Published Ahead of Print 1 April 2009. 10.1128/JCM.02245-08.
2009, 47(6):1931. DOI:J. Clin. Microbiol. and Elisabeth Nicand
Audrey Mérens, Philippe Jean Guérin, Jean-Paul Guthmann
Dried Blood Spots
Rever Transcription-PCR Analysis of Darfur, Sudan: Effectiveness of Real-Time Outbreak of Hepatiti
s E Virus Infection in jcm.asm/content/47/6/1931Updated information and rvices can be found at: The include:
REFERENCES
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Outbreak of Hepatitis E Virus Infection in Darfur,Sudan: Ef fectiveness of Real-Time Rever Transcription-PCR
Analysis of Dried Blood Spotsᰔ
Audrey Me´rens,1*Philippe Jean Gue´rin,2Jean-Paul Guthmann,2and Elisabeth Nicand1
National Reference Laboratory for Hepatitis E,Hospital Val-de-Graˆce,Paris,France,1and Epicentre,Paris,France2 Received21November2008/Returned for modification3February2009/Accepted23March2009 Biological samples collected in refugee camps during an outbreak of hepatitis E were ud to compare the
accuracy of hepatitis E virus RNA amplification by real-time rever transcription-PCR(RT-PCR)for ra and
dried blood spots(concordance of90.6%).Biological profiles(RT-PCR and rology)of asymptomatic indi-
viduals were also analyzed.
Hepatitis E virus(HEV)is a spherical,nonenveloped,sin-gle-stranded RNA virus(2,16)that belongs to the new genus, Hepevirus(14).This pathogen is responsible for at least50%of acute non-A non-B hepatitis in developing countries,where it caus sporadic infection but also large epidemics usually as-sociated with fecal contamination of water(4–6,10,11,15).A large outbreak of hepatitis E was reported in June2004in the internally displaced population camps of Darfur,in West Su-dan,and across the border in Chad:at least5,000HEV infec-tions were recorded from June to December2004(9).A task force,t up by the nongovernmental organizations Me´decins Sans Frontie`res(Doctors Without Borders)and Epicentre in refugee camps in Darfur and by the World Health Organiza-tion and Centers for Dia Control in refugee camps in Chad,conducted afield study of this large epidemic.The samples were drawn from both clinical patients and asymptom-atic 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 asss the feasibility of amplifying HEV RNA from dried blood spots(DBS)in comparison with amplification from rum and t
o describe the biological profiles of patients and asymptomatic individuals.
Eighty-nine displaced persons living in the Chad Goz Amer camp(median age,28years;range,7to65years)and92 persons in the Sudane Mornay camp(median age,25years; range,3to75years)were investigated in August and Septem-ber2004.Two groups were defined in each camp.Thefirst was compod of patients considered to be HEV , patients prenting or having prented jaundice since1July 2004and with negative test results for a malarial diagnosis(nϭ36in Goz Amer;nϭ56in Mornay).The time before ont of jaundice was reported by the physicians of medical teams after investigation of patient histories.The cond group included individuals that were asymptomatic from the start of the out-break(nϭ53in Goz Amer;nϭ36in 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 ra.Aliquots were conrved betweenϩ4andϩ8°C in thefield and atϪ80°C at CNR. Second,finger-prick whole-blood samples were collected on filter paper.Thefilter papers were thoroughly air dried and were stored at ambient local temperatures(28to40°C)in individual paper bags to prevent contamination and prerve long-term stability.As various medical teams were involved, two types offilter paper were ud:Whatman paper in Goz Amer and Isocode Stix in Mornay(Schleicher and Schuell Bioscience,Inc.).
Samples were shipped to France,and rum 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 am-plified from DBS and rum samples from each refugee.Paper sample areas were eluted in220l of phosphate-buffered saline–Tween buffer.Afinal elution or rum volume of200l was ud for nucleic acid extraction with a MagnA pure RNA isolation kit(Roche Diagnostics).After a retrotranscription step,HEV RNA was amplified and detected by connsus TaqMan real-time rever transcription-PCR(RT-PCR)per-formed on a LightCycler as described previously(8).Samples with a crossing point inferior to43,without amplification of the negative control,were considered positive(8).Samples with discordant results for rum and DBS were tested in duplicate in two independent real-time PCR assays(retrotranscription and PCR).HEV RNA amplification results for ra and DBS were concordant in90.6%of cas(Table1),with no statisti-cally significant differences(McNemar chi-square test).The kappa correlation coefficient was calculated as0.808.The re-sults of amplification from DBS and ra were in agreement at 86.5%for Whatman paper and94.6%for Isocode Stix.No significant difference between the two papers was obrved in this study(Pϭ0.1).Thus,DBS are easy to collect and store and have been successfully ud for the amplification of HEV RNA by real-time RT-PCR.However,in nine cas,HEV RNA was amplified in rum samples but not in DBS samples. Six of the nine samples were stored on Whatm
an paper,and the amount of blood on thefilter was less than50l.Three of the nine samples were collected on Isocode Stix in suitable
*Corresponding author.Mailing address:Hoˆpital d’Instruction
des Arme´es Be´gin,69Avenue de Paris,94160Saint-Mande´Cedex, France.Phone:00(33)1-40-51-46-30.Fax:00(33)1-43-98-53-36.E-mail:
发展英文*******************.
ᰔPublished ahead of print on1April2009.
1931 on October 9, 2014 by guest jcm.asm/ Downloaded from
volumes,and discrepancies may be explained by persistence of natural PCR inhibitors in the paper matrix or by RNas in-troduced by finger contact when handling the papers during collection.HEV amplification nsitivity is dependent on stan-dardizing the quantity of blood and on the careful handling of filter papers with gloves,especially for this RNA virus.In eight cas,DBS samples were positive and rum samples were negative for HEV RNA amplification;the cas occurred in samples from patients with clinical and rological evidence of acute hepatitis (n ϭ3)and in samples f
rom asymptomatic refugees in which anti-HEV IgM was detected in the rum (n ϭ4).The cas,which provided the best results for HEV amplification from DBS samples,may be explained by the degradation of RNA in ra due to an abnce of cooler con-ditions during transport (less than Ϫ20°C).
The cond objective of this study was to describe the vari-ous biological profiles identified.Among the group defined as hepatitis E cas,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 cas;among the,51of 58had detectable anti-HEV IgM.The Abbott IgM anti-HEV assay had a nsitivity of 86%in comparison with the results of RT-PCR.The results are consistent with data reported under outbreak conditions (7,12).Interestingly,HEV RNA amplification was positive in 7of 12(58.3%)DBS specimens with negative anti-HEV IgM re-sults,allowing the diagnosis of HEV infection.Converly,among the cas in which HEV RNA amplification was nega-tive (34.9%),26of 31(83.9%)had detectable anti-HEV IgM.The obrvations underline the importance of using different
biological tests for the HEV diagnosis,as none of the tests are sufficiently nsitive for u alone.Interestingly,HEV viremia was detectable with TaqMan HEV RNA real-time RT-PCR for more t
han 39days after the ont 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 cas (Table 3).No statistically significant differences were obrved when comparing the ages of individuals with icteric infections and tho with asymptomatic HEV infections;however,clinical obrvations have documented the frequency of symptoms among older populations (1).An accurate eval-uation of the spread of dia and the infection attack rate also requires biological samples from a cross ction of asymp-tomatic individuals.
DBS have already been ud for PCR detection of hepato-tropic virus,but this study is the first report using real-time nucleic acid amplification of DBS collected under tropical con-ditions in refugee camps during an outbreak.DBS requires only 50l of blood,which is an advantage for children.DBS samples do not require a centrifuge and can be mailed unre-frigerated with a low biohazard risk.Thus,RT-PCR of DBS samples may provide an accurate and reliable tool for conduct-ing field studies of outbreaks in developing countries.
We thank the local medical teams for collecting specimens and Gregory Armstrong from the Centers for Dia Control and Pre-vention,Atlanta,GA.We also thank Vincent Enouf and Me ´lanie Caron fo
r 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.
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TABLE 1.Comparison of results of real-time HEV RNA RT-PCR
amplification from rum and from whole blood collected
on filter paper a
RT-PCR result for rum samples (n ϭ181)
No.of DBS samples (n ϭ181)with
RT-PCR result on:Whatman paper
(n ϭ89)Isocode Stix (n ϭ92)Positive
Negative
麻鹩
Positive
Negative
Positive 316393Negative
646248
a
举杯祝福
Samples were collected from 181refugees in the Goz Amer and Mornay internally displaced population camps.
TABLE 2.Results of real-time HEV RT-PCR and detection of specific anti-HEV antibodies for individuals with jaundice a
Antibody results No.(%)of samples with indicated RT-PCR result Total no.(%)
Positive
Negative
IgG and IgM positive
51(57.2)24(27)75(84.2)IgG positive,IgM negative 7(7.8)5(5.6)12(13.4)IgG negative,IgM positive 02(2.2)2(2.2)IgG negative,IgM negative 000Total
58(65)
31(35)
高楼大厦怎么形容89(100)
a
Serum samples were collected from 89individuals in the Goz Amer and Mornay camps.
TABLE 3.Results of real-time HEV RT-PCR and detection of specific anti-HEV antibodies for asymptomatic individuals a
Antibody results No.(%)of samples with indicated RT-PCR result Total no.(%)
Positive
Negative
IgG and IgM positive
9(9.8)9(9.8)18(19.6)IgG positive,IgM negative 10(10.9)27(29.3)37(40.2)IgG negative,IgM positive 1(1.1)2(2.2)3(3.3)IgG negative,IgM negative 9(9.8)25(27.2)34(37)Total
29(31.5)
63(68.5)
92(100)
a
Serum samples were collected from 92asymptomatic individuals in the Goz Amer and Mornay camps.
金厢滩1932NOTES J.C LIN .M ICROBIOL .
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