easily interpretable.
The performance of the new rapid test was not validated against the commercial kits currently available for the diagnosis of acute HEV infection. Additional evaluations in other HEV-endemic regions are necessary to confirm that this rapid test is appropriate for universal use with all strains and does not have regional limitations. However such limitations appear unlikely because the test is based on a recombinant antigen and monoclonal antibody shown to be widely reactive with different HEV strains.21,22 Because of the promising results obtained in the present study, a large field evaluation of the rapid test in a disease-endemic area might be undertaken.
Although not the intended use of the test, according to the manufacturer’s instructions, the sensitivity of the rapid test was also evaluated with convalescent-phase serum samples from patients with hepatitis E (n = 70). Due to an obvious decrease in IgM levels, the sensitivity of the rapid test in detecting convalescent-phase cases was lower (72.9%, 95% CI = 60.9–82.8%) than for acute-phase samples. However, all of these cases tested negative in the RT-PCR and 8 (11.4%, 95% CI = 5.1–21.3%) cases also had IgM levels not exceeding 100 U/mL and would therefore also test negative in an IgM ELISA. More importantly, the rapid test showed no cross-reactivity with RF serum, which commonly causes interference in serologic tests. Only 1 of 20 EBV sera showed a false-positive result. The rapid test was not evaluated with acute-phase secondary cases of hepatitis E with a predominant IgG antibody response. Also, the sensitivity of the rapid test could not be evaluated with onset of illness because this information was missing in most cases.
The rapid test is also ideal for use in developing countries and rural settings because this requires no laboratory infrastructure or highly skilled personnel, and can also be used under field or outbreak conditions and in situations where appropriate diagnostic facilities are lacking. Also, it does not require extensive training of the operator or cold storage and can be conducted with whole blood samples. We conclude that this new chromatographic test will be a reliable and rapid assay for the clinician or public health worker in developing countries, as well as in outbreak investigations.
Received January 27, 2005. Accepted for publication May 19, 2005.
Acknowledgments: We thank Kittinun Hussem for excellent technical support, and the National Institute of Health Research and Development, the Indonesian Ministry of Health, and the Naval Medical Research Unit 2 for providing us with the HEV outbreak sera from Indonesia.
Financial support: This work was supported by the U.S. Army Medical Research and Materiel Command.
Disclaimer: The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the U.S. Department of the Army or the U.S. Department of Defense. The use of trade names is for identification only and does not constitute endorsement by the U.S. Government or the Government of the Kingdom of Thailand.
* Address correspondence to Khin S. A. Myint, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok 10400, Thailand. E-mail: myintk{at}afrims.org 
Authors’ addresses: Khin S. A. Myint, Harald Noedl, and Mammen P. Mammen, Jr., Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok 10400, Thailand, Telephone: 66-2-644-4674, Fax: 66-2-644-4760, E-mails: myintk{at}afrims.org, harald.noedl{at}afrims.org, and mammenmp{at}afrims.org. Ming Guan, Hsiao Ying Chen, and Yang Lu, MP Biomedicals Asia Pacific Pte. Ltd. (formerly Genelabs Diagnostics), 85 Science Park Drive, #04-01, Singapore 118259, Telephone: 65-6775-0008, Fax: 65-6775-4536, E-mails: ming.guan{at}mpbio.com, hsiaoying.chen{at}mpbio.com, and yang.lu{at}mpbio.com. David Anderson, Select Vaccines, Suit 2, 233 High Street, Armadale, Victoria 3143, Australia, Telephone: 61-3-9824-8166, Fax: 61-3-9824-8161, E-mail: anderson{at}burnet.edu.au. Teresa Howard, The Macfarlane Burnet Institute, GPO Box 2284, Melbourne, Victoria 3004, Australia, Telephone: 61-3-9282-2128, Fax: 61-3-9282-2100, E-mail: toward{at}burnet.edu.au.
Reprint requests: Khin Saw Aye Myint, U.S. Army Medical Component–Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok 10400, Thailand.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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