High prevalence of Hepatitis delta virus amongst Cameroonian HBsAg positive specimens
577
Rodgers
1 MA ,
1Infectious
Coller
1 K,
Luk
1 KC ,
Butler
1 E,
Fuhrman
1 J,
Krilich
Barnaby
2 D,
Ndembi
3 N,
Kaptue
4 L,
Gavin
1 Cloherty
Diseases Research, Abbott Diagnostics, Abbott Park IL, Institute for Science and Health, Franciscan University of Steubenville, Steubenville, OH, 3Institute for Human Virology Nigeria, Abuja, Nigeria, 4Laboratory of Hematology and Immunology, University of Montagnes, Bangangte, Cameroon
Abstract
Methods
Background Hepatitis Delta (HDV) is a defective RNA virus that requires Hepatitis B 1Infectious Diseases Research, Abbott surface antigen (HBsAg) for productive infection. An estimated 15-20 million people are infected with HDV worldwide; however, limited data are available on the prevalence of this virus in sub-Saharan Africa where HIV infection rates are high. HDV/HBV/HIV coinfection confers a greater risk for accelerated progression to liver disease and death. To determine the prevalence of HDV in HIV positive and negative populations in Cameroon, HDV antibodies and RNA were characterized in 1701 HBsAg positive specimens, of which 846 (49.7%) individuals were co-infected with HIV.
Figure 2: HDV serology assay
Methodology Plasma specimens were received from consenting subjects participating in surveillance studies in Cameroon collected over 8 years from 2007 – 2015. Samples were initially screened for antibodies (IgG) to HDV using a prototype HDV serology assay developed on the Abbott ARCHITECT. HDV reactive specimens with remaining volume were diluted 1:10 or 1:100 as necessary and screened using a prototype HDV RNA viral load assay with a calculated limit of detection of 5 IU/ml as calibrated by the HDV RNA WHO standard on the Abbott m2000 instrument. HDV RNA positive specimens with viral load >4.5 Log10 IU/ml were selected for complete genome Sanger sequencing of 3 overlapping regions, and a subset of specimens were selected for HIV or HBV sequencing for classification. All viral sequences were classified by phylogenetic analysis. Results HDV IgG antibodies were detected in 683 (40.2%) specimens, and a majority of samples exhibited evidence of chronic infection with HDV RNA detected in 68% (n=455) of the 669 tested samples with available volume. The rate of chronic HDV infection may be underestimated due to sample dilution during processing. The seropositive rate of HIV/HBV/HDV co-infection was 16.9% (288/1701), with 61.3% (176/287, 1 insufficient volume specimen excluded) positive for HDV RNA. HDV/HBV/HIV co-infected specimens included HIV subtypes A, CRF02, CRF11, D, and G and HBV genotypes A and E. HDV genotypes 1, 6, and 7 were present in this population. Conclusions HDV seroprevalence is high in HBsAg positive Cameroon individuals (40.2%), indicating that a large portion of HBV patients in Cameroon are at elevated risk for severe hepatitis and death. Screening and diagnosis of HDV in HBV/HIV-1 carriers in Cameroon might identify individuals at increased risk for developing liver disease.
2Franciscan
2 E,
Results - Sequencing
Diagnostics, Abbott Park, IL; BT-peptides Biotin-peptides
2University
of Yaoundé I, Yaoundé,
3Université Indirect IgG assay on Abbott Cameroon; des,ARCHITECT Cameroon; 4Institute
+
-Selection of antigenic peptides based on consensus sequence of HDV large antigen genotypes 1-8
Streptavidin microparticle
-Prototype solution phase capture assay utilizes a blend of 3 peptides
+
Antibodies in specimen
Step 1 : 18 mins Mix streptavidin microparticles, sample, and biotinylated (BT)peptides
Step 2 : 4 mins Add acridinylated antihuman IgG (or IgM) conjugate
Figure 3: HDV molecular assay
Figure 5: HDV genomic phylogenetic tree
of Human Virology Nigeria, Abuja, Nigeria
Quantitative assay on Abbott m2000 instruments
45
-Total nucleic acid extracted from 1ml of plasma
29
-Primers/probe located in conserved regions upstream of the large HDAg ORF were designed to detect genotypes 1-8
60
-Screening with n=68 US and n=115 HBsAg negative Cameroon donors indicates 100% specificity -Calibration to the WHO standard resulted in a limit of detection of 5 IU/ml
100
Results - Surveillance Figure 4: Screening summary
All HDV seropositive specimens
1701 HBsAg samples
N821 K4928 SUS363 S3141 U20694 1-AM902180 (Cameroon) K2663 SUS78 S3157 1-AM902171 (Cameroon) B583 U22731 1-AM902164 (Cameroon) SUE574A K2885 K1280 U22972 SUE423D S1273 D2240 K1224 J153 K2126 SUE752A K1370 SUE08D D2248 1-U81989 (Ethiopia) N406 K1299 SUE1048A2 D2943 A176 SUE584D K911 K6027 K5972 SUE635D D2344 S1086 S3925 D5604 S4268 J1030 D2313 F665 S4910 S5154 1-AM779576 (Madagascar) 1-U81988 (Somalia) 1-HM046802 (China) 1-M21012 (woodchuck HDV-1) 1-AJ000558 (CAR) 1-AF098261 (Canada) 1-NC 00165 1-EF514907 (Turkey) 1-EF514903 (Turkey) 1-EF514904 (Turkey) 1-EF514905 (Turkey) 1-AM902168 (Ivory Coast) 1-M84917 (Lebanon) 1-AM779575 (Cameroon) 100 1-AM779577 (CAR) S3893 71 J2001 F886 U24119 J2142 S3046 S993 SUS325 SUS109 D3703 U20635 K3820 S3327 J2183 S996 S3446 V73 J2112 100 SUE876A 7-AM183333 (CAR) 31 SUS380 76 D6591 51 SUE446V S4656 D2195 D4039 SUE238V F930 SUE10D S5018 D4068 SUE586D D1031 S3848 U20765 K1827 K2167 SUK243 K936 D2992 S3277 V190 B466 N557 7-JA417541 (Cameroon) GT-8 U-177114 6-AM183332 (CAR) SUE22D 100 D2198 K1572 SUE1032A 6-AJ584847 (Cameroon) GT-5 GT-4 GT-2 GT-3
GT-1
-Screening with n=100 US and n=100 HBsAg negative Cameroon donors indicates 100% specificity
Detection of immunocomplex
HIV/HBV/HDV seropositive specimens
(n=846, 49.7% HIV positive)
n=455 HDV RNA positive samples
n=168 specimens with viral load >4.5 log10 IU/ml selected for sequencing
n=96 genome sequences >1kb long
HDV genome classification Genotype 1 Genotype 6 Genotype 7
n
%
65 5 26
67.7 5.2 27.1
GT-7
Study Population Figure 1: Map of Cameroon and collection sites
Contact Information: Gavin Cloherty Abbott Diagnostics 100 Abbott Park Rd Abbott Park IL 60064-6015 Email:
[email protected]
HDV RNA negative
Primary screen: HDV IgG
HDV RNA negative
(n=214, 32%)
(n=683, 40.2% seropositive, n=669 with remaining volume)
HDV RNA positive
(n=455, 68%)
Secondary screen: HDV RNA
(n=112, 39%)
HDV RNA positive
GT-6
(n=176, 61%)
(n=455, 68% RNA positive)
Sanger sequencing -Primers target 3 overlapping regions of the 1.6kb genome -Partial or complete genomes were obtained for the majority of specimens sequenced -Sequences were analyzed using PHYLIP 3.5c (J. Felsenstein, University of Washington, Seattle) to generate the phylogenetic tree with bootstrap labels from the consensus tree
0.05
B ip in d i K rib i Cam po
Conclusions
L o lo d o rf E b o lo w a S a n g m é lim a D jo u m
Specimens were collected from a variety of study participants in Douala, Yaoundé, and South Cameroon villages and towns: - Blood donors - Antenatal clinics - Voluntary testing campaigns - Hospital patients - Door to door testing - Chest clinics
Serology result
n
%
HBV+HDV-HIVHBV+HDV+HIVHBV+HDV-HIV+ HBV+HDV+HIV+
460 395 558 288
27.1 23.2 32.8 16.9
HDV prevalence -40% of HBsAg positive specimens were HDV IgG positive -68% of HDV IgG positive specimens were also RNA positive -RNA prevalence may be underestimated due to sample dilution of 1:10 or 1:100
-
HDV seroprevalence is high in HBsAg positive Cameroon individuals (40.2%)
-
A majority of HDV seropositive specimens were RNA positive (68%), suggesting active infections
-
HDV genotype 1 was most prevalent, followed by genotypes 7 and 6, respectively
-
96 new HDV genomic sequences from this study will be added to Genbank, which currently contains 305 HDV genomes
