Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JCM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Clinical Microbiology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JCM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Virology

Outbreak of Hepatitis E Virus Infection in Darfur, Sudan: Effectiveness of Real-Time Reverse Transcription-PCR Analysis of Dried Blood Spots

Audrey Mérens, Philippe Jean Guérin, Jean-Paul Guthmann, Elisabeth Nicand
Audrey Mérens
1National Reference Laboratory for Hepatitis E, Hospital Val-de-Grâce, Paris, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: merens-a@wanadoo.fr
Philippe Jean Guérin
2Epicentre, Paris, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jean-Paul Guthmann
2Epicentre, Paris, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Elisabeth Nicand
1National Reference Laboratory for Hepatitis E, Hospital Val-de-Grâce, Paris, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/JCM.02245-08
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Biological samples collected in refugee camps during an outbreak of hepatitis E were used to compare the accuracy of hepatitis E virus RNA amplification by real-time reverse transcription-PCR (RT-PCR) for sera and dried blood spots (concordance of 90.6%). Biological profiles (RT-PCR and serology) of asymptomatic individuals were also analyzed.

Hepatitis E virus (HEV) is a spherical, nonenveloped, single-stranded RNA virus (2, 16) that belongs to the new genus, Hepevirus (14). This pathogen is responsible for at least 50% of acute non-A non-B hepatitis in developing countries, where it causes sporadic infection but also large epidemics usually associated with fecal contamination of water (4-6, 10, 11, 15). A large outbreak of hepatitis E was reported in June 2004 in the internally displaced population camps of Darfur, in West Sudan, and across the border in Chad: at least 5,000 HEV infections were recorded from June to December 2004 (9). A task force, set up by the nongovernmental organizations Médecins Sans Frontières (Doctors Without Borders) and Epicentre in refugee camps in Darfur and by the World Health Organization and Centers for Disease Control in refugee camps in Chad, conducted a field study of this large epidemic. The samples were drawn from both clinical patients and asymptomatic individuals (3, 9, 13). The French National Reference Laboratory (CNR) for hepatitis E monitored the biological and virological investigations. The aims of this study were to assess the feasibility of amplifying HEV RNA from dried blood spots (DBS) in comparison with amplification from serum and to describe the biological profiles of patients and asymptomatic individuals.

Eighty-nine displaced persons living in the Chad Goz Amer camp (median age, 28 years; range, 7 to 65 years) and 92 persons in the Sudanese Mornay camp (median age, 25 years; range, 3 to 75 years) were investigated in August and September 2004. Two groups were defined in each camp. The first was composed of patients considered to be HEV infected, i.e., patients presenting or having presented jaundice since 1 July 2004 and with negative test results for a malarial diagnosis (n = 36 in Goz Amer; n = 56 in Mornay). The time before onset of jaundice was reported by the physicians of medical teams after investigation of patient histories. The second group included individuals that were asymptomatic from the start of the outbreak (n = 53 in Goz Amer; n = 36 in Mornay). Two types of biological samples were collected for each individual. First, whole-blood samples were collected by venipuncture and were centrifuged locally to isolate sera. Aliquots were conserved between +4 and +8°C in the field and at −80°C at CNR. Second, finger-prick whole-blood samples were collected on filter paper. The filter papers were thoroughly air dried and were stored at ambient local temperatures (28 to 40°C) in individual paper bags to prevent contamination and preserve long-term stability. As various medical teams were involved, two types of filter paper were used: Whatman paper in Goz Amer and Isocode Stix in Mornay (Schleicher and Schuell Bioscience, Inc.).

Samples were shipped to France, and serum samples were tested for anti-HEV immunoglobulin G (IgG) and IgM with enzyme-linked immunosorbent assay HEV IgG and HEV IgM kits (Genelabs Diagnostics and Abbott). HEV RNA was amplified from DBS and serum samples from each refugee. Paper sample areas were eluted in 220 μl of phosphate-buffered saline-Tween buffer. A final elution or serum volume of 200 μl was used for nucleic acid extraction with a MagnA pure RNA isolation kit (Roche Diagnostics). After a retrotranscription step, HEV RNA was amplified and detected by consensus TaqMan real-time reverse transcription-PCR (RT-PCR) performed on a LightCycler as described previously (8). Samples with a crossing point inferior to 43, without amplification of the negative control, were considered positive (8). Samples with discordant results for serum and DBS were tested in duplicate in two independent real-time PCR assays (retrotranscription and PCR). HEV RNA amplification results for sera and DBS were concordant in 90.6% of cases (Table 1), with no statistically significant differences (McNemar chi-square test). The kappa correlation coefficient was calculated as 0.808. The results of amplification from DBS and sera were in agreement at 86.5% for Whatman paper and 94.6% for Isocode Stix. No significant difference between the two papers was observed in this study (P = 0.1). Thus, DBS are easy to collect and store and have been successfully used for the amplification of HEV RNA by real-time RT-PCR. However, in nine cases, HEV RNA was amplified in serum samples but not in DBS samples. Six of the nine samples were stored on Whatman paper, and the amount of blood on the filter was less than 50 μl. Three of the nine samples were collected on Isocode Stix in suitable volumes, and discrepancies may be explained by persistence of natural PCR inhibitors in the paper matrix or by RNases introduced by finger contact when handling the papers during collection. HEV amplification sensitivity is dependent on standardizing the quantity of blood and on the careful handling of filter papers with gloves, especially for this RNA virus. In eight cases, DBS samples were positive and serum samples were negative for HEV RNA amplification; these cases occurred in samples from patients with clinical and serological evidence of acute hepatitis (n = 3) and in samples from asymptomatic refugees in which anti-HEV IgM was detected in the serum (n = 4). These cases, which provided the best results for HEV amplification from DBS samples, may be explained by the degradation of RNA in sera due to an absence of cooler conditions during transport (less than −20°C).

The second objective of this study was to describe the various biological profiles identified. Among the group defined as hepatitis E cases, all the specimens were positive for at least one of the HEV markers HEV RNA, anti-HEV IgG, and anti-HEV IgM (Table 2). HEV RNA was detected in 65.1% of cases; among these, 51 of 58 had detectable anti-HEV IgM. The Abbott IgM anti-HEV assay had a sensitivity of 86% in comparison with the results of RT-PCR. These results are consistent with data reported under outbreak conditions (7, 12). Interestingly, HEV RNA amplification was positive in 7 of 12 (58.3%) DBS specimens with negative anti-HEV IgM results, allowing the diagnosis of HEV infection. Conversely, among the cases in which HEV RNA amplification was negative (34.9%), 26 of 31 (83.9%) had detectable anti-HEV IgM. These observations underline the importance of using different biological tests for the HEV diagnosis, as none of the tests are sufficiently sensitive for use alone. Interestingly, HEV viremia was detectable with TaqMan HEV RNA real-time RT-PCR for more than 39 days after the onset of jaundice. Another point to underline is the large proportion of asymptomatic individuals displaying a biological profile consistent with acute hepatitis E infection (43.5%), with a detectable viremia for 31.5% of cases (Table 3). No statistically significant differences were observed when comparing the ages of individuals with icteric infections and those with asymptomatic HEV infections; however, clinical observations have documented the frequency of symptoms among older populations (1). An accurate evaluation of the spread of disease and the infection attack rate also requires biological samples from a cross section of asymptomatic individuals.

DBS have already been used for PCR detection of hepatotropic viruses, but this study is the first report using real-time nucleic acid amplification of DBS collected under tropical conditions in refugee camps during an outbreak. DBS requires only 50 μl of blood, which is an advantage for children. DBS samples do not require a centrifuge and can be mailed unrefrigerated with a low biohazard risk. Thus, RT-PCR of DBS samples may provide an accurate and reliable tool for conducting field studies of outbreaks in developing countries.

View this table:
  • View inline
  • View popup
TABLE 1.

Comparison of results of real-time HEV RNA RT-PCR amplification from serum and from whole blood collected on filter papera

View this table:
  • View inline
  • View popup
TABLE 2.

Results of real-time HEV RT-PCR and detection of specific anti-HEV antibodies for individuals with jaundicea

View this table:
  • View inline
  • View popup
TABLE 3.

Results of real-time HEV RT-PCR and detection of specific anti-HEV antibodies for asymptomatic individualsa

ACKNOWLEDGMENTS

We thank the local medical teams for collecting specimens and Gregory Armstrong from the Centers for Disease Control and Prevention, Atlanta, GA. We also thank Vincent Enouf and Mélanie Caron for their technical contributions to this study.

Declaration of interest: we do not have a financial interest in any organization that could be perceived as a real or apparent conflict of interest in the context of the material submitted here. There was no grant support for the work.

FOOTNOTES

    • Received 21 November 2008.
    • Returned for modification 3 February 2009.
    • Accepted 23 March 2009.
  • Copyright © 2009 American Society for Microbiology

REFERENCES

  1. 1.↵
    Aggarwal, R., H. Shahi, and K. Nai. 1997. Evidence in favour of high infection rate with hepatitis E among young children in India. J. Hepatol.26:1425-1426.
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.↵
    Balayan, M. S., A. G. Andjaparidze, S. S. Savinskaya, E. S. Ketiladze, D. M. Braginsky, A. P. Savinov, and V. F. Poleschuk. 1983. Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal-oral route. Intervirology20:23-31.
    OpenUrlCrossRefPubMedWeb of Science
  3. 3.↵
    Boccia, D., J. P. Guthmann, H. Klovstad, N. Hamid, M. Tatay, I. Ciglenecki, J. Y. Nizou, E. Nicand, and P. J. Guerin. 2006. High mortality associated with an outbreak of hepatitis E among displaced persons in Darfur, Sudan. Clin. Infect. Dis.42:1679-1684.
    OpenUrlCrossRefPubMedWeb of Science
  4. 4.↵
    Bryan, J. P., M. Iqbal, S. Tsarev, I. A. Malik, J. F. Duncan, A. Ahmed, A. Khan, A. Khan, A. R. Rafiqui, R. H. Purcell, and L. J. Legters. 2002. Epidemic of hepatitis E in a military unit in Abbotrabad, Pakistan. Am. J. Trop. Med. Hyg.67:662-668.
    OpenUrlAbstract
  5. 5.
    Bryan, J. P., S. A. Tsarev, M. Iqbal, J. Ticehurst, S. Emerson, A. Ahmed, J. Duncan, A. R. Rafiqui, I. A. Malik, R. H. Purcell, et al. 1994. Epidemic hepatitis E in Pakistan: patterns of serologic response and evidence that antibody to hepatitis E virus protects against disease. J. Infect. Dis.170:517-521.
    OpenUrlCrossRefPubMedWeb of Science
  6. 6.↵
    Buisson, Y., M. Grandadam, E. Nicand, P. Cheval, H. van Cuyck-Gandre, B. Innis, P. Rehel, P. Coursaget, R. Teyssou, and S. Tsarev. 2000. Identification of a novel hepatitis E virus in Nigeria. J. Gen. Virol.81:903-909.
    OpenUrlCrossRefPubMedWeb of Science
  7. 7.↵
    El-Sayed Zaki, M., M. H. El-Deen Zaghloul, and O. El Sayed. 2006. Acute sporadic hepatitis E in children: diagnostic relevance of specific immunoglobulin M and immunoglobulin G compared with nested reverse transcriptase PCR. FEMS Immunol. Med. Microbiol.48:16-20.
    OpenUrlCrossRefPubMed
  8. 8.↵
    Enouf, V., G. Dos Reis, J. P. Guthmann, P. J. Guerin, M. Caron, V. Marechal, and E. Nicand. 2006. Validation of single real-time TaqMan PCR assay for the detection and quantitation of four major genotypes of hepatitis E virus in clinical specimens. J. Med. Virol.78:1076-1082.
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    Guthmann, J. P., H. Klovstad, D. Boccia, N. Hamid, L. Pinoges, J. Y. Nizou, M. Tatay, F. Diaz, A. Moren, R. F. Grais, I. Ciglenecki, E. Nicand, and P. J. Guerin. 2006. A large outbreak of hepatitis E among a displaced population in Darfur, Sudan, 2004: the role of water treatment methods. Clin. Infect. Dis.42:1685-1691.
    OpenUrlCrossRefPubMedWeb of Science
  10. 10.↵
    Iqbal, M., A. Ahmed, A. Qamar, K. Dixon, J. F. Duncan, N. U. Islam, A. Rauf, J. P. Bryan, I. A. Malik, and L. J. Legters. 1989. An outbreak of enterically transmitted non-A, non-B hepatitis in Pakistan. Am. J. Trop. Med. Hyg.40:438-443.
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    Isaacson, M., J. Frean, J. He, J. Seriwatana, and B. L. Innis. 2000. An outbreak of hepatitis E in northern Namibia, 1983. Am. J. Trop. Med. Hyg.62:619-625.
    OpenUrlAbstract
  12. 12.↵
    Myint, K. S. A., T. P. Endy, R. V. Gibbons, K. Laras, M. P. Mammen, Jr., E. R. Sedyaningsih, J. Seriwatana, J. S. Glass, S. Narupiti, and A. L. Corwin. 2006. Evaluation of diagnostic assays for hepatitis E virus in outbreak settings. J. Clin. Microbiol.44:1581-1583.
    OpenUrlAbstract/FREE Full Text
  13. 13.↵
    Nicand, E., G. L. Armstrong, V. Enouf, J. P. Guthmann, J. P. Guerin, M. Caron, J. Y. Nizou, and R. Andraghetti. 2005. Genetic heterogeneity of hepatitis E virus in Darfur, Sudan, and neighboring Chad. J. Med. Virol.77:519-521.
    OpenUrlCrossRefPubMedWeb of Science
  14. 14.↵
    Okamoto, H. 2007. Genetic variability and evolution of hepatitis E virus. Virus Res.127:216-228.
    OpenUrlCrossRefPubMedWeb of Science
  15. 15.↵
    Schlauder, G. G., G. J. Dawson, I. K. Mushahwar, A. Ritter, R. Sutherland, A. Moaness, and M. A. Kamel. 1993. Viraemia in Egyptian children with hepatitis E virus infection. Lancet341:378.
    OpenUrlPubMed
  16. 16.↵
    Tam, A. W., M. M. Smith, M. E. Guerra, C. C. Huang, D. W. Bradley, K. E. Fry, and G. R. Reyes. 1991. Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome. Virology185:120-131.
    OpenUrlCrossRefPubMedWeb of Science
View Abstract
PreviousNext
Back to top
Download PDF
Citation Tools
Outbreak of Hepatitis E Virus Infection in Darfur, Sudan: Effectiveness of Real-Time Reverse Transcription-PCR Analysis of Dried Blood Spots
Audrey Mérens, Philippe Jean Guérin, Jean-Paul Guthmann, Elisabeth Nicand
Journal of Clinical Microbiology May 2009, 47 (6) 1931-1933; DOI: 10.1128/JCM.02245-08

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Journal of Clinical Microbiology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Outbreak of Hepatitis E Virus Infection in Darfur, Sudan: Effectiveness of Real-Time Reverse Transcription-PCR Analysis of Dried Blood Spots
(Your Name) has forwarded a page to you from Journal of Clinical Microbiology
(Your Name) thought you would be interested in this article in Journal of Clinical Microbiology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Outbreak of Hepatitis E Virus Infection in Darfur, Sudan: Effectiveness of Real-Time Reverse Transcription-PCR Analysis of Dried Blood Spots
Audrey Mérens, Philippe Jean Guérin, Jean-Paul Guthmann, Elisabeth Nicand
Journal of Clinical Microbiology May 2009, 47 (6) 1931-1933; DOI: 10.1128/JCM.02245-08
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

blood
Desiccation
Disease Outbreaks
Hepatitis E
hepatitis E virus
Reverse Transcriptase Polymerase Chain Reaction
Specimen Handling

Related Articles

Cited By...

About

  • About JCM
  • Editor in Chief
  • Board of Editors
  • Editor Conflicts of Interest
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Resources for Clinical Microbiologists
  • Ethics
  • Contact Us

Follow #JClinMicro

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

 

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0095-1137; Online ISSN: 1098-660X