Molecular Detection and Prevalence of Human Herpesvirus 6 (HHV-6) Among Male Blood Donors in Kassala, Sudan

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E. Musa, Abdelrhman A. Mohammed, Yasir B. Ahmed, Duaa H. Ibrahim, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8043922/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Human herpesvirus 6 (HHV-6) is a latent beta-herpesvirus with potential transfusion-transmissible relevance, particularly in immunocompromised recipients. Data on HHV-6 viremia among Sudanese blood donors are extremely limited. This study aimed to determine the molecular prevalence of HHV-6 DNA among blood donors in Kassala, Sudan. Methods: A descriptive cross-sectional study was conducted at Kassala Teaching Hospital. Whole-blood samples (n = 180) were screened for HHV-6 DNA using real-time PCR, followed by multiplex PCR for genotyping attempts. Fisher’s exact and Mann–Whitney U tests were used where applicable; however, statistical analyses were interpreted cautiously due to the small number of positive cases. Results: HHV-6 DNA was detected in 2.78% (5/180) of donors. No significant associations were found between HHV-6 positivity and age group, region, or ABO/Rh blood groups. Hematological parameters showed no significant differences between HHV-6–positive and negative donors. Genotyping was unsuccessful for all positive samples, likely due to low viral load (Ct values 38.7–39.9). Conclusion: HHV-6 viremia was infrequent among blood donors in Kassala. Given the very small number of positive cases and limited genotyping capacity, the findings should be considered preliminary. Larger multicenter studies incorporating more sensitive molecular methods are recommended to characterize the epidemiology of HHV-6 in Sudan. HHV-6 Blood donors Prevalence Real-time PCR Kassala Transfusion safety Introduction Blood transfusion plays a critical role in saving millions of lives each year; however, transfusion-transmitted infections (TTIs) remain a major concern, particularly in low-resource settings [ 1 ]. Screening donated blood is therefore essential to reduce the risk of transmitting infectious agents to recipients [ 2 ]. Human herpesvirus 6 (HHV-6) is a double-stranded DNA virus belonging to the Betaherpesvirinae subfamily [ 3 ]. First identified by Gallo and colleagues in 1986 in patients with AIDS and lymphoproliferative disorders [ 4 ], HHV-6 exists as two distinct species, HHV-6A and HHV-6B [ 5 ]. Primary infection typically occurs in early childhood, and HHV-6B is the etiological agent of exanthema subitum (roseola infantum) [ 6 – 10 ]. After primary infection, the virus establishes lifelong latency in various cells—such as monocytes, macrophages, bone marrow progenitors, and T lymphocytes—and may reactivate under conditions of immunosuppression [ 11 ]. HHV-6 reactivation is usually asymptomatic in immunocompetent individuals but can cause severe complications in immunocompromised hosts, including encephalitis, pneumonitis, bone marrow suppression, graft rejection, and increased morbidity and mortality following hematopoietic stem cell transplantation (HSCT) or solid organ transplantation (SOT) [ 12 – 15 ]. A unique biological feature of HHV-6 is its ability to integrate into human chromosomes, producing chromosomally integrated HHV-6 (ciHHV-6), which affects approximately 1% of the population [ 16 – 18 ]. Individuals with ciHHV-6 exhibit persistently high viral DNA loads, potentially complicating clinical interpretation and leading to misdiagnosis. Although HHV-6 is primarily transmitted through saliva, transmission via blood transfusion and organ transplantation has also been reported [ 19 , 20 ]. In sub-Saharan Africa, blood safety challenges remain significant, and targeted studies on HHV-6 in the transfusion context are scarce. Previous investigations have reported variable HHV-6 prevalence: 6.1% in Burkina Faso [ 22 ], dominant HHV-6A infections in Zambia [ 21 ], 3.49% in Greece [ 26 ], 24.3% in Qatar [ 20 ], and 16.2% in China [ 24 ]. These variations may be attributed to geographical differences, population immunity, methodological approaches, and resource limitations in molecular diagnostics. In Sudan, despite the clinical relevance of HHV-6, no molecular data are available regarding its prevalence among blood donors. This study was therefore conducted to determine the molecular prevalence of HHV-6 DNA among blood donors in Kassala, Sudan, and to attempt genotype differentiation for positive samples. Methods Study Design and Setting A descriptive cross-sectional study was conducted at Kassala Teaching Hospital, Kassala State, Sudan. The study was carried out as part of a research protocol approved in 2022, and blood samples were collected during the study period in accordance with the approved ethical clearance. Study Population and Rationale for Inclusion of Male Donors The study population consisted exclusively of male blood donors. In Kassala, female blood donation rates are extremely low due to cultural, social, and physiological factors, resulting in insufficient female donor availability during the study period. Therefore, only male donors met the eligibility criteria, allowing for consistent recruitment without introducing sampling bias. Sample Size and Sampling Technique A total of 180 donors were enrolled using a convenience sampling technique. This method was selected due to donor availability, logistical limitations, and the need for timely laboratory processing. While convenience sampling limits generalizability, it remains appropriate for descriptive virological studies in similar settings. Eligibility Criteria The inclusion criteria were: male blood donors aged 18–55 years, clinically healthy at the time of donation, meeting WHO donor criteria, and providing informed consent [ 23 ]. Exclusion criteria included seropositivity for HBV, HCV, HIV, or syphilis; recent febrile illness; immunosuppressive therapy; and insufficient blood sample volume. Sample Collection and Processing Due to the war and the loss of some initial samples, the sample collection process was repeated. All samples included in the final analysis were collected between March 2024 and July 2024, and all collections occurred within the validity period of the approved ethical clearance. Five milliliters of venous blood were drawn into EDTA tubes. Samples were divided into two parts: one for CBC analysis and the other for DNA extraction. Plasma was separated by centrifugation at 3,000 rpm for 5 minutes and stored at − 20°C until testing. Complete Blood Count (CBC) Analysis CBC was performed using the Mindray BC-30s automated hematology analyzer with tri-level quality control. Standard adult reference ranges were used for WBCs, RBCs, hemoglobin, hematocrit, platelets, and differential counts. Manual differential assessment for monocytes was performed when required. DNA Extraction Viral DNA was extracted from plasma using a commercial kit (Beijing Origin Gene-Tech Biotechnology Co., Ltd., China). An extraction blank was included with each batch, and strict separation was maintained between pre-PCR and post-PCR areas. Filtered pipette tips were used to prevent contamination. Real-Time PCR Detection of HHV-6 DNA HHV-6 DNA was detected using a TaqMan real-time PCR assay targeting a conserved genomic region, following the method described by Zheng et al. (2021). Macrogen synthesized primers and probes. β-globin amplification served as an internal control to assess extraction efficiency and potential PCR inhibition. PCR Reaction Setup and Cycling Conditions Each reaction consisted of 5 µL extracted DNA within a total volume of 25 µL, including primers, probe, dNTP mix, MgCl₂, Taq polymerase, and PCR buffer. Reactions were performed on the LineGene 9600 platform with an initial denaturation at 94°C for 5 minutes, followed by 40 cycles of 94°C for 30 seconds and 60°C for 1 minute. Samples were run in duplicate, with positive, negative, and extraction controls included in every run. Samples with Ct ≤ 40 were considered positive. Multiplex PCR for HHV-6 Genotyping Genotyping was attempted using a multiplex TaqMan assay adapted from Leibovitch et al. (2014), targeting the U57 gene with FAM-labeled probe for HHV-6A and HEX-labeled probe for HHV-6B. The same cycling conditions as those used in the real-time PCR assay were employed. Samples without amplification for either genotype were labeled “Undetermined.” Statistical Analysis Data analysis was performed using SPSS version 28. Categorical variables were expressed as frequencies and percentages, while continuous variables were reported as mean ± standard error. Fisher’s exact test was used for categorical comparisons, and the Mann-Whitney U test for nonparametric continuous variables. Due to the small number of HHV-6–positive samples, results were interpreted cautiously as exploratory. Results A total of 180 male donors were included in the analysis. HHV-6 DNA was detected in 5 donors (2.78%). As shown in Table 1 , no statistically significant associations were observed between HHV-6 detection and age group, region of residence, or ABO/Rh blood groups (all p > 0.05, Fisher’s exact test). Four positive cases occurred among donors aged 19–35 years (2.6%), and one case occurred among donors aged 36–52 years (3.6%). All positive donors originated from Kassala, whereas no cases were detected in donors from other regions; however, the number of donors from these regions was minimal. These findings suggest no clear demographic pattern of HHV-6 positivity in this cohort, although the small number of positive samples limits interpretation. Table 1 Association of HHV-6 Detection with Demographic and Blood Group Characteristics among Blood Donors Variable Category Total Samples HHV-6 Positive n (%) Fisher’s exact test P-value Age Group (years) 19–35 152 4 (2.6%) 0.575 36–52 28 1 (3.6%) Region Kassala 175 5 (2.9%) 1.000 Khartoum 2 0 (0.0%) Al-Jazeera 2 0 (0.0%) Darfur 1 0 (0.0%) Blood Group O+ 85 2 (2.4%) 0.639 B+ 60 2 (3.3%) A+ 25 1 (4.0%) AB+ 5 0 (0.0%) Rh− (O−, B−) 5 0 (0.0%) A comparison of hematological parameters between HHV-6 RT-PCR–negative blood donors showed no statistically significant differences across all measured indices. Total white blood cell counts were slightly lower in the positive group (4.58 ± 0.59) than in the negative group (5.10 ± 0.13), yielding a small mean difference of − 0.52 (t = 0.727, p = 0.409). Neutrophil counts displayed a higher mean in the positive donors (34.52 ± 3.58) than in the negative donors (43.27 ± 0.96), with a mean difference of − 8.75 (t = 1.526, p = 0.105). Lymphocyte levels were also moderately elevated among HHV-6 positive donors (54.98 ± 3.10) compared with the negative group (46.00 ± 0.93), resulting in a mean difference of 8.98 (t = 1.617, p = 0.057). Monocyte values were similar between the groups, with a negligible mean difference of − 0.08 (t = 0.036, p = 0.680). Platelet counts were slightly higher in positive donors (247.6 ± 43.19) than in negative donors (236.6 ± 7.0), with a mean difference of 11.0 (t = 0.267, p = 0.659). For all parameters, the 95% confidence intervals of the mean differences crossed zero, and the corresponding Mann–Whitney U values further confirmed the absence of statistically significant variation between groups ( Table 2 ). Table 2 Analysis of hematological parameters in HHV-6 positive and negative blood donors Parameter Positive-RT-PCR MEAN ± SE Negative-RT-PCR MEAN ± SE Mean Diff. T 95% CI of diff Mann-Whitney U P-value TWBCs 4.580 ± 0.5928 5.105 ± 0.1260 -0.5255 0.7270 -2.243to1.192 322.5 0.4091 Neutrophils 34.52 ± 3.583 43.27 ± 0.9626 -8.746 1.526 -22.60 to 5.104 245.0 0.1051 Lymphocytes 54.98 ± 3.103 46.00 ± 0.9327 8.976 1.617 -4.438to22.39 213.5 0.0573 Monocytes 8.200 ± 2.577 8.284 ± 0.3577 -0.08363 0.03565 -4.713to4.545 381.0 0.6803 Platelets 247.6 ± 43.19 236.6 ± 6.994 11.00 0.2670 -70.27 to 92.27 377.5 0.6594 All five HHV-6–positive samples failed to yield type-specific amplification for HHV-6A or HHV-6B in the multiplex assay. This was consistent with their high cycle threshold (Ct) values (38.7–39.9), indicating low viral DNA levels that may be below the detection limit for reliable genotyping ( Table 3 ). Table 3 HHV-6 genotyping results and corresponding viral load (Ct values) of positive samples Total Positive Samples Genotype A Genotype B Ct Range 5 Undetermined Undetermined 38.7–39.9 Overall, HHV-6 DNA was detected at a low prevalence of 2.78% among male blood donors in Kassala. No significant demographic, geographic, or hematological patterns were associated with HHV-6 positivity, and genotyping could not be completed due to low viral load. Discussion The present study provides the first molecular evidence of HHV-6 DNA detection among blood donors in Sudan, revealing a low prevalence of 2.78%. This finding aligns with reports from low-endemic regions such as Greece (3.49%) [ 26 ] and Burkina Faso (6.1%) [ 22 ], but remains lower than the higher prevalence rates documented in Qatar (24.3%) [ 20 ], China (16.2%) [ 24 ], and Zambia, where HHV-6A predominated [ 21 ]. These variations may reflect geographical differences, climatic and environmental factors, population immunity, socioeconomic conditions, and importantly, differences in methodological sensitivity across studies. In the current study, HHV-6 detection showed no significant association with donor age, geographic region, or ABO/Rh blood groups. The small number of positive cases limits the ability to detect subtle associations, and the uneven representation of donors from regions outside Kassala may have further influenced these observations. Nevertheless, the absence of demographic correlations is consistent with prior literature suggesting that most adults have been exposed to HHV-6 early in life, resulting in a relatively uniform prevalence across adult age groups [ 20 ]. Similarly, no established biological mechanism links HHV-6 susceptibility to ABO or Rh blood group antigens, and previous epidemiological studies rarely report any such association. Hematological findings among HHV-6–positive donors showed a pattern of slightly reduced total WBC counts and modest lymphocytosis compared with negative donors. Although these differences were statistically insignificant, they resemble transient hematological alterations described during HHV-6 reactivation or low-level viremia [ 15 ]. However, given the very small number of positive donors and the wide confidence intervals, these findings should be interpreted cautiously and considered exploratory rather than indicative of a true biological effect. Larger studies with more robust analytical power are needed to clarify whether mild hematological changes accompany HHV-6 viremia in asymptomatic adults. Genotyping attempts for HHV-6A and HHV-6B were unsuccessful for all five positive samples, most likely due to the low viral loads reflected by high Ct values (38.7–39.9). This phenomenon has been described in previous studies, where samples with low-level viremia or inherited chromosomally integrated HHV-6 (ciHHV-6) show limited amplification efficiency, leading to “Undetermined” genotype results [ 25 , 17 ]. Advanced molecular approaches such as droplet digital PCR, nested PCR, or next-generation sequencing may be required in future research to improve detection sensitivity and enable reliable genotype differentiation in low-copy samples. Although HHV-6 is not routinely screened in blood donation programs, the virus has recognized clinical significance in immunocompromised individuals. HHV-6 reactivation has been associated with severe complications, including encephalitis, pneumonitis, delayed engraftment, and graft-versus-host disease, especially among hematopoietic stem-cell transplant (HSCT) recipients [ 12 – 15 ]. Studies from Egypt and Germany have documented high reactivation rates and clinically significant outcomes in transplant settings [ 15 , 27 ]. While the present study does not assess transfusion transmission directly, detecting HHV-6 DNA in blood donors highlights a potential concern for high-risk recipients. Accordingly, these results support the need for further research to determine whether HHV-6 viremia in donors may play a role in transfusion-related infection or reactivation in vulnerable patients. The findings also contribute to the broader understanding of transfusion-transmissible infections (TTIs) in Sudan, where data on emerging viral pathogens remain limited. Strengthening molecular surveillance—especially in high-risk recipient groups—may enhance early detection and support evidence-based policy development. Implementing pilot screening strategies, cost-effectiveness analyses, and integration of more sensitive molecular assays into TTI monitoring systems may ultimately improve transfusion safety in resource-limited settings. Overall, although the prevalence observed in this study is low, the detection of HHV-6 DNA among asymptomatic donors represents an important initial step in establishing baseline epidemiological data for Sudan. Future multicenter studies incorporating larger sample sizes, serological testing, and more sensitive molecular methods are warranted to better characterize the epidemiology and transmission potential of HHV-6 in the Sudanese population. Limitations This study had several limitations. First, the relatively small number of HHV-6–positive cases (n = 5) limits statistical power and prevents meaningful subgroup analysis. Second, the use of convenience sampling at a single center may reduce the generalizability of the findings to other regions in Sudan, especially rural areas, because the samples are unlikely to represent the diverse socioeconomic and healthcare profiles of the broader population. Third, the exclusive inclusion of male donors does not reflect the prevalence in the general population and may skew the results, particularly when it comes to health and risk factors. Fourth, the inability to genotype HHV-6A and HHV-6B was likely due to the low viral load and limited sensitivity of the multiplex PCR assay used. Finally, the absence of serological testing prevented the distinction between active, latent, and chromosomally integrated HHV-6. Conclusion HHV-6 DNA was detected at a low prevalence among blood donors in Kassala, Sudan. The absence of genotype identification and the small number of positive cases reflect the need for larger multicenter studies using more sensitive molecular methods such as digital PCR or nested PCR. Future investigations incorporating both molecular and serological approaches would improve understanding of HHV-6 epidemiology and help determine its relevance to transfusion safety in low-resource settings. Abbreviations aGVHD Acute Graft-versus-Host Disease CBC Complete Blood Count ciHHV-6 Chromosomally Integrated HHV-6 Ct Cycle Threshold DNA Deoxyribonucleic Acid HHV-6 Human Herpesvirus 6 RT-PCR Real-Time Polymerase Chain Reaction TTIs Transfusion-Transmitted Infections Declarations Acknowledgements None Author contributions DM and NA conceived and designed the study. DM, YA, DI, LA, GM, AA, and NA contributed to the data collection, analysis, and interpretation of the results. NA drafted and revised the manuscript and performed statistical analyses. All authors critically reviewed and approved the final version of this manuscript for publication. Funding This research received no funding from public, commercial, or non-profit sectors. Availability of data and materials The research data are available upon request. Declarations Ethics approval and consent to participate The study protocol was reviewed and approved by the Ethical Review Board of the Karary University Postgraduate Ethics Committee under Protocol No. [KU-PG-2022-011] and the Sudan Ministry of Health Research Ethics Committee under Reference Number: [MOH-ERC-2022-045]. All participants were informed of the purpose, procedures, and potential benefits of the study. Written informed consent was obtained from each donor before participation. The confidentiality of donor information was strictly maintained, and all data were used solely for research purposes. 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Coinfection of human herpesviruses 6A (HHV-6A) and HHV-6B as demonstrated by novel digital droplet PCR assay. PLoS One . 2014;9(3):e92328. Published 2014 Mar 24. doi:10.1371/journal.pone.0092328 Rouka E, Kyriakou D. Molecular epidemiology of human Herpesviruses types 1-6 and 8 among Greek blood donors. Transfus Med . 2015;25(4):276-279. doi:10.1111/tme.12202 Hentrich M, Oruzio D, Jäger G, et al. Impact of human herpesvirus-6 after haematopoietic stem cell transplantation. Br J Haematol . 2005;128(1):66-72. doi:10.1111/j.1365-2141.2004.05254.x Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8043922","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":560301631,"identity":"7a8d22c7-c39e-4a5f-9046-062f14728676","order_by":0,"name":"Doaa M. E. 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Ibrahim","email":"","orcid":"","institution":"Kassala University","correspondingAuthor":false,"prefix":"","firstName":"Duaa","middleName":"H.","lastName":"Ibrahim","suffix":""},{"id":560301638,"identity":"66dbf9a6-6922-4f2d-81ea-a30ccb5cb1df","order_by":4,"name":"Leila M. A. Abdelgader","email":"","orcid":"","institution":"Shendi University","correspondingAuthor":false,"prefix":"","firstName":"Leila","middleName":"M. A.","lastName":"Abdelgader","suffix":""},{"id":560301640,"identity":"466ec13d-e760-455f-be43-13ef0f7121da","order_by":5,"name":"Ghanem M. Mahjaf","email":"","orcid":"","institution":"Shendi University","correspondingAuthor":false,"prefix":"","firstName":"Ghanem","middleName":"M.","lastName":"Mahjaf","suffix":""},{"id":560301641,"identity":"8ff54a7d-187c-4f91-bbe8-81f3fb7e5e58","order_by":6,"name":"Alhaj S. M. A. Ali","email":"","orcid":"","institution":"Shendi University","correspondingAuthor":false,"prefix":"","firstName":"Alhaj","middleName":"S. M. A.","lastName":"Ali","suffix":""},{"id":560301643,"identity":"209e3ab8-23c5-418a-a1a7-171b917dfa69","order_by":7,"name":"Nadir M. Abuzeid","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYHACgwMMDEDEwNgg8YFBAiwCxMzEaZGcQawWBogWBgZpHoQIbi3m7Ic3Hq6ouZM4v7258bZNhUViA3vzNgmGP9Y4tVj2pBUcPHPsWWJjz8Fm65wzEokNPMfKJBjb0vF4JMfgYAPb4cRmicQ26dw2oBaJHDMJxobDuLWcfwPU8u9wYpv8wzZpS5AW+TdmQIfh0XIDaEtj2+HEHqBjpBnBtvAAtbDh0/Ks4GBj3zPjGTyJzZY9ZySM23jSii0S8fnlfPLmjw3f7sjObz/+8MaPijrZfmAY3viAJ8QwARuISCBBwygYBaNgFIwCTAAAzrBbEOOKDEUAAAAASUVORK5CYII=","orcid":"","institution":"Delta College of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Nadir","middleName":"M.","lastName":"Abuzeid","suffix":""}],"badges":[],"createdAt":"2025-11-06 05:23:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8043922/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8043922/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98412277,"identity":"7e3d059b-05e2-4157-a611-c8198b4e5891","added_by":"auto","created_at":"2025-12-17 14:06:41","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":52148,"visible":true,"origin":"","legend":"","description":"","filename":"RevisedManuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8043922/v1/761d853c9c0a4ebc29bb67e6.docx"},{"id":98441686,"identity":"2ed667b1-1587-468b-8af4-8089180e8130","added_by":"auto","created_at":"2025-12-17 17:05:42","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8806,"visible":true,"origin":"","legend":"","description":"","filename":"1c13abf2bf7f45b1b3682c6be62b8069.json","url":"https://assets-eu.researchsquare.com/files/rs-8043922/v1/d2c2bf303a23f7aa86b70914.json"},{"id":98412282,"identity":"d980db49-c1bb-45c2-84a9-0bca36a7c3d5","added_by":"auto","created_at":"2025-12-17 14:06:41","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82765,"visible":true,"origin":"","legend":"","description":"","filename":"1c13abf2bf7f45b1b3682c6be62b80691enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8043922/v1/2351f4f77e8d2f6aa74db85d.xml"},{"id":98412279,"identity":"0d04cb0d-2259-49ba-a084-2f1f31726a79","added_by":"auto","created_at":"2025-12-17 14:06:41","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":80392,"visible":true,"origin":"","legend":"","description":"","filename":"1c13abf2bf7f45b1b3682c6be62b80691structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8043922/v1/2698669b4da706da534690c8.xml"},{"id":98412281,"identity":"d9407f4c-779e-4e3d-82db-d05adc95d24b","added_by":"auto","created_at":"2025-12-17 14:06:41","extension":"html","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":94395,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8043922/v1/28bf50cda5e8da42cd6d6ded.html"},{"id":102822456,"identity":"f9530d36-a0ad-48c5-9f78-7b3f2e0f3564","added_by":"auto","created_at":"2026-02-17 07:56:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":800982,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8043922/v1/670d2c90-699d-4724-819e-d9f8b99ddb1b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular Detection and Prevalence of Human Herpesvirus 6 (HHV-6) Among Male Blood Donors in Kassala, Sudan","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBlood transfusion plays a critical role in saving millions of lives each year; however, transfusion-transmitted infections (TTIs) remain a major concern, particularly in low-resource settings [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Screening donated blood is therefore essential to reduce the risk of transmitting infectious agents to recipients [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Human herpesvirus 6 (HHV-6) is a double-stranded DNA virus belonging to the \u003cem\u003eBetaherpesvirinae\u003c/em\u003e subfamily [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. First identified by Gallo and colleagues in 1986 in patients with AIDS and lymphoproliferative disorders [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], HHV-6 exists as two distinct species, HHV-6A and HHV-6B [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Primary infection typically occurs in early childhood, and HHV-6B is the etiological agent of exanthema subitum (roseola infantum) [\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. After primary infection, the virus establishes lifelong latency in various cells\u0026mdash;such as monocytes, macrophages, bone marrow progenitors, and T lymphocytes\u0026mdash;and may reactivate under conditions of immunosuppression [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. HHV-6 reactivation is usually asymptomatic in immunocompetent individuals but can cause severe complications in immunocompromised hosts, including encephalitis, pneumonitis, bone marrow suppression, graft rejection, and increased morbidity and mortality following hematopoietic stem cell transplantation (HSCT) or solid organ transplantation (SOT) [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. A unique biological feature of HHV-6 is its ability to integrate into human chromosomes, producing chromosomally integrated HHV-6 (ciHHV-6), which affects approximately 1% of the population [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Individuals with ciHHV-6 exhibit persistently high viral DNA loads, potentially complicating clinical interpretation and leading to misdiagnosis. Although HHV-6 is primarily transmitted through saliva, transmission via blood transfusion and organ transplantation has also been reported [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In sub-Saharan Africa, blood safety challenges remain significant, and targeted studies on HHV-6 in the transfusion context are scarce. Previous investigations have reported variable HHV-6 prevalence: 6.1% in Burkina Faso [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], dominant HHV-6A infections in Zambia [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], 3.49% in Greece [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], 24.3% in Qatar [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and 16.2% in China [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These variations may be attributed to geographical differences, population immunity, methodological approaches, and resource limitations in molecular diagnostics. In Sudan, despite the clinical relevance of HHV-6, no molecular data are available regarding its prevalence among blood donors. This study was therefore conducted to determine the molecular prevalence of HHV-6 DNA among blood donors in Kassala, Sudan, and to attempt genotype differentiation for positive samples.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design and Setting\u003c/h2\u003e \u003cp\u003eA descriptive cross-sectional study was conducted at Kassala Teaching Hospital, Kassala State, Sudan. The study was carried out as part of a research protocol approved in 2022, and blood samples were collected during the study period in accordance with the approved ethical clearance.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy Population and Rationale for Inclusion of Male Donors\u003c/h3\u003e\n\u003cp\u003eThe study population consisted exclusively of male blood donors. In Kassala, female blood donation rates are extremely low due to cultural, social, and physiological factors, resulting in insufficient female donor availability during the study period. Therefore, only male donors met the eligibility criteria, allowing for consistent recruitment without introducing sampling bias.\u003c/p\u003e\n\u003ch3\u003eSample Size and Sampling Technique\u003c/h3\u003e\n\u003cp\u003eA total of 180 donors were enrolled using a convenience sampling technique. This method was selected due to donor availability, logistical limitations, and the need for timely laboratory processing. While convenience sampling limits generalizability, it remains appropriate for descriptive virological studies in similar settings.\u003c/p\u003e\n\u003ch3\u003eEligibility Criteria\u003c/h3\u003e\n\u003cp\u003eThe inclusion criteria were: male blood donors aged 18\u0026ndash;55 years, clinically healthy at the time of donation, meeting WHO donor criteria, and providing informed consent [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Exclusion criteria included seropositivity for HBV, HCV, HIV, or syphilis; recent febrile illness; immunosuppressive therapy; and insufficient blood sample volume.\u003c/p\u003e\n\u003ch3\u003eSample Collection and Processing\u003c/h3\u003e\n\u003cp\u003eDue to the war and the loss of some initial samples, the sample collection process was repeated. All samples included in the final analysis were collected between March 2024 and July 2024, and all collections occurred within the validity period of the approved ethical clearance. Five milliliters of venous blood were drawn into EDTA tubes. Samples were divided into two parts: one for CBC analysis and the other for DNA extraction. Plasma was separated by centrifugation at 3,000 rpm for 5 minutes and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until testing.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eComplete Blood Count (CBC) Analysis\u003c/h2\u003e \u003cp\u003eCBC was performed using the Mindray BC-30s automated hematology analyzer with tri-level quality control. Standard adult reference ranges were used for WBCs, RBCs, hemoglobin, hematocrit, platelets, and differential counts. Manual differential assessment for monocytes was performed when required.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA Extraction\u003c/h3\u003e\n\u003cp\u003eViral DNA was extracted from plasma using a commercial kit (Beijing Origin Gene-Tech Biotechnology Co., Ltd., China). An extraction blank was included with each batch, and strict separation was maintained between pre-PCR and post-PCR areas. Filtered pipette tips were used to prevent contamination.\u003c/p\u003e\n\u003ch3\u003eReal-Time PCR Detection of HHV-6 DNA\u003c/h3\u003e\n\u003cp\u003eHHV-6 DNA was detected using a TaqMan real-time PCR assay targeting a conserved genomic region, following the method described by Zheng et al. (2021). Macrogen synthesized primers and probes. β-globin amplification served as an internal control to assess extraction efficiency and potential PCR inhibition.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePCR Reaction Setup and Cycling Conditions\u003c/h2\u003e \u003cp\u003eEach reaction consisted of 5 \u0026micro;L extracted DNA within a total volume of 25 \u0026micro;L, including primers, probe, dNTP mix, MgCl₂, Taq polymerase, and PCR buffer. Reactions were performed on the LineGene 9600 platform with an initial denaturation at 94\u0026deg;C for 5 minutes, followed by 40 cycles of 94\u0026deg;C for 30 seconds and 60\u0026deg;C for 1 minute. Samples were run in duplicate, with positive, negative, and extraction controls included in every run. Samples with Ct\u0026thinsp;\u0026le;\u0026thinsp;40 were considered positive.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMultiplex PCR for HHV-6 Genotyping\u003c/h2\u003e \u003cp\u003eGenotyping was attempted using a multiplex TaqMan assay adapted from Leibovitch et al. (2014), targeting the U57 gene with FAM-labeled probe for HHV-6A and HEX-labeled probe for HHV-6B. The same cycling conditions as those used in the real-time PCR assay were employed. Samples without amplification for either genotype were labeled \u0026ldquo;Undetermined.\u0026rdquo;\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData analysis was performed using SPSS version 28. Categorical variables were expressed as frequencies and percentages, while continuous variables were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Fisher\u0026rsquo;s exact test was used for categorical comparisons, and the Mann-Whitney U test for nonparametric continuous variables. Due to the small number of HHV-6\u0026ndash;positive samples, results were interpreted cautiously as exploratory.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 180 male donors were included in the analysis. HHV-6 DNA was detected in 5 donors (2.78%). As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, no statistically significant associations were observed between HHV-6 detection and age group, region of residence, or ABO/Rh blood groups (all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fisher\u0026rsquo;s exact test). Four positive cases occurred among donors aged 19\u0026ndash;35 years (2.6%), and one case occurred among donors aged 36\u0026ndash;52 years (3.6%). All positive donors originated from Kassala, whereas no cases were detected in donors from other regions; however, the number of donors from these regions was minimal. These findings suggest no clear demographic pattern of HHV-6 positivity in this cohort, although the small number of positive samples limits interpretation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAssociation of HHV-6 Detection with Demographic and Blood Group Characteristics among Blood Donors\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCategory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal Samples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHHV-6 Positive n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFisher\u0026rsquo;s exact test P-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eAge Group (years)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19\u0026ndash;35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e152\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.575\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u0026ndash;52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1 (3.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cb\u003eRegion\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKassala\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e175\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5 (2.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKhartoum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0 (0.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAl-Jazeera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0 (0.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDarfur\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0 (0.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003eBlood Group\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eO+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2 (2.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e0.639\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2 (3.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1 (4.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0 (0.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRh\u0026minus; (O\u0026minus;, B\u0026minus;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0 (0.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA comparison of hematological parameters between HHV-6 RT-PCR\u0026ndash;negative blood donors showed no statistically significant differences across all measured indices. Total white blood cell counts were slightly lower in the positive group (4.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59) than in the negative group (5.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13), yielding a small mean difference of \u0026minus;\u0026thinsp;0.52 (t\u0026thinsp;=\u0026thinsp;0.727, p\u0026thinsp;=\u0026thinsp;0.409). Neutrophil counts displayed a higher mean in the positive donors (34.52\u0026thinsp;\u0026plusmn;\u0026thinsp;3.58) than in the negative donors (43.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96), with a mean difference of \u0026minus;\u0026thinsp;8.75 (t\u0026thinsp;=\u0026thinsp;1.526, p\u0026thinsp;=\u0026thinsp;0.105). Lymphocyte levels were also moderately elevated among HHV-6 positive donors (54.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.10) compared with the negative group (46.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93), resulting in a mean difference of 8.98 (t\u0026thinsp;=\u0026thinsp;1.617, p\u0026thinsp;=\u0026thinsp;0.057). Monocyte values were similar between the groups, with a negligible mean difference of \u0026minus;\u0026thinsp;0.08 (t\u0026thinsp;=\u0026thinsp;0.036, p\u0026thinsp;=\u0026thinsp;0.680). Platelet counts were slightly higher in positive donors (247.6\u0026thinsp;\u0026plusmn;\u0026thinsp;43.19) than in negative donors (236.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.0), with a mean difference of 11.0 (t\u0026thinsp;=\u0026thinsp;0.267, p\u0026thinsp;=\u0026thinsp;0.659). For all parameters, the 95% confidence intervals of the mean differences crossed zero, and the corresponding Mann\u0026ndash;Whitney U values further confirmed the absence of statistically significant variation between groups \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis of hematological parameters in HHV-6 positive and negative blood donors\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive-RT-PCR\u003c/p\u003e \u003cp\u003eMEAN\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative-RT-PCR\u003c/p\u003e \u003cp\u003eMEAN\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean Diff.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e95% CI of diff\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMann-Whitney U\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTWBCs\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.580\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5928\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.105\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.5255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.7270\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-2.243to1.192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e322.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.4091\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNeutrophils\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e34.52\u0026thinsp;\u0026plusmn;\u0026thinsp;3.583\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e43.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9626\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-8.746\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.526\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-22.60 to 5.104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e245.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.1051\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLymphocytes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e54.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e46.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9327\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.976\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-4.438to22.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e213.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0573\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMonocytes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.200\u0026thinsp;\u0026plusmn;\u0026thinsp;2.577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e8.284\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.08363\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.03565\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-4.713to4.545\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e381.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.6803\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePlatelets\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e247.6\u0026thinsp;\u0026plusmn;\u0026thinsp;43.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e236.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.994\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.2670\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-70.27 to 92.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e377.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.6594\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll five HHV-6\u0026ndash;positive samples failed to yield type-specific amplification for HHV-6A or HHV-6B in the multiplex assay. This was consistent with their high cycle threshold (Ct) values (38.7\u0026ndash;39.9), indicating low viral DNA levels that may be below the detection limit for reliable genotyping \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHHV-6 genotyping results and corresponding viral load (Ct values) of positive samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Positive Samples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenotype A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGenotype B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCt Range\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUndetermined\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUndetermined\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.7\u0026ndash;39.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOverall, HHV-6 DNA was detected at a low prevalence of 2.78% among male blood donors in Kassala. No significant demographic, geographic, or hematological patterns were associated with HHV-6 positivity, and genotyping could not be completed due to low viral load.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study provides the first molecular evidence of HHV-6 DNA detection among blood donors in Sudan, revealing a low prevalence of 2.78%. This finding aligns with reports from low-endemic regions such as Greece (3.49%) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and Burkina Faso (6.1%) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], but remains lower than the higher prevalence rates documented in Qatar (24.3%) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], China (16.2%) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and Zambia, where HHV-6A predominated [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These variations may reflect geographical differences, climatic and environmental factors, population immunity, socioeconomic conditions, and importantly, differences in methodological sensitivity across studies. In the current study, HHV-6 detection showed no significant association with donor age, geographic region, or ABO/Rh blood groups. The small number of positive cases limits the ability to detect subtle associations, and the uneven representation of donors from regions outside Kassala may have further influenced these observations. Nevertheless, the absence of demographic correlations is consistent with prior literature suggesting that most adults have been exposed to HHV-6 early in life, resulting in a relatively uniform prevalence across adult age groups [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Similarly, no established biological mechanism links HHV-6 susceptibility to ABO or Rh blood group antigens, and previous epidemiological studies rarely report any such association. Hematological findings among HHV-6\u0026ndash;positive donors showed a pattern of slightly reduced total WBC counts and modest lymphocytosis compared with negative donors. Although these differences were statistically insignificant, they resemble transient hematological alterations described during HHV-6 reactivation or low-level viremia [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, given the very small number of positive donors and the wide confidence intervals, these findings should be interpreted cautiously and considered exploratory rather than indicative of a true biological effect. Larger studies with more robust analytical power are needed to clarify whether mild hematological changes accompany HHV-6 viremia in asymptomatic adults. Genotyping attempts for HHV-6A and HHV-6B were unsuccessful for all five positive samples, most likely due to the low viral loads reflected by high Ct values (38.7\u0026ndash;39.9). This phenomenon has been described in previous studies, where samples with low-level viremia or inherited chromosomally integrated HHV-6 (ciHHV-6) show limited amplification efficiency, leading to \u0026ldquo;Undetermined\u0026rdquo; genotype results [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Advanced molecular approaches such as droplet digital PCR, nested PCR, or next-generation sequencing may be required in future research to improve detection sensitivity and enable reliable genotype differentiation in low-copy samples. Although HHV-6 is not routinely screened in blood donation programs, the virus has recognized clinical significance in immunocompromised individuals. HHV-6 reactivation has been associated with severe complications, including encephalitis, pneumonitis, delayed engraftment, and graft-versus-host disease, especially among hematopoietic stem-cell transplant (HSCT) recipients [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Studies from Egypt and Germany have documented high reactivation rates and clinically significant outcomes in transplant settings [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. While the present study does not assess transfusion transmission directly, detecting HHV-6 DNA in blood donors highlights a potential concern for high-risk recipients. Accordingly, these results support the need for further research to determine whether HHV-6 viremia in donors may play a role in transfusion-related infection or reactivation in vulnerable patients. The findings also contribute to the broader understanding of transfusion-transmissible infections (TTIs) in Sudan, where data on emerging viral pathogens remain limited. Strengthening molecular surveillance\u0026mdash;especially in high-risk recipient groups\u0026mdash;may enhance early detection and support evidence-based policy development. Implementing pilot screening strategies, cost-effectiveness analyses, and integration of more sensitive molecular assays into TTI monitoring systems may ultimately improve transfusion safety in resource-limited settings. Overall, although the prevalence observed in this study is low, the detection of HHV-6 DNA among asymptomatic donors represents an important initial step in establishing baseline epidemiological data for Sudan. Future multicenter studies incorporating larger sample sizes, serological testing, and more sensitive molecular methods are warranted to better characterize the epidemiology and transmission potential of HHV-6 in the Sudanese population.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eThis study had several limitations. First, the relatively small number of HHV-6\u0026ndash;positive cases (n\u0026thinsp;=\u0026thinsp;5) limits statistical power and prevents meaningful subgroup analysis. Second, the use of convenience sampling at a single center may reduce the generalizability of the findings to other regions in Sudan, especially rural areas, because the samples are unlikely to represent the diverse socioeconomic and healthcare profiles of the broader population. Third, the exclusive inclusion of male donors does not reflect the prevalence in the general population and may skew the results, particularly when it comes to health and risk factors. Fourth, the inability to genotype HHV-6A and HHV-6B was likely due to the low viral load and limited sensitivity of the multiplex PCR assay used. Finally, the absence of serological testing prevented the distinction between active, latent, and chromosomally integrated HHV-6.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eHHV-6 DNA was detected at a low prevalence among blood donors in Kassala, Sudan. The absence of genotype identification and the small number of positive cases reflect the need for larger multicenter studies using more sensitive molecular methods such as digital PCR or nested PCR. Future investigations incorporating both molecular and serological approaches would improve understanding of HHV-6 epidemiology and help determine its relevance to transfusion safety in low-resource settings.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eaGVHD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAcute Graft-versus-Host Disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCBC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eComplete Blood Count\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eciHHV-6\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChromosomally Integrated HHV-6\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCt\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCycle Threshold\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDNA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDeoxyribonucleic Acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHHV-6\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman Herpesvirus 6\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRT-PCR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReal-Time Polymerase Chain Reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTTIs\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransfusion-Transmitted Infections\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDM and NA conceived and designed the study. DM, YA, DI, LA, GM, AA, and NA contributed to the\u0026nbsp;data collection, analysis, and interpretation of the results. NA drafted and revised the manuscript and performed statistical analyses. All authors critically reviewed and approved the final version of this manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no funding from public, commercial, or non-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research data are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was reviewed and approved by the Ethical Review Board of the\u0026nbsp;Karary University Postgraduate Ethics Committee under Protocol No.\u003cstrong\u003e[KU-PG-2022-011]\u003c/strong\u003e and the Sudan Ministry of Health Research Ethics Committee under Reference Number: \u003cstrong\u003e[MOH-ERC-2022-045].\u003c/strong\u003e All participants were informed of the purpose, procedures, and potential benefits of the study. Written informed consent was obtained from each donor before participation. The confidentiality of donor information was strictly maintained, and all data were used solely for research purposes. All sample collection procedures were conducted between March 2024 and July 2024, entirely within the approved ethical clearance period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors provide consent for publication of the findings presented in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCheema S, Rana V, Kulhari K, Yadav A, Sachdeva A. Prevalence of transfusion transmissible infections and associated factors among healthy blood donors in the North Indian population\u0026ndash;4-year experience of licensed blood bank at a tertiary care hospital. Journal of Marine Medical Society. 2022 Jul 1;24(Suppl 1):S47-52.\u003c/li\u003e\n\u003cli\u003eWorld Health Organization (WHO). Screening donated blood for transfusion-transmissible infections: recommendations. Geneva: WHO; 2009.\u003c/li\u003e\n\u003cli\u003eNakayama H, Yamazaki R, Kato J, Koda Y, Sakurai M, Mori T. Impact of Specific Antibody Level on Human Herpesvirus 6 Reactivation after Allogeneic Stem Cell Transplantation. \u003cem\u003eTransplant Cell Ther\u003c/em\u003e. 2021;27(2):174.e1-174.e5. doi:10.1016/j.jtct.2020.10.011.\u003c/li\u003e\n\u003cli\u003eSalahuddin SZ, Ablashi DV, Markham PD, et al. Isolation of a new virus, HBLV, in patients with lymphoproliferative disorders. \u003cem\u003eScience\u003c/em\u003e. 1986;234(4776):596-601. doi:10.1126/science.2876520\u003c/li\u003e\n\u003cli\u003eCaselli E, D\u0026apos;Accolti M, Caccuri F, et al. The U94 Gene of Human Herpesvirus 6: A Narrative Review of Its Role and Potential Functions. \u003cem\u003eCells\u003c/em\u003e. 2020;9(12):2608. Published 2020 Dec 4. doi:10.3390/cells9122608.\u003c/li\u003e\n\u003cli\u003eAgut H, Bonnafous P, Gautheret-Dejean A. Laboratory and clinical aspects of human herpesvirus 6 infections. \u003cem\u003eClin Microbiol Rev\u003c/em\u003e. 2015;28(2):313-335. doi:10.1128/CMR.00122-14.\u003c/li\u003e\n\u003cli\u003eYamanishi K, Okuno T, Shiraki K, et al. Identification of human herpesvirus-6 as a causal agent for exanthem subitum. \u003cem\u003eLancet\u003c/em\u003e. 1988;1(8594):1065-1067. doi:10.1016/s0140-6736(88)91893-4.\u003c/li\u003e\n\u003cli\u003ePhan TL, Carlin K, Ljungman P, et al. Human Herpesvirus-6B Reactivation Is a Risk Factor for Grades II to IV Acute Graft-versus-Host Disease after Hematopoietic Stem Cell Transplantation: A Systematic Review and Meta-Analysis. \u003cem\u003eBiol Blood Marrow Transplant\u003c/em\u003e. 2018;24(11):2324-2336. doi:10.1016/j.bbmt.2018.04.021.\u003c/li\u003e\n\u003cli\u003eCaserta MT, Mock DJ, Dewhurst S. Human herpesvirus 6. \u003cem\u003eClin Infect Dis\u003c/em\u003e. 2001;33(6):829-833. doi:10.1086/322691.\u003c/li\u003e\n\u003cli\u003eMullins TB, Krishnamurthy K. Roseola Infantum. [Updated 2023 Jul 4]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK448190. \u003c/li\u003e\n\u003cli\u003ePantry SN, Medveczky PG. Latency, Integration, and Reactivation of Human Herpesvirus-6. \u003cem\u003eViruses\u003c/em\u003e. 2017;9(7):194. Published 2017 Jul 24. doi:10.3390/v9070194.\u003c/li\u003e\n\u003cli\u003eKing O, Syed HA, Al Khalili Y. Human Herpesvirus 6. In: StatPearls. Treasure Island (FL): StatPearls Publishing; May 5, 2025. \u003c/li\u003e\n\u003cli\u003eYoshihara S, Kato R, Inoue T, et al. Successful treatment of life-threatening human herpesvirus-6 encephalitis with donor lymphocyte infusion in a patient who had undergone human leukocyte antigen-haploidentical nonmyeloablative stem cell transplantation. \u003cem\u003eTransplantation\u003c/em\u003e.2004;77(6):835-838. doi:10.1097/01.tp.0000119603.59880.47. \u003c/li\u003e\n\u003cli\u003eWang X, Patel SA, Haddadin M, Cerny J. Post-allogeneic hematopoietic stem cell transplantation viral reactivations and viremias: a focused review on human herpesvirus-6, BK virus and adenovirus. \u003cem\u003eTher Adv Infect Dis\u003c/em\u003e. 2021;8:20499361211018027. Published 2021 May 24. doi:10.1177/20499361211018027.\u003c/li\u003e\n\u003cli\u003eRaouf MME, Ouf NM, Elsorady MAS, Ghoneim FM. Human herpesvirus-6 in hematopoietic stem cell transplant recipients: a prospective cohort study in Egypt. \u003cem\u003eVirol J\u003c/em\u003e. 2023;20(1):20. Published 2023 Feb 4. doi:10.1186/s12985-023-01980-w.\u003c/li\u003e\n\u003cli\u003ePellett PE, Ablashi DV, Ambros PF, et al. Chromosomally integrated human herpesvirus 6: questions and answers. Rev Med Virol. 2012;22(3):144-155. doi:10.1002/rmv.715.\u003c/li\u003e\n\u003cli\u003eGabrielli L, Balboni A, Borgatti EC, et al. Inherited Chromosomally Integrated Human Herpesvirus 6: Laboratory and Clinical Features. Microorganisms. 2023;11(3):548. Published 2023 Feb 21. doi:10.3390/microorganisms11030548.\u003c/li\u003e\n\u003cli\u003eLuppi M, Barozzi P, Bosco R, et al. Human herpesvirus 6 latency characterized by high viral load: chromosomal integration in many, but not all, cells. J Infect Dis. 2006;194(7):1020-1023. doi:10.1086/506952.\u003c/li\u003e\n\u003cli\u003eLautenschlager I, Razonable RR. Human herpesvirus-6 infections in kidney, liver, lung, and heart transplantation: review. Transpl Int. 2012;25(5):493-502. doi:10.1111/j.1432-2277.2012.01443.x.\u003c/li\u003e\n\u003cli\u003eAl-Sadeq DW, Zedan HT, Aldewik N, et al. Human herpes simplex virus-6 (HHV-6) detection and seroprevalence among Qatari nationals and immigrants residing in Qatar. IJID Reg. 2021;2:90-95. Published 2021 Dec 16. doi:10.1016/j.ijregi.2021.12.005.\u003c/li\u003e\n\u003cli\u003eBates M, Monze M, Bima H, et al. Predominant human herpesvirus 6 variant A infant infections in an HIV-1 endemic region of Sub-Saharan Africa. \u003cem\u003eJ Med Virol\u003c/em\u003e. 2009;81(5):779-789. doi:10.1002/jmv.21455\u003c/li\u003e\n\u003cli\u003eTraore L, Tao I, Bisseye C, et al. Molecular diagnosis of cytomegalovirus, Epstein-Barr virus, and Herpes virus 6 infections among blood donors by multiplex real-time PCR in Ouagadougou, Burkina Faso. \u003cem\u003ePan Afr Med J\u003c/em\u003e. 2016;24:298. Published 2016 Aug 3. doi:10.11604/pamj.2016.24.298.6578\u003c/li\u003e\n\u003cli\u003eBlood Donor Selection: Guidelines on Assessing Donor Suitability for Blood Donation. Geneva: World Health Organization; 2012. 4, General donor assessment. Available from: https://www.ncbi.nlm.nih.gov/books/NBK138219/\u003c/li\u003e\n\u003cli\u003eZheng Y, Zhao Y, Wang Y, Rao J. A multiplex real-time PCR quantitation of human herpesvirus-6, 7, 8 viruses: application in blood transfusions. \u003cem\u003eVirol J\u003c/em\u003e. 2021;18(1):38. Published 2021 Feb 18. doi:10.1186/s12985-021-01510-6\u003c/li\u003e\n\u003cli\u003eLeibovitch EC, Brunetto GS, Caruso B, et al. Coinfection of human herpesviruses 6A (HHV-6A) and HHV-6B as demonstrated by novel digital droplet PCR assay. \u003cem\u003ePLoS One\u003c/em\u003e. 2014;9(3):e92328. Published 2014 Mar 24. doi:10.1371/journal.pone.0092328\u003c/li\u003e\n\u003cli\u003eRouka E, Kyriakou D. Molecular epidemiology of human Herpesviruses types 1-6 and 8 among Greek blood donors. \u003cem\u003eTransfus Med\u003c/em\u003e. 2015;25(4):276-279. doi:10.1111/tme.12202\u003c/li\u003e\n\u003cli\u003eHentrich M, Oruzio D, J\u0026auml;ger G, et al. Impact of human herpesvirus-6 after haematopoietic stem cell transplantation. \u003cem\u003eBr J Haematol\u003c/em\u003e. 2005;128(1):66-72. doi:10.1111/j.1365-2141.2004.05254.x\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HHV-6, Blood donors, Prevalence, Real-time PCR, Kassala, Transfusion safety","lastPublishedDoi":"10.21203/rs.3.rs-8043922/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8043922/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Human herpesvirus 6 (HHV-6) is a latent beta-herpesvirus with potential transfusion-transmissible relevance, particularly in immunocompromised recipients. Data on HHV-6 viremia among Sudanese blood donors are extremely limited. This study aimed to determine the molecular prevalence of HHV-6 DNA among blood donors in Kassala, Sudan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eA descriptive cross-sectional study was conducted at Kassala Teaching Hospital. Whole-blood samples (n = 180) were screened for HHV-6 DNA using real-time PCR, followed by multiplex PCR for genotyping attempts. Fisher’s exact and Mann–Whitney U tests were used where applicable; however, statistical analyses were interpreted cautiously due to the small number of positive cases.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eHHV-6 DNA was detected in 2.78% (5/180) of donors. No significant associations were found between HHV-6 positivity and age group, region, or ABO/Rh blood groups. Hematological parameters showed no significant differences between HHV-6–positive and negative donors. Genotyping was unsuccessful for all positive samples, likely due to low viral load (Ct values 38.7–39.9).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eHHV-6 viremia was infrequent among blood donors in Kassala. Given the very small number of positive cases and limited genotyping capacity, the findings should be considered preliminary. Larger multicenter studies incorporating more sensitive molecular methods are recommended to characterize the epidemiology of HHV-6 in Sudan.\u003c/p\u003e","manuscriptTitle":"Molecular Detection and Prevalence of Human Herpesvirus 6 (HHV-6) Among Male Blood Donors in Kassala, Sudan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-17 14:06:36","doi":"10.21203/rs.3.rs-8043922/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"85f7eea5-12bb-44c3-8863-c45e32a13bbb","owner":[],"postedDate":"December 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-17T07:55:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-17 14:06:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8043922","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8043922","identity":"rs-8043922","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}