IJID Regions 14 (2025) 100560 Contents lists available at ScienceDirect IJID Regions journal homepage: www.elsevier.com/locate/ijregi Prevalence and molecular characterization of hepatitis delta virus infection among hepatitis B virus surface antigen positive students and pregnant women in N’djamena, Chad Nalda Debsikréo 1 , 2 , 3 , ∗ , Maire Dehainsala 4 , Odan Debsikréo 5 , Nafissatou Leye 2 , Gora LO 2 , Aminata Dia 2 , Makoutchouang Nzonde Biscotine Flore 1 , 2 , Ndeye Aminata Diaw 2 , Ndeye Dieynaba Diouf 2 , Isaac Darko Otchere 6 , 7 , Rayana Maryse Toyé8 , Isabelle Chemin 8 , Ali Mahamat Moussa 3 , 4 , Ndèye Coumba Toure-Kane 2 , Françoise Lunel-Fabiani 9 1 Cheikh Anta Diop University, Dakar, Senegal 2 Institut de Recherche en Santé, de Surveillance Épidémiologique et de Formation, Dakar, Sénégal 3 University of N’Djamena, N’Djamena, Chad 4 Centre Hospitalier Universitaire la Référence, N’Djamena, Chad 5 University of Félix Houphouët Boigny Abidjan, Abidjan, Côte d’ivore 6 Medical Research Council Unit the Gambia at London School of Hygiene and Tropical Medicine, Fajara, the Gambia 7 Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Accra, Ghana 8 Centre de Recherche en Cancérologie de Lyon INSERM U1052, CNRS UMR5286, Université de Lyon, Lyon, France 9 Centre Hospitalier Universitaire Angers, BAT IBS-4 rue Larrey-49000 ANGERS, Laboratoire HIFIH, UFR Santé département Médecine, SFR 4208-UPRES EA3859, Université d’Angers, Angers Cedex, France a r t i c l e i n f o Keywords: Hepatitis D virus Genotype Prevalence Students Pregnant women Chad and Africa a b s t r a c t Objectives: This study sought to determine the prevalence of hepatitis B virus (HBV)-hepatitis D virus (HDV) co-infection and to characterize isolates of both viruses in a Chadian population of HBV surface antigen (HBsAg)- positive pregnant women and students. Methods: This was a cross-sectional retrospective study using archived samples from pregnant women and stu- dents in N’djamena who had been systematically screened for HBsAg between April and August 2021. HBsAg- positive samples were tested for the presence of HDV antibodies (Ab) and were screened for the presence of both HBV and HDV (in anti-HDV Ab-positive samples) viral load estimations. Genome sequencing of the viruses was used for both genotyping and phylogenetic analysis. Results: A total of 94 participants were included in this study. The mean age was 24 ± 4.89 years (range: 18-42 years). Anti-HDV Ab were found in 9.57% (9/94) of the participants. The prevalence of anti-HDV Ab positivity among students (6.45% [4/62]) was lower than the 15.63% (5/32) observed among pregnant women. HDV-RNA was detected in 7/9 (77.77%) confirmed anti-HDV-positive participants. Most HDV-RN-positive participants had very low HBV DNA viral loads. All HBV sequences belonged to genotype E and all HDV sequences to genotype 1. Conclusions: Hepatitis D is a potential public health challenge in Chad, which requires active surveillance and public education in the country for proper control. This surveillance should be supported with mass immunization against HBV. I c m h r h l [ m c u a p h R 2 l ntroduction Hepatitis D virus (HDV) is a hepatotropic virus, a negative-sense cir- ular single-stranded RNA virus of approximately 1.7 kb, and is the only ember of the Deltaviridae family (genus Deltavirus) [ 1 ]. HDV requires epatitis B virus (HBV) surface proteins (HBsAg) to form infectious vi- al particles [ 2 ]. Without HBV, the HDV is incapable of penetrating epatocytes and replicating itself [ 3 ]. HDV causes acute and chronic ∗ Corresponding author: E-mail address: ndaldaeliane@gmail.com (N. Debsikréo) . ttps://doi.org/10.1016/j.ijregi.2024.100560 eceived 4 November 2024; Received in revised form 22 December 2024; Accepted 2 772-7076/© 2024 The Authors. Published by Elsevier Ltd on behalf of Internationa icense ( http://creativecommons.org/licenses/by/4.0/ ) iver disease [ 4 ] and an increased risk of hepatocellular carcinoma 5 ]. Delta hepatitis is still a major global health problem affecting 48-60 illion individuals worldwide [ 6 ]. It is estimated that 7.33% of HBsAg arriers are co-infected or superinfected with HDV in the general pop- lation, whereas 9.57% of people with liver disease in West Africa are lso infected with HDV [ 7 ]. In Central Africa, it is 25.64% of the general opulation and 37.77% among people with liver disease [ 7 , 8 ]. 4 December 2024 l Society for Infectious Diseases. This is an open access article under the CC BY https://doi.org/10.1016/j.ijregi.2024.100560 http://www.ScienceDirect.com http://www.elsevier.com/locate/ijregi http://crossmark.crossref.org/dialog/?doi=10.1016/j.ijregi.2024.100560&domain=pdf mailto:ndaldaeliane@gmail.com https://doi.org/10.1016/j.ijregi.2024.100560 http://creativecommons.org/licenses/by/4.0/ N. Debsikréo, M. Dehainsala, O. Debsikréo et al. IJID Regions 14 (2025) 100560 t M d t r g o i g E t r s d C b p t i p o o H o t a r u s t M S m t f e [ t f i s d o d t a S I i q A N c S a f t 2 v Q c d S P 1 fi a e D o e Q C a t g C t p R F p d S a r t p m l s r J K M L A H t K D A M A G F S i In addition to Central Africa, regions, including Northern Africa [ 7 ], he Amazon basin, Eastern Europe, the Mediterranean countries, the iddle East, and certain regions of Asia, are regions with a high en- emicity of HDV [ 9 ]. Besides its co-existence with HBV, the HDV geno- ypes or subtypes may have variable levels of virulence, such as the isk of developing chronic infection. Currently, HDV has eight distinct enotypes, each with two to four subtypes, with a sequence disparity f approximately 35% between genotypes. The predominant genotype n a geographic region varies with genetic diversity between and within enotypes [ 10 ]. Genotype 1 is the most widespread and predominates in urope and North America. Genotype 2 is most widespread in Asia and he Middle East. Compared with genotype 2, genotype 1 has a higher isk of adverse effects and lower remission rates [ 11 ]. Genotype 3 is as- ociated with severe liver disease, found in the Amazon basin, and is ifferent from the other genotypes. Genotype 4 is found in Taiwan and hina. Genotypes 5, 6, 7, and 8 are predominant in Africa and can also e found in Europe [ 12 ]. HDV is mainly transmitted parenterally through blood exposure, un- rotected sexual intercourse, or blood products, making mother-to-child ransmission very rare [ 13 ]. A person may either be simultaneously co- nfected with the two hepatitis viruses or be a carrier of HBV prior to ex- osure to HDV, which is referred to as superinfection [ 14 ]. Co-infection r superinfection may lead to fulminant forms or severe chronic course f the disease. It can change the course of asymptomatic or inactive BV infection into chronic active hepatitis, favoring the development f severe liver lesions and rapid progression to liver cirrhosis and hepa- ocellular carcinoma [ 15 ]. Even though seroprevalence of HDV antibodies (anti-HDV) is 14% mong chronic HBsAg carriers in N’Djamena hospitals [ 16 ], information egarding the circulating genotypes of HDV is scarcely available in Chad. Therefore, we sought to determine the prevalence of HDV in a pop- lation of HBsAg-positive pregnant women and students using archived amples from previous studies in Chad [ 17 , 18 ] and characterize the de- ected HBV and HDV. ethods tudy population This was a retrospective cross-sectional survey of 457 students (both ales and females) from the University of N’djamena (public institu- ion) and Emi Koussi University (private institution in N’djamena) Chad rom July 3 to July 23, 2021 [ 17 ] and 458 pregnant women from ight gynecology and obstetrics departments of hospitals in N’djamena 18 ]. The inclusion criteria for recruitment are already described in he earlier publications [ 17 , 18 ]. We used only the archived samples rom these studies for our study. After taking out seven samples with nsufficient sample volume, 94 frozen sera samples positive for HB- Ag from 47 males (all students) and 47 females (comprising 15 stu- ents and 32 pregnant women) stored at − 80 °C in the biobank lab- ratory of the National Reference Hospital were sent to the “Institut e Recherche en Santé, de Surveillance Epidémiologique et de Forma- ion ” (IRESSEF) in Senegal and INSERM U1052 in France for further nalysis. erologic assays Serologic tests were performed at the virology laboratory of the RESSEF. HBsAg positivity was confirmed using the Abbott Architect 1000SR analyzer (Abbott Diagnostics, Abbott Park, Illinois, USA), and uantification of HDV antibodies (HDV-Ab) was determined using ETI- B-DELTAK-2 kits (Diasorin, Italy). ucleic acid extraction DNA was extracted using the MagMAXTM Viral/Pathogen II Nu- leic Acid Kit on the KingFisherTM Flex automated system from Thermo 2 cientificTM , according to the manufacturer’s instructions. HBV-DNA nd HDV-RNA extractions from samples received at INSERM were per- ormed using the High Pure viral nucleic acid kit (Roche), according o the manufacturer’s instructions. The technique requires a volume of 00 μl of serum or plasma, and the nucleic acids were eluted in a final olume of 50 μl. uantification of hepatitis B virus and hepatitis B virus genotyping HBV viral load was determined using the GeneProof HBV polymerase hain reaction (PCR) kit with sensitivity up to 13.9 IU/ml and 100% iagnostic sensitivity [ 19 ]. Viral DNA of HBV samples had 1200 nt HBV gene amplified by semi-nested PCR, using the GoTaq® G2 Flexi DNA olymerase Kit. Two primer pairs, F8 /R5 and F3/R8, were used for PCR and PCR 2 amplification, respectively. The cycling conditions were ve initial denaturation at 94 °C, followed by 40 cycles of denaturation t 94 °C for 30 seconds, annealing at 42 °C for 30 seconds, and primer xtension at 72 °C for 1 minute before final extension of the amplified NA at 72 °C for 7 minutes. The PCR products were then visualized n 1.5% agarose gel under ultra violet illumination after staining with thidium bromide. uantification of hepatitis D virus and hepatitis D virus genotyping HDV viral loads (VL) were determined at the Centre de Recherche en ancérologie de Lyon INSERM U1052 laboratory (France) using ddPCR nd reverse transcription-ddPCR (Biorad). One-step quantitative reverse ranscription-PCR was performed using the Quantitect Virus kit (Qia- en, Courtaboeuf, France), following the manufacturer’s instructions. o-amplification of CI and HDV RNAs occurred in the same tube. CI de- ection was performed using the Quasar 670 labeled probe and primers rovided in the kit (Simplexa Extraction and Amplification Control Set- NA; Focus Diagnostics, Cypress, California, and Eurobio, Les Ulis, rance) using two direct primers (AgD-F1, AgD-F2) and one reverse rimer (AgD-R), following the previously described amplification con- itions [ 20 ]. equencing and phylogenetic analyses To determine HVB and HDV isolates, the HBV S gene (1200 nt) nd the HDV R0 region (400 nt) were sequenced by Sanger from pu- ified PCR fragments. The sequenced genetic elements were mapped o respective HBV and HDV reference sequences using the multi- le alignment fast Fourier transform (MAFFT) tool. We performed a odel test and constructed a phylogenetic tree based on the maximum ikelihood method with 1000 bootstrap replicates using the MEGA 7 oftware. For alignment and HDV genotyping, prototype HDV-sequences etrieved from the NCBI GenBank were used HDV-1: LS482933, X888110, JX888098, JX888105, JX888108, U81989, LT604946, J744227, MH457142, AM779579, L22066, AM902175, MG711717, G711801, JX888109, MG711713, MG711667, JN187436; HDV-2: 19598, LT604953; HDV-3: L22063, KC590319; HDV-4: AB118841, B088679; HDV-5: JX888107, U19598; HDV-6: JX888102, LT604968; DV-7: AM183333, LT604972, and HDV-8: LT604973, LT604974. For alignment and HBV-genotyping, HBV-genotype sequences re- rieved from the NCBI GenBank were used HBV genotype A: EU694385, P168423, GQ331046, KM606737, AM184126, FJ692601; genotype B: 23679, JQ801514; genotype C: DQ089781, KC774182; genotype D: B222711, MF925358; genotype E: KF170742, MT426116, MG621119, G821154, GQ161817, GQ161772, DQ060824, JQ000008, DQ060822, J605008, AM494689; genotype F: HM585194, DQ899143; genotype : AB056513; genotype H: FJ356715, and genotype I: AB562463; J023669. tatistical analysis The obtained data were entered into Microsoft Excel and imported nto STATA version 12.1 for data analysis. Quantitative variables were N. Debsikréo, M. Dehainsala, O. Debsikréo et al. IJID Regions 14 (2025) 100560 Table 1 Participant’s sociodemographic characteristics and history. Variable Pregnant women n = 32 Student n = 62 Total cohort n = 94 Age Mean = 25 Mean = 23 Mean = 24.39 ( ± 4.89) 18-24 15 (46.88) 42/62 (67.74) 57 (60.64) 25-34 11 (34.38) 20/62 (32.26) 31 (32.98) ≥ 35 6 (18.75) 0/62 (0.00) 6 (6.38) Gender Male 0 (00.00%) 47 (75.81) 47 (50.00) Female 32/32 (100.00) 15 (24.19) 47 (50.00) Marital status Single 0/32 (0.00) 46/62 (74.19) 46 (48.94) Married 32/32 (100.00) 16/62 (25.81) 48 (51.06) Potential risk factors Blood transfusions 12/32 (12.50) 10/62 (16.13) 14/94 (14.89) Hospital admission 8/32 (25.00) 13/62 (20.97) 21/62 (22.34) Mother hepatitis B surface antigen positive 4/32 (12.50) 21/62 (33.87° 25/94 (26.60) Family history hepatitis B virus 2/32 (6.25) 13/62 (20.97) 15/94 (15.96) Transcutaneous medical examinations 0/32 (00.00) 16/62 (25.81) 16/94 (17.02) Unprotected sex 31/32 (96.88) 20/62 (64.52) 71/94 (75.53) Injection drugs 0/32 (00.00) 1/62 (1.61) 1/94 (1.06) Jail 1/32 (3.13) 5/62 (8.06) 6/94 (6.38) e a w t v a t R C a ± xpressed as mean ± SD. Categoric variables were expressed as numbers nd percentages and compared using chi-square or Fisher’s exact test hen appropriate, whereas the independent t -test was used to compare he means of continuous variables. When normal distribution or equal ariance could not be assumed, the Mann-Whitney U test was used. With 95% confidence interval (CI), P -values < 0.05 were considered statis- ically significant. Table 2 Prevalence of hepatitis D virus among participants. Variable Categories Anti-hepatit Negative Cohort Pregnant women 27 (84.38) Student 58 (93.55) Age 18-24 52 (91.23) 25-34 28 (90.32) ≥ 35 5 (83.33) Gender Female 42 (89.36) Male 43 (91.49) Marital situation Single 42 (91.30) Married 43 (89.58) Blood transfusions No 73 (91.25) Yes 12 (85.71) Hospital admission No 65 (89.04) Yes 20 (95.24) Hepatitis B surface antigen positive mother No 55 (91.67) Yes 22 (88.00) Family history hepatitis B virus No 46 (85.19) Yes 15 (100.00) Transcutaneous medical examinations No 69 (88.46) Yes 16 (100.00) Unprotected sex No 22 (95.65) Yes 63 (88.73) Injection drugs No 85 (91.40) Yes 0 (0.00) Jail No 79 (89.77) Yes 6 (100.00) 3 esults haracteristics of the study population As shown below ( Table 1 ), a total of 94 samples from 62 students nd 32 pregnant women described elsewhere [ 17 , 18 ] (mean age 24.39 4.89 years) were included in this study. In addition to the 32 pregnant is D virus antibodies Crude odds ratio P (95% confidence interval) Positive 5 (15.63) 0.37 0.265 (0.09-1.49) 4 (6.45) 1.31 0.607 (0.45-3.77) 5 (8.77) 3 (9.68) 1 (16.67) 0.78 1.000 (0.19-3.11) 5 (10.64) 4 (8.51) 1.22 1.000 (0.30-4.86) 4 (8.70) 5 (10.42) 1.73 0.618 (0.32-9.38) 7 (8.75) 2 (9.57) 0.40 0.678 (0.04-3.44) 8 (10.96) 1 (4.76) 1.25 0.656 (0.47-3.33) 5 (8.33) 3 (12.00) 0.37 0.191 (0.11-1.28) 8 (14.81) 0 (0.00) NA NA 9(11.54) 0 (0.00) 2.79 0.445 (0.33-23.62) 1 (4.35) 8 (11.27) NA NA 8 (8.60) 1 (100.00) NA NA 9 (10.23) 0 (0.00) N. Debsikréo, M. Dehainsala, O. Debsikréo et al. IJID Regions 14 (2025) 100560 Figure 1. Flow chart of study participants. HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HDV, hepatitis D virus. w u w S ( f s A 9 w D R ( p ( c c H c m B u T e s d q E C n t w omen, there were 15 females among the students whose samples were sed for this study, given a 1:1 ratio. Whereas all the pregnant (100%) omen were married, only 16 (25.8%) of the students were married. eventy-one (75.53%) of the participants had had unprotected sex, 25 26.60%) had HBsAg-positive mothers, and other potential risk factors or HBV/HDV infection, including family history of HBV, blood transfu- ions, transcutaneous medical examinations, and injecting drugs. nti-hepatitis D virus antibodies seroprevalence and associated risk factors The HDV prevalence (anti-HDV antibodies) of the total cohort was .57% (9/64), whereas that of the students and the pregnant women as 6.45% (4/62) and 15.63% (5/32), respectively ( Table 2 ). etection and quantification of hepatitis B virus DNA and hepatitis D virus NA HBV DNA was detected in 52 of the 94 HBsAg-positive cases 55.31%) and HDV RNA in 7/9 (77.77%) of the anti-HDV-Ab-positive articipants. The median HDV viral load (VL) was 2.63 log10 copies/ml 1.18-3.22), with only one sample having a VL greater than 3 log10 4 opies/ml with three samples having a VL greater than 3 log10 opies/ml ( Figure 1 ). epatitis B virus and hepatitis D virus genotypes HBV genotyping was successfully performed for three samples, in- luding those from one pregnant woman and two male students. The odel test showed that the best substitution model with the lowest ayesian information criterion (BIC) score was the Kimura-2 parameter sing a discrete Gamma distribution with five rate categories (K2 + G). he phylogenetic tree comprised our three sequences and 31 HBV ref- rence sequences from all genotypes (34 sequences in total). Figure 2 hows that all HBV sequences from Chad belong to genotype E. In- eed, our sequences clustered close to HBV genotype E reference se- uences with a very high bootstrap value (99%) and closer to genotype sequences from neighboring countries, including Sudan, Nigeria, and ameroon. HDV genotyping was successful in six samples, including three preg- ant women, one female student, and two male students. The model est showed that the best substitution model with the lowest BIC score as the Tamura-3 parameter using a discrete Gamma distribution with N. Debsikréo, M. Dehainsala, O. Debsikréo et al. IJID Regions 14 (2025) 100560 Figure 2. Maximum likelihood phylogenetic tree of Chadian HBV. Isolates (981 nucleotides) and HBV genotypes A to J reference sequences retrieved from the GenBank database. The evolutionary history was inferred using the maximum likelihood method based on the Kimura 2-parameter (K2) model. Samples sequenced in this study are identified by the prefix TC. Reference sequences are identified by their accession number, country of origin, and (sub) genotype. The numbers at each node correspond to the bootstrap values ( > 70%) obtained with 1000 replicates. HBV, hepatitis B virus. fi s ( C H ( t G m n w P D a t p p i s l t H h ve rate categories (T92 + G). The phylogenetic tree comprised our ix sequences and 33 HDV reference sequences from all genotypes 39 sequences in total). Figure 3 shows that all HDV sequences from had belong to genotype 1. Indeed, our sequences clustered close to DV genotype 1 reference sequences with a very high bootstrap value 98%) and closer to genotype 1 sequences from neighboring coun- ries, including Cameroon, Nigeria, Central African Republic (CAR), and abon. Two patients (an 18-year-old pregnant woman and a 20-year-old ale student) were both co-infected with the HBV-E and HDV-1 combi- ation. The HBV and HDV nucleotide sequences generated from this study ere deposited in the GenBank database accession numbers PQ252377- Q252379 and PQ112532- PQ112537, respectively. 5 iscussion Co-infection of HBV and HDV poses a significant public health threat, nd there is limited data on the prevalence of HDV in Chad. Therefore, his study sought to estimate the prevalence of HDV infection among regnant women and students in Chad. Notwithstanding the small sam- le size used in this study, these populations are important because it s possible to limit the spread of infection through protective measures uch as vaccination and transmission of mother to child. The seropreva- ence of anti-HDV Ab was searched in a cohort of 94 individuals who ested positive for HBsAg, and molecular characterization of HBV and DV was performed in the HBsAg-positive and HDV-positive samples. The overall prevalence of anti-HDV Ab in the HBsAg-positive co- ort was 9.57%, which was low compared with the study carried out in N. Debsikréo, M. Dehainsala, O. Debsikréo et al. IJID Regions 14 (2025) 100560 Figure 3. Maximum likelihood phylogenetic tree of Chadian HDV. Isolates (367 nucleotides) and HDV genotypes 1 to 8 reference sequences retrieved from the GenBank database. The evolutionary history was inferred using the maximum likelihood method based on the Tamura 3-parameter (T92) model. Samples sequenced in this study are identified by the prefix TC. Reference sequences are identified by their accession number and country of origin. The numbers at each node correspond to the bootstrap values ( > 70%) obtained with 1000 replicates. HDV, hepatitis D virus. N b p i A o c n t m [ w G a l ’Djamena hospitals, which reported an overall prevalence of 14% [ 16 ] ut consistent with other reports from other African countries with a revalence between 4% and 15% [ 7 , 21 ]. Our results show that Chad s an HDV-endemic country, which aligns with the report that most frican countries have a high HDV endemicity [ 7 ]. The prevalence f anti-HDV Ab among pregnant women was higher (15.63% [5/32]) ompared with students (6.45% [4/62]), although the difference was ot significant ( P = 0.265, 95% confidence interval 0.09-1.49). Never- 6 heless, this finding highlights that HDV infection in pregnant women ight be a significant health problem, consistent with earlier reports 22 , 23 ]. The prevalence of HDV antibodies rate of 15.63% among pregnant omen in this study is consistent with the results of a study conducted in abon, which reported a prevalence of 15.6% in pregnant women [ 24 ] nd another in Mauritania with 14.7% prevalence [ 25 ]. However, it is ower than the 20.63% reported in Pakistan [ 26 ] but higher than the N. Debsikréo, M. Dehainsala, O. Debsikréo et al. IJID Regions 14 (2025) 100560 7 p a a i p d H p t a t a r b a s a [ p b p m h p d a i s o H t f N H l q s t L t o r t d C h ( H g p i p w m D F c E i t ( l ( f o E t a t A a A c t A d t R w f R [ .3% reported in Cameroon [ 27 ]. Similarly, the observed 6.45% HDV revalence among students in this study compares to the 5.4% reported mong Central African students [ 28 ]. It can also be said that students re younger and less often exposed to nosocomial contamination, which s often the mode of contamination for HDV [ 25 ]. HDV RNA load was detected in 77.77% of anti-HDV-positive partici- ants, of whom the majority had very low HBV DNA load. HBV DNA was etected in 55.32% of the HBsAg-positive individuals, confirming active BV infection. Interestingly, only 87.5% of anti-HDV-positive partici- ants also tested positive for HBV DNA. Moreover, HDV RNA was de- ected in 77.77% of anti-HDV-positive participant samples. These results re consistent with the available literature showing that HDV replica- ion is found in approximately 50% of anti-HDV-positive participants nd also agree with the assertion that HDV infection suppresses HBV eplication [ 2 ]. These findings highlight the importance of testing for oth HBV DNA and HDV RNA to accurately assess the status of HBV nd HDV infection. HDV can be transmitted by drug injection practices, including poorly terilized needles and medical instruments, re-use of disposable needles nd syringes, history of transfusion, and unprotected sexual intercourse 15 ]. Although several of such risk factors for HDV infection were ex- lored in this study, we did not find statistically significant associations etween these factors and HDV seropositivity among our small sam- le size. This discovery may explain further why pregnant women were ore likely to be HDV-positive compared with the students, with the ypothesis that the pregnant women were more likely to engage in un- rotected sex compared with the students who, even if sexually active, o engage in protected sex. A second hypothesis can also be that students re younger and less often exposed to nosocomial contamination, which s often the mode of contamination for HDV. However, these hypothe- es call for further research with a larger cohort for better exploration f the mentioned risks and other potential risk factors associated with DV infection in Chad. All the HBV sequences generated in our study belonged to geno- ype E, which is found almost exclusively in Sub-Saharan Africa. HBV rom our study clustered with HBV from neighboring countries (Sudan, igeria, and Cameroon), indicating the circulation of region-specific BV genotypes. All the HDV sequences generated in our study be- onged to the globally ubiquitous genotype 1. Interestingly, our se- uences clustered into two distinct groups within HDV-1, comprising equences isolated from the same country (Chad) or neighboring coun- ries (Cameroon, Nigeria, CAR, and Gabon). imitations and future directions Our study was limited by the small sample size, which may affect he generalizability of the results. In addition, the retrospective nature of ur study made it impossible to, unfortunately, get clinical data. Further esearch, including longitudinal studies with larger cohorts, is needed o better understand the epidemiology of HDV infection in Chad and to evelop effective prevention and control strategies. onclusion Despite the small sample size, the results of this study confirm the igh prevalence of HDV, particularly among pregnant Chadian women > 10%). In addition, HBV and HDV genotyping showed that HBV-E and DV-1 circulate in this region as expected. Finally, a combination of enotypes of both viruses was found in young adults. This study re- orted a prevalence of HDV of 15.63% in pregnant women and 6.45% n students. HDV viral RNA was detectable in 77.77% of the partici- ants. In Chad, there is certainly a need to test HBsAg-positive pregnant omen for total antibodies against HDV, as positive ones might present ore severe liver disease. 7 eclarations of competing interest The authors have no competing interests to declare. unding This research did not receive any specific grant from funding agen- ies in the public, commercial, or not-for-profit sectors. thics approval and consent to participate This study was approved by the National Bioethics Committee n Chad on November 8, 2020 (201/PR/MESRI/DG/CNBT/2020), he UCAD Research Ethics Committee on December 7, 2021 CER /UCAD/AD/MSN/050/2020), and the Ministry of Pub- ic Health and National Solidarity on February 18, 2021 N°294/PR/MSPSN/SE/DGM/DGTPC /DPERO2021). Informed consent rom all participants was obtained in accordance with the approval f the Chad National Bioethics Committee and the UCAD Research thics Committee. The objectives of the survey were presented to he participants. A unique anonymous study identifier number was ssigned to every participant. Each participant had been informed of he results of their serology and advised on the measures to be taken. ll experiments were performed in accordance with relevant guidelines nd regulations. cknowledgments The authors express their sincere thanks to Dr Isabelle CHEMIN, Can- er Research Centre INSERM U1052 of Lyon, for his help in quantifying he HDV viral load. uthor contributions ND, FLF, AMM, and NCT, conceived the project; ND oversaw the ata collection; ND, MD, OD GL, NL, MD, OD and IDO analyzed and in- erpreted the quantitative data, and, NL, GL, NAD and IDO, NAD, MNB, MT and IC processed the sample. 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PLOS Negl Trop Dis 2018; 12 :e0006377. doi: 10.1371/journal.pntd.0006377 . https://doi.org/10.1053/j.gastro.2006.01.035 https://doi.org/10.3748/wjg.v27.i42.7271 https://www.who.int/news-room/fact-sheets/detail/hepatitis-d https://doi.org/10.1128/JVI.01936-19 https://doi.org/10.3390/v13050830 https://doi.org/10.4236/ojgas.2022.129022 https://doi.org/10.1371/journal.pone.0273589 https://doi.org/10.26502/fjhs.123 http://www.geneproof.com/geneproof-hepatitis-b-virus-hbv-pcr-kit/p1093 https://doi.org/10.1128/JCM.06829-11 https://doi.org/10.1016/j.jhep.2020.04.008 https://doi.org/10.1016/j.ijregi.2024.100447 https://doi.org/10.1371/journal.pone.0287491 https://doi.org/10.1128/JCM.02142-07 https://doi.org/10.1002/jmv.23336 https://doi.org/10.1089/vim.2019.0048 https://doi.org/10.1371/journal.pone.0080346 https://doi.org/10.1371/journal.pntd.0006377 Prevalence and molecular characterization of hepatitis delta virus infection among hepatitis B virus surface antigen positive students and pregnant women in N’djamena, Chad Introduction Methods Study population Serologic assays Nucleic acid extraction Quantification of hepatitis B virus and hepatitis B virus genotyping Quantification of hepatitis D virus and hepatitis D virus genotyping Sequencing and phylogenetic analyses Statistical analysis Results Characteristics of the study population Anti-hepatitis D virus antibodies seroprevalence and associated risk factors Detection and quantification of hepatitis B virus DNA and hepatitis D virus RNA Hepatitis B virus and hepatitis D virus genotypes Discussion Limitations and future directions Conclusion Declarations of competing interest Funding Ethics approval and consent to participate Acknowledgments Author contributions References